From 009c5deb67c2be74ea057f75a42b9dae0728c9b8 Mon Sep 17 00:00:00 2001 From: DW <49634738+zd105@users.noreply.github.com> Date: Mon, 30 Sep 2019 17:08:11 +0800 Subject: [PATCH] Add files via upload --- RRBS.v1.1.pipeline | 363 +++++ config.template | 91 ++ database/Control.fa | 972 +++++++++++++ samples.map.template | 19 + script/00_FQ_trim.2.pl | 44 + script/01_FQ_pas.pl | 62 + script/02_FQ_clean.2.pl | 138 ++ script/02_FQ_stat.pl | 49 + script/BS_CleanFastqc_batch.v1.0.pl | 79 + script/BS_DMRannotate.v1.1.pl | 141 ++ script/BS_DMRdetect.v1.1.pl | 162 +++ script/BS_ElementMethy_batch.v1.1.pl | 121 ++ script/BS_ElementMethy_batch.v1.2.pl | 132 ++ script/BS_GlobalMethy_batch.v1.0.pl | 79 + script/BS_Globalpattern_batch.v1.0.pl | 108 ++ script/BS_bedgraph_batch.v1.0.pl | 80 ++ script/BS_makeMatrix.v1.2.pl | 137 ++ script/BS_qc_stat.v1.0.pl | 83 ++ script/BS_stackbar.v1.0.pl | 84 ++ script/CGI.element_finder.v1.0.sh | 40 + script/CGI.element_finder_forUCSC.v1.0.sh | 40 + script/Draw_venn.pl | 264 ++++ script/EAGE_function.v1.0.pl | 70 + script/HCLUST.R | 22 + script/PCA.R | 79 + script/PCC.R | 48 + script/PVCLUST.R | 38 + script/RawFastqc_batch.v1.0.pl | 78 + script/RawFastqc_batch.v1.1.pl | 78 + script/Venn.pl | 170 +++ script/averge_methyl.barplot.R | 24 + script/cut_rrbs.pl | 66 + script/cut_rrbs.v1.0.pl | 139 ++ script/disposeOverlap.pl | 1503 ++++++++++++++++++++ script/makeMatrix_cluster.pl | 169 +++ script/makeMatrix_merge.pl | 177 +++ script/merge_methy_stat.v1.0.sh | 31 + script/methy_stack_bar.v1.1.R | 112 ++ script/methy_type.pieplot.R | 39 + script/metilene_output.v_p-value_filter.R | 105 ++ script/metilene_output.v_p-value_filter.pl | 201 +++ script/metilene_output.v_q-value_filter.R | 105 ++ script/metilene_output.v_q-value_filter.pl | 199 +++ script/p01.DMR-detector.v1.2.pl | 200 +++ script/p02.DMR-annotator.pl | 120 ++ script/p03.DMR-ploter.pl | 406 ++++++ script/parse_NCBI_gff3.sh | 97 ++ script/parse_UCSC_refGene.sh | 240 ++++ script/parse_UCSC_repeats.sh | 12 + script/plot_DMR.annotate.R | 89 ++ script/plot_element_methy.R | 42 + script/plot_element_methy.v1.0.pl | 82 ++ script/plot_group_element_methy.v1.0.R | 30 + script/plot_sample_element_methy.v1.0.R | 30 + script/pointTopoint.pl | 26 + script/qc_stat.v1.0.sh | 31 + script/rrbs_control_batch.v1.0.pl | 115 ++ script/rrbs_cout_batch.v1.0.pl | 76 + script/rrbs_final.stat.v1.1.sh | 153 ++ script/rrbs_fq_clean.v1.1.pl | 116 ++ script/rrbs_fq_clean.v1.2.pl | 116 ++ script/rrbs_mapping_batch.v1.0.pl | 132 ++ script/stat-fa_GC_detect.pl | 60 + script/venn.main.v1.0.pl | 52 + script/z01.accu_cover.R | 28 + script/z01.dep_coverage.pl | 131 ++ script/z01.methyl_distr.R | 32 + script/z02.get_seq.pl | 115 ++ script/z02.mC_seqlogo.sh | 22 + script/z02.seqlogo.R | 30 + script/z03.global_pattern.R | 46 + script/z03.global_pattern.pl | 88 ++ script/z04.elements_methyl.R | 83 ++ script/z04.elements_methyl.pl | 90 ++ script/z04.elements_methyl.repeats.R | 90 ++ script/z04.elements_methyl.sh | 67 + script/z04.elements_methyl.v1.1.sh | 75 + software/bsmap-2.73.zip | Bin 0 -> 2982558 bytes software/metilene_v0.2-7.zip | Bin 0 -> 2459215 bytes 79 files changed, 9863 insertions(+) create mode 100644 RRBS.v1.1.pipeline create mode 100644 config.template create mode 100644 database/Control.fa create mode 100644 samples.map.template create mode 100644 script/00_FQ_trim.2.pl create mode 100644 script/01_FQ_pas.pl create mode 100644 script/02_FQ_clean.2.pl create mode 100644 script/02_FQ_stat.pl create mode 100644 script/BS_CleanFastqc_batch.v1.0.pl create mode 100644 script/BS_DMRannotate.v1.1.pl create mode 100644 script/BS_DMRdetect.v1.1.pl create mode 100644 script/BS_ElementMethy_batch.v1.1.pl create mode 100644 script/BS_ElementMethy_batch.v1.2.pl create mode 100644 script/BS_GlobalMethy_batch.v1.0.pl create mode 100644 script/BS_Globalpattern_batch.v1.0.pl create mode 100644 script/BS_bedgraph_batch.v1.0.pl create mode 100644 script/BS_makeMatrix.v1.2.pl create mode 100644 script/BS_qc_stat.v1.0.pl create mode 100644 script/BS_stackbar.v1.0.pl create mode 100644 script/CGI.element_finder.v1.0.sh create mode 100644 script/CGI.element_finder_forUCSC.v1.0.sh create mode 100644 script/Draw_venn.pl create mode 100644 script/EAGE_function.v1.0.pl create mode 100644 script/HCLUST.R create mode 100644 script/PCA.R create mode 100644 script/PCC.R create mode 100644 script/PVCLUST.R create mode 100644 script/RawFastqc_batch.v1.0.pl create mode 100644 script/RawFastqc_batch.v1.1.pl create mode 100644 script/Venn.pl create mode 100644 script/averge_methyl.barplot.R create mode 100644 script/cut_rrbs.pl create mode 100644 script/cut_rrbs.v1.0.pl create mode 100644 script/disposeOverlap.pl create mode 100644 script/makeMatrix_cluster.pl create mode 100644 script/makeMatrix_merge.pl create mode 100644 script/merge_methy_stat.v1.0.sh create mode 100644 script/methy_stack_bar.v1.1.R create mode 100644 script/methy_type.pieplot.R create mode 100644 script/metilene_output.v_p-value_filter.R create mode 100644 script/metilene_output.v_p-value_filter.pl create mode 100644 script/metilene_output.v_q-value_filter.R create mode 100644 script/metilene_output.v_q-value_filter.pl create mode 100644 script/p01.DMR-detector.v1.2.pl create mode 100644 script/p02.DMR-annotator.pl create mode 100644 script/p03.DMR-ploter.pl create mode 100644 script/parse_NCBI_gff3.sh create mode 100644 script/parse_UCSC_refGene.sh create mode 100644 script/parse_UCSC_repeats.sh create mode 100644 script/plot_DMR.annotate.R create mode 100644 script/plot_element_methy.R create mode 100644 script/plot_element_methy.v1.0.pl create mode 100644 script/plot_group_element_methy.v1.0.R create mode 100644 script/plot_sample_element_methy.v1.0.R create mode 100644 script/pointTopoint.pl create mode 100644 script/qc_stat.v1.0.sh create mode 100644 script/rrbs_control_batch.v1.0.pl create mode 100644 script/rrbs_cout_batch.v1.0.pl create mode 100644 script/rrbs_final.stat.v1.1.sh create mode 100644 script/rrbs_fq_clean.v1.1.pl create mode 100644 script/rrbs_fq_clean.v1.2.pl create mode 100644 script/rrbs_mapping_batch.v1.0.pl create mode 100644 script/stat-fa_GC_detect.pl create mode 100644 script/venn.main.v1.0.pl create mode 100644 script/z01.accu_cover.R create mode 100644 script/z01.dep_coverage.pl create mode 100644 script/z01.methyl_distr.R create mode 100644 script/z02.get_seq.pl create mode 100644 script/z02.mC_seqlogo.sh create mode 100644 script/z02.seqlogo.R create mode 100644 script/z03.global_pattern.R create mode 100644 script/z03.global_pattern.pl create mode 100644 script/z04.elements_methyl.R create mode 100644 script/z04.elements_methyl.pl create mode 100644 script/z04.elements_methyl.repeats.R create mode 100644 script/z04.elements_methyl.sh create mode 100644 script/z04.elements_methyl.v1.1.sh create mode 100644 software/bsmap-2.73.zip create mode 100644 software/metilene_v0.2-7.zip diff --git a/RRBS.v1.1.pipeline b/RRBS.v1.1.pipeline new file mode 100644 index 0000000..b17d29d --- /dev/null +++ b/RRBS.v1.1.pipeline @@ -0,0 +1,363 @@ +#!/usr/bin/sh +############################################################################################################################## +#### This is a pipeline for RRBS #################### +#### version: v1.1 +#### author: zhangdu +#### date: 2018-08-08 +############################################################################################################################## + +######## 00 data preparation ############### + +# 0.1 Put raw fastq data in $Pj_path/00_rawdata +# 0.2 Creat "samples.map" in $Pj_path/: format: #Group SampleID fq1 fq2 +# 0.3 Modify the "config.template" file according to your project +# 0.4 Run a screen window to config a new project + +######## 01 RRBS data clean ########### +### 1.1 raw fastqc check #### +mkdir -p $Pj_path/01_cleandata/01_raw_fastqc +perl $myscr_path/RawFastqc_batch.v1.1.pl -i $map -p $Pj_path/00_rawdata/ -o $Pj_path/01_cleandata/01_raw_fastqc/ -fastqc $fastqc -t 4 +#|---result files: +# /jobs/ +# all.pbs +cd $Pj_path/01_cleandata/01_raw_fastqc/jobs +sh all.sge +#|---result files: +# sampleid_R1/2._fastqc.html +# sampleid_R1/2._fastqc.zip + +### 1.2 clean ### +perl $myscr_path/rrbs_fq_clean.v1.2.pl -i $map -p $Pj_path/00_rawdata/ -o $Pj_path/01_cleandata/02_clean/ -m 0 -py3 $python3 -cut $cutadapt + +#|---result files: +# /jobs/ +# all.pbs + +# run clean +cd $Pj_path/01_cleandata/02_clean/jobs +sh all.sge +#|---result files: +# sampleid.clean.1/2.fq.gz +# many fq.gz temple files ------delete it after following fq stat step + +### 1.3 fastqc check #### +mkdir $Pj_path/01_cleandata/03_clean_fastqc +perl $myscr_path/BS_CleanFastqc_batch.v1.0.pl -i $map -p $Pj_path/01_cleandata/02_clean/ -o $Pj_path/01_cleandata/03_clean_fastqc/ -fastqc $fastqc -t 4 +#|---result files: +# /jobs/ +# all.pbs +cd $Pj_path/01_cleandata/03_clean_fastqc/jobs +sh all.sge +#|---result files: +# sampleid.clean.1/2._fastqc.html +# sampleid.clean.1/2._fastqc.zip + +### 1.4 data clean stat ### +mkdir $Pj_path/01_cleandata/04_qc_stat +perl $myscr_path/BS_qc_stat.v1.0.pl -i $map -p $Pj_path/01_cleandata/02_clean/ -o $Pj_path/01_cleandata/04_qc_stat/ +#|---result files: +# /jobs/ +# all.pbs +cd $Pj_path/01_cleandata/04_qc_stat/jobs +sh all.sge +#|---result files: +# sampleid.raw.xls +# sampleid.noAdapter.xls +# sampleid.clean.xls + +cd $Pj_path/01_cleandata//04_qc_stat/ +# "YNRBS" is the comon prefix of samples id +sh $myscr_path/qc_stat.v1.0.sh $Pj_path/01_cleandata/04_qc_stat/ YNRBS +#|---result files: +# QC_result.xls + +########### 02 methylation level calling for C site ############## +mkdir $Pj_path/02_methycalling + +######### 2.1 mapping ########### +mkdir $Pj_path/02_methycalling/01_bsmap +#-m :p for pair-end; s for single end. +perl $myscr_path/rrbs_mapping_batch.v1.0.pl -i $map -m p -r $ref_fa -p $Pj_path/01_cleandata/02_clean/ \ +-o $Pj_path/02_methycalling/01_bsmap -t 4 -soft 0 -softdir $bsmap +#|---result files: +# /jobs/ +# all.sge +# all.sge +cd $Pj_path/02_methycalling/01_bsmap/jobs +# for SGE job sub: +sh all.sge +#|---result files: +# sampleid.merge.bam +# sampleid.mapping.stat --------simple mapping result stat + +######## 2.2 generate cout ############ +mkdir $Pj_path/02_methycalling/02_cout +perl $myscr_path/rrbs_cout_batch.v1.0.pl -i $map -r $ref_fa -b $Pj_path/02_methycalling/01_bsmap/ \ +-o $Pj_path/02_methycalling/02_cout/ -py2 $python2 -bsmap $bsmap +#|---result files: +# /jobs/ +# all.sge +# all.pbs +cd $Pj_path/02_methycalling/02_cout/jobs +sh all.sge +#|---result files: +# sampleid.cout.gz + +######## 2.3 methylation calling for lamda DNA ############ +mkdir $Pj_path/02_methycalling/03_control +perl $myscr_path/rrbs_control_batch.v1.0.pl -i $map -m p -r $Control -p $Pj_path/01_cleandata/02_clean/ \ +-o $Pj_path/02_methycalling/03_control/ -bsmap $bsmap -t 8 +#|---result files: +# /jobs/ +# all.sge +# all.pbs + +cd $Pj_path/02_methycalling/03_control/jobs +sh all.sge +#|---result files: +# sampleid.control.cout.gz + +######## 2.4 stat ############### +# "YNRBS" is the prefix of sample name +# "YES" for lamda DNA CT convertion rate stat,else "NO" +mkdir $Pj_path/02_methycalling/04_stat +sh $myscr_path/rrbs_final.stat.v1.1.sh $Pj_path YNRBS YES +#|---result files: +# 01.QC-mapping_stat.xls + + +######## 03 global methylation profile ########## +##### 3.1 coverage and depth ####### +mkdir $Pj_path/03_global_methy +mkdir $Pj_path/03_global_methy/01_coverage_depth +perl $myscr_path/BS_GlobalMethy_batch.v1.0.pl -i $map -r $ref_fa -c $Pj_path/02_methycalling/02_cout/ -o $Pj_path/03_global_methy/01_coverage_depth/ -d 30 -Rscript $Rscript +#|---result files: +# /jobs/ +# all.sge +# all.pbs +cd $Pj_path/03_global_methy/01_coverage_depth/jobs +sh all.sge +#|---result files: +# ./sampleid/04.sampleid_cover_depth.stat.xls +# ./sampleid/sampleid.accu_cover.pdf +# ./sampleid/sampleid.averge_methyl.xls +# ./sampleid/sampleid.coverage_depth.xls +# ./sampleid/sampleid.mCG.seqlogo.pdf +# ./sampleid/sampleid.mCHG.seqlogo.pdf +# ./sampleid/sampleid.mCHH.seqlogo.pdf + +## merge stat result +cd $Pj_path/03_global_methy/01_coverage_depth +sh $myscr_path/merge_methy_stat.v1.0.sh $Pj_path YNRBS $Rscript +#|---result files: +# 04.all.averge_methyl.xls +# 05.all.coverage_depth.xls +# all.averge_methyl.pdf +# global_methy_type.pieplot.pdf + +#### 3.2 global methylation pattern in chr ##### +mkdir $Pj_path/03_global_methy/02_global_pattern +# set the -m to desision the min plot chr length. +perl $myscr_path/BS_Globalpattern_batch.v1.0.pl -i $map -r $ref_fa -c $Pj_path/02_methycalling/02_cout/ \ +-o $Pj_path/03_global_methy/02_global_pattern/ -m 500000 -Rscript $Rscript +#|---result files:/jobs/ + # all.sge +# all.pbs +cd $Pj_path/03_global_methy/02_global_pattern/jobs +sh all.sge +#|---result files: +# genome.500000.chr_list ------------ chr list to plot +# genome.GC_stat ------------genome GC stat +# chr*.global_pattern.xls +# chr*.global_pattern.pdf --------CG,CHG,CHH methylation pattern in chr* + +#### 3.3 gene element methylation level ###### +mkdir $Pj_path/03_global_methy/03_element_methy +perl $myscr_path/BS_ElementMethy_batch.v1.2.pl -i $map -db $ref_db -r $ref_fa -anno $ref_anno -cgi $ref_cgi -repeat $ref_rep -c $Pj_path/02_methycalling/02_cout \ +-o $Pj_path/03_global_methy/03_element_methy -Rscript $Rscript +#|---result files:/jobs/ +# all.sge +# all.pbs +cd $Pj_path/03_global_methy/03_element_methy/jobs +sh all.sge +#|---result files: +# sampleid.genome.cgi/cgi_shore/cgi_shelf/promoter/down1k/down2k/exon/genebody/intergenic/intron/up2k/up1k/exon1/intron1/cds.methy.txt +# sampleid.genome.CG.violin_plot.pdf +# sampleid.genome.genetic.violin_plot.pdf +# sampleid.genome.repeats.violin_plot.pdf + +#plot +perl $myscr_path/plot_element_methy.v1.0.pl -i $map -p $Pj_path/03_global_methy/03_element_methy/ -o $Pj_path/03_global_methy/03_element_methy/ \ +-e cgi,cgi_shore,cgi_shelf,promoter,down1k,down2k,exon,genebody,intergenic,intron,up2k,up1k,exon1,intron1,cds,5utr,3utr,DNA,LINE,LTR,Retroposon,RNA,Satellite,SINE,Low_complexity,Simple_repeat -Rscript $Rscript +#|---result files: +# all.sample.genome.cgi.methy.txt +# all.sample.genome.cgi.methy_vioplot.pdf +# all.group.genome.cgi.methy_vioplot.pdf + + +#### 3.4 global/element methylation change between samples/groups ###### +mkdir $Pj_path/03_global_methy/04_pattern_change + + + + +############# 04 clustering ############## +mkdir $Pj_path/04_Cluster_analyses + +##### 4.1 CG depth filtering and generate .bedgragh ####### +mkdir $Pj_path/04_Cluster_analyses/01_bedgraph +# filtering CG by >= 5x in depth +perl $myscr_path/BS_bedgraph_batch.v1.0.pl -i $map -c $Pj_path/02_methycalling/02_cout -o $Pj_path/04_Cluster_analyses/01_bedgraph -bedtools $bedtools -d 5 +#|---result files:/jobs/ +# all.sge +# all.pbs +cd $Pj_path/04_Cluster_analyses/01_bedgraph/jobs +sh all.sge +#|---result files: +# sampleid.5xCG.bedgraph + +##### 4.2 clustering ##### +mkdir $Pj_path/04_Cluster_analyses/02_clustering +# generate the standard methylation matrix +# PCA, Hierarchical clustering and PCC +perl $myscr_path/BS_makeMatrix.v1.2.pl -i $map -b $Pj_path/04_Cluster_analyses/01_bedgraph -o $Pj_path/04_Cluster_analyses/02_clustering \ +-d 5 -t CG -bedtools $bedtools -Rscript $Rscript +#|---result files: +# /mC/ +# /mCG/ +# /mCHG/ +# /mCHH/ +# 5xC/CG/CHG/CHH.methy_Matrix.txt +# 5xC/CG/CHG/CHH.methy_Matrix.txt.PCC.pdf +# 5xC/CG/CHG/CHH.methy_Matrix.txt.PCA.pdf +# 5xC/CG/CHG/CHH.methy_Matrix.txt.hclust.pdf +# 5xC/CG/CHG/CHH.methy_Matrix.txt.pvclust.pdf + +## a suplymentary methylation stacked bar plot for samples and groups +mkdir $Pj_path/03_global_methy/05_stackbar +perl $myscr_path/BS_stackbar.v1.0.pl -i $map -b $Pj_path/04_Cluster_analyses/01_bedgraph -o $Pj_path/03_global_methy/05_stackbar \ +-t CG,CHG,CHH -d 5 -bedtools $bedtools -Rscript $Rscript +#|---result files: +# all_samples/CON-AraC.5xC/CG/CHG/CHH_stackbar.pdf + +########## 05 DMC finding ########### +mkdir -p $Pj_path/05_DMC_analyses/01_DMC_detection +##### 5.1 DMR detection ##### +# you could use p-value or q-value to filter DMRs +# specify X,Y as you sample number + + + + +######### 06 DMR findng ############ +mkdir -p $Pj_path/06_DMR_analyses/01_DMR_detection +##### 6.1 DMR detection ##### +# you could use p-value or q-value to filter DMRs +# specify X,Y as you sample number +perl $myscr_path/BS_DMRdetect.v1.1.pl -i $map -b $Pj_path/04_Cluster_analyses/01_bedgraph -o $Pj_path/06_DMR_analyses/01_DMR_detection -thread 8 \ +-t CG -d 5 -q 0.05 -maxdist 300 -minc 5 -ele global -diff 0.1 -X 1 -Y 1 -pairwise CON-AraC,CON-DAC -Rscript $Rscript -bedtools $bedtools -metilene $metilene +#|---result files: +# ./mCG/ +# ./CON-AraC/ +# ./CON-DAC/ +# ./global-5x-5CG-dist300-D0.1-Q0.05/ +# CON-AraC.dmr.filter_qval.0.05.bedgraph +# CON-AraC.dmr.filter_qval.0.05.DMR_difference_histogram.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_mC_length_distribution.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_methylation_distribution.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_nt_length_distribution.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_nt-mC_number.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_q_val-difference.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_scatter_plot.pdf +# CON-AraC.dmr.filter_qval.0.05.DMR_violin_plot.pdf +# CON-AraC.dmr.filter_qval.0.05.out.xls +# CON-AraC.dmr.xls --------------DMR information table + +##### 6.2 DMR annotation ##### +mkdir $Pj_path/06_DMR_analyses/02_DMR_annotation +export group="CON-AraC" && export rules="global-5x-5CG-dist300-D0.1-Q0.05" +perl $myscr_path/BS_DMRannotate.v1.1.pl -i $Pj_path/06_DMR_analyses/01_DMR_detection/mCG_DMR/$group/$rules/$group.dmr.filter_qval.0.05.out.xls \ +-p $Pj_path -o $Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group/$rules/ -ele promoter,genebody +#|---result files: +# default.annolist +# $group.dmr.filter_qval.0.05.out.xls.annotate.xls +# $group.dmr.filter_qval.0.05.out.xls.annotate.genebody.genelist.txt +# $group.dmr.filter_qval.0.05.out.xls.annotate.promoter.genelist.txt + +## annotation stat and plot +$Rscript $myscr_path/plot_DMR.annotate.R $Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group/$rules/$group.dmr.filter_qval.0.05.out.xls.annotate.xls \ +$Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group/$rules/$group.dmr.filter_qval.0.05.out.annotate +#|---result files: +# $group.dmr.filter_qval.0.05.out.annotate.stat.xls +# $group.dmr.filter_qval.0.05.out.annotate_barplot.pdf +# $group.dmr.filter_qval.0.05.out.annotate_roseplot.pdf + +##### 6.3 DMG function ##### +mkdir $Pj_path/06_DMR_analyses/03_DMG_function +# mkdir according to you DMR detection type,pairwise group and rule: /mCG_DMR/$group/$rules/ +mkdir -p $Pj_path/06_DMR_analyses/03_DMG_function/mCG_DMR/$group/$rules/ +perl $EAGE/EAGE_function.v1.2.pl \ +-i $Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group/$rules/$group.dmr.filter_qval.0.05.out.xls.annotate.xls.promoter.genelist.txt \ +-o $Pj_path/06_DMR_analyses/03_DMG_function/mCG_DMR/$group/$rules/ -s mmu -q 0.05 + +##### 6.4 DMR plot ##### +mkdir $Pj_path/06_DMR_analyses/04_DMR_plot +mkdir -p $Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/$rules/ +cd $Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/$rules/ + +# select DMR you want to plot in "*.dmr.filter_qval.0.05.out" file, and make a file contain DMRs in the same format as "*.dmr.filter_qval.0.05.out" file. +sort -k4,4g $Pj_path/06_DMR_analyses/01_DMR_detection/mCG_DMR/$group/$rules/$group.dmr.filter_qval.0.05.out.xls | head -21 > \ +$Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/$rules/$group.top20.dmr.list + +vi $Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/plot.sample.list +#== "plot.list" file format: +#CON 2,3,4 +#CASE 5,6,7 + +perl $myscr_path/p03.DMR-ploter.pl \ +--I $Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/$rules/$group.top20.dmr.list \ +--E 1000 --C $Pj_path/06_DMR_analyses/01_DMR_detection/mCG_DMR/$group/$rules/$group.input \ +--F $Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/plot.sample.list \ +--R $ref_fa \ +--G $Pj_path/03_global_methy/03_element_methy/element/genome.promoter.bed \ +--O $Pj_path/06_DMR_analyses/04_DMR_plot/mCG_DMR/$group/$rules/$group.dmr_plot \ +--Rscript $Rscript + + +##### 6.5 DMR ovelap analysis ##### +# DMR venn +export group1="Control-Case1" && export group2="Control-Case2" && export venn="Control-Case1_vs_Control-Case2" +mkdir $Pj_path/06_DMR_analyses/05_DMR_overlap +mkdir -p $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMR/$venn/ + +perl -alne 'next if($.==1);print join "\:",@F[0..2]' $Pj_path/06_DMR_analyses/01_DMR_detection/mCG_DMR/$group1/$rules/$group1.dmr.filter_qval.0.05.out.xls > $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMR/$venn/$group1.Q0.05.dmr.list + +perl -alne 'next if($.==1);print join "\:",@F[0..2]' $Pj_path/06_DMR_analyses/01_DMR_detection/mCG_DMR/$group2/$rules/$group2.dmr.filter_qval.0.05.out.xls > $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMR/$venn/$group2.Q0.05.dmr.list + +perl $myscr_path/venn.main.v1.0.pl \ +-I $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMR/$venn/$group1.Q0.05.dmr.list,$Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMR/$venn/$group2.Q0.05.dmr.list \ +-N $group1,$group2 -Rscript $Rscript \ +-O $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMR/$venn/$venn.DMR + +mkdir -p $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMG/$venn + +# promoter-DMG venn +perl $myscr_path/venn.main.v1.0.pl \ +-I $Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group1/$rules/$group1.dmr.filter_qval.0.05.out.xls.annotate.xls.promoter.genelist.txt,$Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group2/$rules/$group2.dmr.filter_qval.0.05.out.xls.annotate.xls.promoter.genelist.txt \ +-N $group1,$group2 -Rscript $Rscript \ +-O $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMG/$venn/$venn.promoter-DMG + +# genebody-DMG venn +perl $myscr_path/venn.main.v1.0.pl \ +-I $Pj_path/06_DMR_analyses/02_DMR_annotation/mCG_DMR/$group1/$rules/$group1.dmr.filter_qval.0.05.out.xls.annotate.xls.genebody.genelist.txt,$Pj_path/06_DMR_analyses/02_DMR_annota tion/mCG_DMR/$group2/$rules/$group2.dmr.filter_qval.0.05.out.xls.annotate.xls.genebody.genelist.txt \ +-N $group1,$group2 -Rscript $Rscript \ +-O $Pj_path/06_DMR_analyses/05_DMR_overlap/$rules/mCG_DMG/$venn/$venn.genebody-DMG + + + +######### + + + + + diff --git a/config.template b/config.template new file mode 100644 index 0000000..97772db --- /dev/null +++ b/config.template @@ -0,0 +1,91 @@ +######################################################## +### CONFIGURE FILE FOR PIPELINE: RRBS.v1.1.pipeline #### +######################################################## + +############# +# USAGE ##### +############# +# 1. Making a project running environment: +# Run a screen window for a new project, +# the screen should not be quit until the +# project run finished. +# 2. Customize your configure file: +# Modify this configure file according to +# your project. +# 3. Configure your project: +# Cat this configure file in the screen of +# your project, copy and run the output in comandline. +######################################################## + +######################################################### +# Project: your must modified for each new project. ##### +######################################################### + +# the new projet path +export Pj_path=/public/agis/gaofei_group/zhangdu/5_program/06_yongnuo3RRBS/ + +# reference sequence file +export ref_fa=/public/agis/gaofei_group/zhangdu/4_DB/01_genome/mmu/UCSC/mmu10.fa + +# reference annotation file +export ref_anno=/public/agis/gaofei_group/zhangdu/4_DB/01_genome/mmu/UCSC/mmu10.refGene.txt.gz + +# reference database name: UCSC, NCBI or esemble +export ref_db=UCSC + +# reference cgi information file +export ref_cgi=/public/agis/gaofei_group/zhangdu/4_DB/01_genome/mmu/UCSC/mmu10.cpgIslandExt.txt.gz + +# reference repeats information file +export ref_rep=/public/agis/gaofei_group/zhangdu/4_DB/01_genome/mmu/UCSC/mmu10.RepeatMaser.txt.gz + +# samples.map file +export map=/public/agis/gaofei_group/zhangdu/5_program/06_yongnuo3RRBS/samples.map + + +############################################################################################# +# Software: modified only when software upgrade or run pipeline in a new computer cluster ### +############################################################################################# + +# Self-developed scripts path +export myscr_path=/public/agis/gaofei_group/zhangdu/5_program/PIPELINE/RRBS/RRBS.v1.1/script + +# Python 2.7.8 or later version +export python2=/public/software/bin/python + +# Python 3.6.4 or later version +export python3=/public/agis/gaofei_group/zhangdu/2_soft/miniconda3/bin/python + +# fastqc +export fastqc=~/mybin/fastqc + +# cutadapt-1.9 or later version +export cutadapt=/public/agis/gaofei_group/zhangdu/2_soft/cutadapt-1.9/bin/cutadapt + +# Rscript 3.4 or later version +# R package reqiured: [ ggplot2;vioplot;ade4;pvclust;seqLogo;corrplot; ] +export Rscript=/public/home/zhangdu/src/R-3.4.2/builddir/bin/Rscript + +## bsmap, version >= 2.73 +export bsmap=/public/agis/gaofei_group/zhangdu/2_soft/bsmap-2.73/ + +# metilene dir, version >= 0.2-6 +export metilene=/public/agis/gaofei_group/zhangdu/2_soft/metilene_v0.2-6/ + +# EAGE program path +export EAGE=/public/agis/gaofei_group/zhangdu/3_script/EAGE/ + +# bedtools +export bedtools=/public/agis/gaofei_group/zhangdu/2_soft/bedtools2/bin/bedtools + +############### +### Database ## +############### + +# lambda DNA sequence +export Control=/public/agis/gaofei_group/zhangdu/3_script/pipeline/Methylome/WGBS-seq-1.0/Example/reference/Control.fa + + +########### +####END#### +########### diff --git a/database/Control.fa b/database/Control.fa new file mode 100644 index 0000000..f8912ca --- /dev/null +++ b/database/Control.fa @@ -0,0 +1,972 @@ +>Control +GGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAA +GGCGTTTCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACC +CTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGGCTTTTTGGCCT +CTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCA +ACAAAAAGCAGCTGGCTGACATTTTCGGTGCGAGTATCCGTACCATTCAG +AACTGGCAGGAACAGGGAATGCCCGTTCTGCGAGGCGGTGGCAAGGGTAA +TGAGGTGCTTTATGACTCTGCCGCCGTCATAAAATGGTATGCCGAAAGGG +ATGCTGAAATTGAGAACGAAAAGCTGCGCCGGGAGGTTGAAGAACTGCGG +CAGGCCAGCGAGGCAGATCTCCAGCCAGGAACTATTGAGTACGAACGCCA +TCGACTTACGCGTGCGCAGGCCGACGCACAGGAACTGAAGAATGCCAGAG +ACTCCGCTGAAGTGGTGGAAACCGCATTCTGTACTTTCGTGCTGTCGCGG +ATCGCAGGTGAAATTGCCAGTATTCTCGACGGGCTCCCCCTGTCGGTGCA +GCGGCGTTTTCCGGAACTGGAAAACCGACATGTTGATTTCCTGAAACGGG +ATATCATCAAAGCCATGAACAAAGCAGCCGCGCTGGATGAACTGATACCG +GGGTTGCTGAGTGAATATATCGAACAGTCAGGTTAACAGGCTGCGGCATT +TTGTCCGCGCCGGGCTTCGCTCACTGTTCAGGCCGGAGCCACAGACCGCC +GTTGAATGGGCGGATGCTAATTACTATCTCCCGAAAGAATCCGCATACCA +GGAAGGGCGCTGGGAAACACTGCCCTTTCAGCGGGCCATCATGAATGCGA +TGGGCAGCGACTACATCCGTGAGGTGAATGTGGTGAAGTCTGCCCGTGTC +GGTTATTCCAAAATGCTGCTGGGTGTTTATGCCTACTTTATAGAGCATAA +GCAGCGCAACACCCTTATCTGGTTGCCGACGGATGGTGATGCCGAGAACT +TTATGAAAACCCACGTTGAGCCGACTATTCGTGATATTCCGTCGCTGCTG +GCGCTGGCCCCGTGGTATGGCAAAAAGCACCGGGATAACACGCTCACCAT +GAAGCGTTTCACTAATGGGCGTGGCTTCTGGTGCCTGGGCGGTAAAGCGG +CAAAAAACTACCGTGAAAAGTCGGTGGATGTGGCGGGTTATGATGAACTT +GCTGCTTTTGATGATGATATTGAACAGGAAGGCTCTCCGACGTTCCTGGG +TGACAAGCGTATTGAAGGCTCGGTCTGGCCAAAGTCCATCCGTGGCTCCA +CGCCAAAAGTGAGAGGCACCTGTCAGATTGAGCGTGCAGCCAGTGAATCC +CCGCATTTTATGCGTTTTCATGTTGCCTGCCCGCATTGCGGGGAGGAGCA +GTATCTTAAATTTGGCGACAAAGAGACGCCGTTTGGCCTCAAATGGACGC +CGGATGACCCCTCCAGCGTGTTTTATCTCTGCGAGCATAATGCCTGCGTC +ATCCGCCAGCAGGAGCTGGACTTTACTGATGCCCGTTATATCTGCGAAAA +GACCGGGATCTGGACCCGTGATGGCATTCTCTGGTTTTCGTCATCCGGTG +AAGAGATTGAGCCACCTGACAGTGTGACCTTTCACATCTGGACAGCGTAC +AGCCCGTTCACCACCTGGGTGCAGATTGTCAAAGACTGGATGAAAACGAA +AGGGGATACGGGAAAACGTAAAACCTTCGTAAACACCACGCTCGGTGAGA +CGTGGGAGGCGAAAATTGGCGAACGTCCGGATGCTGAAGTGATGGCAGAG +CGGAAAGAGCATTATTCAGCGCCCGTTCCTGACCGTGTGGCTTACCTGAC +CGCCGGTATCGACTCCCAGCTGGACCGCTACGAAATGCGCGTATGGGGAT +GGGGGCCGGGTGAGGAAAGCTGGCTGATTGACCGGCAGATTATTATGGGC +CGCCACGACGATGAACAGACGCTGCTGCGTGTGGATGAGGCCATCAATAA +AACCTATACCCGCCGGAATGGTGCAGAAATGTCGATATCCCGTATCTGCT +GGGATACTGGCGGGATTGACCCGACCATTGTGTATGAACGCTCGAAAAAA +CATGGGCTGTTCCGGGTGATCCCCATTAAAGGGGCATCCGTCTACGGAAA +GCCGGTGGCCAGCATGCCACGTAAGCGAAACAAAAACGGGGTTTACCTTA +CCGAAATCGGTACGGATACCGCGAAAGAGCAGATTTATAACCGCTTCACA +CTGACGCCGGAAGGGGATGAACCGCTTCCCGGTGCCGTTCACTTCCCGAA +TAACCCGGATATTTTTGATCTGACCGAAGCGCAGCAGCTGACTGCTGAAG +AGCAGGTCGAAAAATGGGTGGATGGCAGGAAAAAAATACTGTGGGACAGC +AAAAAGCGACGCAATGAGGCACTCGACTGCTTCGTTTATGCGCTGGCGGC +GCTGCGCATCAGTATTTCCCGCTGGCAGCTGGATCTCAGTGCGCTGCTGG +CGAGCCTGCAGGAAGAGGATGGTGCAGCAACCAACAAGAAAACACTGGCA +GATTACGCCCGTGCCTTATCCGGAGAGGATGAATGACGCGACAGGAAGAA +CTTGCCGCTGCCCGTGCGGCACTGCATGACCTGATGACAGGTAAACGGGT +GGCAACAGTACAGAAAGACGGACGAAGGGTGGAGTTTACGGCCACTTCCG +TGTCTGACCTGAAAAAATATATTGCAGAGCTGGAAGTGCAGACCGGCATG +ACACAGCGACGCAGGGGACCTGCAGGATTTTATGTATGAAAACGCCCACC +ATTCCCACCCTTCTGGGGCCGGACGGCATGACATCGCTGCGCGAATATGC +CGGTTATCACGGCGGTGGCAGCGGATTTGGAGGGCAGTTGCGGTCGTGGA +ACCCACCGAGTGAAAGTGTGGATGCAGCCCTGTTGCCCAACTTTACCCGT +GGCAATGCCCGCGCAGACGATCTGGTACGCAATAACGGCTATGCCGCCAA +CGCCATCCAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCA +GTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGT +GCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGA +CTGCTGCTGCATTGACGTTGAGCGAAAACGCACGTTTACCATGATGATTC +GGGAAGGTGTGGCCATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCC +ACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGT +CAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACT +GCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTAC +GTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGAT +ACCCCGTGAGTTACCCGGCGGGCGCGCCTCGTTCATTCACGTTTTTGAAC +CCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATG +GAGCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTGCAGAGCGC +CATTGTGAAGGCGATGTATGCCGCCACCATTGAGAGTGAGCTGGATACGC +AGTCAGCGATGGATTTTATTCTGGGCGCGAACAGTCAGGAGCAGCGGGAA +AGGCTGACCGGCTGGATTGGTGAAATTGCCGCGTATTACGCCGCAGCGCC +GGTCCGGCTGGGAGGCGCAAAAGTACCGCACCTGATGCCGGGTGACTCAC +TGAACCTGCAGACGGCTCAGGATACGGATAACGGCTACTCCGTGTTTGAG +CAGTCACTGCTGCGGTATATCGCTGCCGGGCTGGGTGTCTCGTATGAGCA +GCTTTCCCGGAATTACGCCCAGATGAGCTACTCCACGGCACGGGCCAGTG +CGAACGAGTCGTGGGCGTACTTTATGGGGCGGCGAAAATTCGTCGCATCC +CGTCAGGCGAGCCAGATGTTTCTGTGCTGGCTGGAAGAGGCCATCGTTCG +CCGCGTGGTGACGTTACCTTCAAAAGCGCGCTTCAGTTTTCAGGAAGCCC +GCAGTGCCTGGGGGAACTGCGACTGGATAGGCTCCGGTCGTATGGCCATC +GATGGTCTGAAAGAAGTTCAGGAAGCGGTGATGCTGATAGAAGCCGGACT +GAGTACCTACGAGAAAGAGTGCGCAAAACGCGGTGACGACTATCAGGAAA +TTTTTGCCCAGCAGGTCCGTGAAACGATGGAGCGCCGTGCAGCCGGTCTT +AAACCGCCCGCCTGGGCGGCTGCAGCATTTGAATCCGGGCTGCGACAATC +AACAGAGGAGGAGAAGAGTGACAGCAGAGCTGCGTAATCTCCCGCATATT +GCCAGCATGGCCTTTAATGAGCCGCTGATGCTTGAACCCGCCTATGCGCG +GGTTTTCTTTTGTGCGCTTGCAGGCCAGCTTGGGATCAGCAGCCTGACGG +ATGCGGTGTCCGGCGACAGCCTGACTGCCCAGGAGGCACTCGCGACGCTG +GCATTATCCGGTGATGATGACGGACCACGACAGGCCCGCAGTTATCAGGT +CATGAACGGCATCGCCGTGCTGCCGGTGTCCGGCACGCTGGTCAGCCGGA +CGCGGGCGCTGCAGCCGTACTCGGGGATGACCGGTTACAACGGCATTATC +GCCCGTCTGCAACAGGCTGCCAGCGATCCGATGGTGGACGGCATTCTGCT +CGATATGGACACGCCCGGCGGGATGGTGGCGGGGGCATTTGACTGCGCTG +ACATCATCGCCCGTGTGCGTGACATAAAACCGGTATGGGCGCTTGCCAAC +GACATGAACTGCAGTGCAGGTCAGTTGCTTGCCAGTGCCGCCTCCCGGCG +TCTGGTCACGCAGACCGCCCGGACAGGCTCCATCGGCGTCATGATGGCTC +ACAGTAATTACGGTGCTGCGCTGGAGAAACAGGGTGTGGAAATCACGCTG +ATTTACAGCGGCAGCCATAAGGTGGATGGCAACCCCTACAGCCATCTTCC +GGATGACGTCCGGGAGACACTGCAGTCCCGGATGGACGCAACCCGCCAGA +TGTTTGCGCAGAAGGTGTCGGCATATACCGGCCTGTCCGTGCAGGTTGTG +CTGGATACCGAGGCTGCAGTGTACAGCGGTCAGGAGGCCATTGATGCCGG +ACTGGCTGATGAACTTGTTAACAGCACCGATGCGATCACCGTCATGCGTG +ATGCACTGGATGCACGTAAATCCCGTCTCTCAGGAGGGCGAATGACCAAA +GAGACTCAATCAACAACTGTTTCAGCCACTGCTTCGCAGGCTGACGTTAC +TGACGTGGTGCCAGCGACGGAGGGCGAGAACGCCAGCGCGGCGCAGCCGG +ACGTGAACGCGCAGATCACCGCAGCGGTTGCGGCAGAAAACAGCCGCATT +ATGGGGATCCTCAACTGTGAGGAGGCTCACGGACGCGAAGAACAGGCACG +CGTGCTGGCAGAAACCCCCGGTATGACCGTGAAAACGGCCCGCCGCATTC +TGGCCGCAGCACCACAGAGTGCACAGGCGCGCAGTGACACTGCGCTGGAT +CGTCTGATGCAGGGGGCACCGGCACCGCTGGCTGCAGGTAACCCGGCATC +TGATGCCGTTAACGATTTGCTGAACACACCAGTGTAAGGGATGTTTATGA +CGAGCAAAGAAACCTTTACCCATTACCAGCCGCAGGGCAACAGTGACCCG +GCTCATACCGCAACCGCGCCCGGCGGATTGAGTGCGAAAGCGCCTGCAAT +GACCCCGCTGATGCTGGACACCTCCAGCCGTAAGCTGGTTGCGTGGGATG +GCACCACCGACGGTGCTGCCGTTGGCATTCTTGCGGTTGCTGCTGACCAG +ACCAGCACCACGCTGACGTTCTACAAGTCCGGCACGTTCCGTTATGAGGA +TGTGCTCTGGCCGGAGGCTGCCAGCGACGAGACGAAAAAACGGACCGCGT +TTGCCGGAACGGCAATCAGCATCGTTTAACTTTACCCTTCATCACTAAAG +GCCGCCTGTGCGGCTTTTTTTACGGGATTTTTTTATGTCGATGTACACAA +CCGCCCAACTGCTGGCGGCAAATGAGCAGAAATTTAAGTTTGATCCGCTG +TTTCTGCGTCTCTTTTTCCGTGAGAGCTATCCCTTCACCACGGAGAAAGT +CTATCTCTCACAAATTCCGGGACTGGTAAACATGGCGCTGTACGTTTCGC +CGATTGTTTCCGGTGAGGTTATCCGTTCCCGTGGCGGCTCCACCTCTGAA +TTTACGCCGGGATATGTCAAGCCGAAGCATGAAGTGAATCCGCAGATGAC +CCTGCGTCGCCTGCCGGATGAAGATCCGCAGAATCTGGCGGACCCGGCTT +ACCGCCGCCGTCGCATCATCATGCAGAACATGCGTGACGAAGAGCTGGCC +ATTGCTCAGGTCGAAGAGATGCAGGCAGTTTCTGCCGTGCTTAAGGGCAA +ATACACCATGACCGGTGAAGCCTTCGATCCGGTTGAGGTGGATATGGGCC +GCAGTGAGGAGAATAACATCACGCAGTCCGGCGGCACGGAGTGGAGCAAG +CGTGACAAGTCCACGTATGACCCGACCGACGATATCGAAGCCTACGCGCT +GAACGCCAGCGGTGTGGTGAATATCATCGTGTTCGATCCGAAAGGCTGGG +CGCTGTTCCGTTCCTTCAAAGCCGTCAAGGAGAAGCTGGATACCCGTCGT +GGCTCTAATTCCGAGCTGGAGACAGCGGTGAAAGACCTGGGCAAAGCGGT +GTCCTATAAGGGGATGTATGGCGATGTGGCCATCGTCGTGTATTCCGGAC +AGTACGTGGAAAACGGCGTCAAAAAGAACTTCCTGCCGGACAACACGATG +GTGCTGGGGAACACTCAGGCACGCGGTCTGCGCACCTATGGCTGCATTCA +GGATGCGGACGCACAGCGCGAAGGCATTAACGCCTCTGCCCGTTACCCGA +AAAACTGGGTGACCACCGGCGATCCGGCGCGTGAGTTCACCATGATTCAG +TCAGCACCGCTGATGCTGCTGGCTGACCCTGATGAGTTCGTGTCCGTACA +ACTGGCGTAATCATGGCCCTTCGGGGCCATTGTTTCTCTGTGGAGGAGTC +CATGACGAAAGATGAACTGATTGCCCGTCTCCGCTCGCTGGGTGAACAAC +TGAACCGTGATGTCAGCCTGACGGGGACGAAAGAAGAACTGGCGCTCCGT +GTGGCAGAGCTGAAAGAGGAGCTTGATGACACGGATGAAACTGCCGGTCA +GGACACCCCTCTCAGCCGGGAAAATGTGCTGACCGGACATGAAAATGAGG +TGGGATCAGCGCAGCCGGATACCGTGATTCTGGATACGTCTGAACTGGTC +ACGGTCGTGGCACTGGTGAAGCTGCATACTGATGCACTTCACGCCACGCG +GGATGAACCTGTGGCATTTGTGCTGCCGGGAACGGCGTTTCGTGTCTCTG +CCGGTGTGGCAGCCGAAATGACAGAGCGCGGCCTGGCCAGAATGCAATAA +CGGGAGGCGCTGTGGCTGATTTCGATAACCTGTTCGATGCTGCCATTGCC +CGCGCCGATGAAACGATACGCGGGTACATGGGAACGTCAGCCACCATTAC +ATCCGGTGAGCAGTCAGGTGCGGTGATACGTGGTGTTTTTGATGACCCTG +AAAATATCAGCTATGCCGGACAGGGCGTGCGCGTTGAAGGCTCCAGCCCG +TCCCTGTTTGTCCGGACTGATGAGGTGCGGCAGCTGCGGCGTGGAGACAC +GCTGACCATCGGTGAGGAAAATTTCTGGGTAGATCGGGTTTCGCCGGATG +ATGGCGGAAGTTGTCATCTCTGGCTTGGACGGGGCGTACCGCCTGCCGTT +AACCGTCGCCGCTGAAAGGGGGATGTATGGCCATAAAAGGTCTTGAGCAG +GCCGTTGAAAACCTCAGCCGTATCAGCAAAACGGCGGTGCCTGGTGCCGC +CGCAATGGCCATTAACCGCGTTGCTTCATCCGCGATATCGCAGTCGGCGT +CACAGGTTGCCCGTGAGACAAAGGTACGCCGGAAACTGGTAAAGGAAAGG +GCCAGGCTGAAAAGGGCCACGGTCAAAAATCCGCAGGCCAGAATCAAAGT +TAACCGGGGGGATTTGCCCGTAATCAAGCTGGGTAATGCGCGGGTTGTCC +TTTCGCGCCGCAGGCGTCGTAAAAAGGGGCAGCGTTCATCCCTGAAAGGT +GGCGGCAGCGTGCTTGTGGTGGGTAACCGTCGTATTCCCGGCGCGTTTAT +TCAGCAACTGAAAAATGGCCGGTGGCATGTCATGCAGCGTGTGGCTGGGA +AAAACCGTTACCCCATTGATGTGGTGAAAATCCCGATGGCGGTGCCGCTG +ACCACGGCGTTTAAACAAAATATTGAGCGGATACGGCGTGAACGTCTTCC +GAAAGAGCTGGGCTATGCGCTGCAGCATCAACTGAGGATGGTAATAAAGC +GATGAAACATACTGAACTCCGTGCAGCCGTACTGGATGCACTGGAGAAGC +ATGACACCGGGGCGACGTTTTTTGATGGTCGCCCCGCTGTTTTTGATGAG +GCGGATTTTCCGGCAGTTGCCGTTTATCTCACCGGCGCTGAATACACGGG +CGAAGAGCTGGACAGCGATACCTGGCAGGCGGAGCTGCATATCGAAGTTT +TCCTGCCTGCTCAGGTGCCGGATTCAGAGCTGGATGCGTGGATGGAGTCC +CGGATTTATCCGGTGATGAGCGATATCCCGGCACTGTCAGATTTGATCAC +CAGTATGGTGGCCAGCGGCTATGACTACCGGCGCGACGATGATGCGGGCT +TGTGGAGTTCAGCCGATCTGACTTATGTCATTACCTATGAAATGTGAGGA +CGCTATGCCTGTACCAAATCCTACAATGCCGGTGAAAGGTGCCGGGACCA +CCCTGTGGGTTTATAAGGGGAGCGGTGACCCTTACGCGAATCCGCTTTCA +GACGTTGACTGGTCGCGTCTGGCAAAAGTTAAAGACCTGACGCCCGGCGA +ACTGACCGCTGAGTCCTATGACGACAGCTATCTCGATGATGAAGATGCAG +ACTGGACTGCGACCGGGCAGGGGCAGAAATCTGCCGGAGATACCAGCTTC +ACGCTGGCGTGGATGCCCGGAGAGCAGGGGCAGCAGGCGCTGCTGGCGTG +GTTTAATGAAGGCGATACCCGTGCCTATAAAATCCGCTTCCCGAACGGCA +CGGTCGATGTGTTCCGTGGCTGGGTCAGCAGTATCGGTAAGGCGGTGACG +GCGAAGGAAGTGATCACCCGCACGGTGAAAGTCACCAATGTGGGACGTCC +GTCGATGGCAGAAGATCGCAGCACGGTAACAGCGGCAACCGGCATGACCG +TGACGCCTGCCAGCACCTCGGTGGTGAAAGGGCAGAGCACCACGCTGACC +GTGGCCTTCCAGCCGGAGGGCGTAACCGACAAGAGCTTTCGTGCGGTGTC +TGCGGATAAAACAAAAGCCACCGTGTCGGTCAGTGGTATGACCATCACCG +TGAACGGCGTTGCTGCAGGCAAGGTCAACATTCCGGTTGTATCCGGTAAT +GGTGAGTTTGCTGCGGTTGCAGAAATTACCGTCACCGCCAGTTAATCCGG +AGAGTCAGCGATGTTCCTGAAAACCGAATCATTTGAACATAACGGTGTGA +CCGTCACGCTTTCTGAACTGTCAGCCCTGCAGCGCATTGAGCATCTCGCC +CTGATGAAACGGCAGGCAGAACAGGCGGAGTCAGACAGCAACCGGAAGTT +TACTGTGGAAGACGCCATCAGAACCGGCGCGTTTCTGGTGGCGATGTCCC +TGTGGCATAACCATCCGCAGAAGACGCAGATGCCGTCCATGAATGAAGCC +GTTAAACAGATTGAGCAGGAAGTGCTTACCACCTGGCCCACGGAGGCAAT +TTCTCATGCTGAAAACGTGGTGTACCGGCTGTCTGGTATGTATGAGTTTG +TGGTGAATAATGCCCCTGAACAGACAGAGGACGCCGGGCCCGCAGAGCCT +GTTTCTGCGGGAAAGTGTTCGACGGTGAGCTGAGTTTTGCCCTGAAACTG +GCGCGTGAGATGGGGCGACCCGACTGGCGTGCCATGCTTGCCGGGATGTC +ATCCACGGAGTATGCCGACTGGCACCGCTTTTACAGTACCCATTATTTTC +ATGATGTTCTGCTGGATATGCACTTTTCCGGGCTGACGTACACCGTGCTC +AGCCTGTTTTTCAGCGATCCGGATATGCATCCGCTGGATTTCAGTCTGCT +GAACCGGCGCGAGGCTGACGAAGAGCCTGAAGATGATGTGCTGATGCAGA +AAGCGGCAGGGCTTGCCGGAGGTGTCCGCTTTGGCCCGGACGGGAATGAA +GTTATCCCCGCTTCCCCGGATGTGGCGGACATGACGGAGGATGACGTAAT +GCTGATGACAGTATCAGAAGGGATCGCAGGAGGAGTCCGGTATGGCTGAA +CCGGTAGGCGATCTGGTCGTTGATTTGAGTCTGGATGCGGCCAGATTTGA +CGAGCAGATGGCCAGAGTCAGGCGTCATTTTTCTGGTACGGAAAGTGATG +CGAAAAAAACAGCGGCAGTCGTTGAACAGTCGCTGAGCCGACAGGCGCTG +GCTGCACAGAAAGCGGGGATTTCCGTCGGGCAGTATAAAGCCGCCATGCG +TATGCTGCCTGCACAGTTCACCGACGTGGCCACGCAGCTTGCAGGCGGGC +AAAGTCCGTGGCTGATCCTGCTGCAACAGGGGGGGCAGGTGAAGGACTCC +TTCGGCGGGATGATCCCCATGTTCAGGGGGCTTGCCGGTGCGATCACCCT +GCCGATGGTGGGGGCCACCTCGCTGGCGGTGGCGACCGGTGCGCTGGCGT +ATGCCTGGTATCAGGGCAACTCAACCCTGTCCGATTTCAACAAAACGCTG +GTCCTTTCCGGCAATCAGGCGGGACTGACGGCAGATCGTATGCTGGTCCT +GTCCAGAGCCGGGCAGGCGGCAGGGCTGACGTTTAACCAGACCAGCGAGT +CACTCAGCGCACTGGTTAAGGCGGGGGTAAGCGGTGAGGCTCAGATTGCG +TCCATCAGCCAGAGTGTGGCGCGTTTCTCCTCTGCATCCGGCGTGGAGGT +GGACAAGGTCGCTGAAGCCTTCGGGAAGCTGACCACAGACCCGACGTCGG +GGCTGACGGCGATGGCTCGCCAGTTCCATAACGTGTCGGCGGAGCAGATT +GCGTATGTTGCTCAGTTGCAGCGTTCCGGCGATGAAGCCGGGGCATTGCA +GGCGGCGAACGAGGCCGCAACGAAAGGGTTTGATGACCAGACCCGCCGCC +TGAAAGAGAACATGGGCACGCTGGAGACCTGGGCAGACAGGACTGCGCGG +GCATTCAAATCCATGTGGGATGCGGTGCTGGATATTGGTCGTCCTGATAC +CGCGCAGGAGATGCTGATTAAGGCAGAGGCTGCGTATAAGAAAGCAGACG +ACATCTGGAATCTGCGCAAGGATGATTATTTTGTTAACGATGAAGCGCGG +GCGCGTTACTGGGATGATCGTGAAAAGGCCCGTCTTGCGCTTGAAGCCGC +CCGAAAGAAGGCTGAGCAGCAGACTCAACAGGACAAAAATGCGCAGCAGC +AGAGCGATACCGAAGCGTCACGGCTGAAATATACCGAAGAGGCGCAGAAG +GCTTACGAACGGCTGCAGACGCCGCTGGAGAAATATACCGCCCGTCAGGA +AGAACTGAACAAGGCACTGAAAGACGGGAAAATCCTGCAGGCGGATTACA +ACACGCTGATGGCGGCGGCGAAAAAGGATTATGAAGCGACGCTGAAAAAG +CCGAAACAGTCCAGCGTGAAGGTGTCTGCGGGCGATCGTCAGGAAGACAG +TGCTCATGCTGCCCTGCTGACGCTTCAGGCAGAACTCCGGACGCTGGAGA +AGCATGCCGGAGCAAATGAGAAAATCAGCCAGCAGCGCCGGGATTTGTGG +AAGGCGGAGAGTCAGTTCGCGGTACTGGAGGAGGCGGCGCAACGTCGCCA +GCTGTCTGCACAGGAGAAATCCCTGCTGGCGCATAAAGATGAGACGCTGG +AGTACAAACGCCAGCTGGCTGCACTTGGCGACAAGGTTACGTATCAGGAG +CGCCTGAACGCGCTGGCGCAGCAGGCGGATAAATTCGCACAGCAGCAACG +GGCAAAACGGGCCGCCATTGATGCGAAAAGCCGGGGGCTGACTGACCGGC +AGGCAGAACGGGAAGCCACGGAACAGCGCCTGAAGGAACAGTATGGCGAT +AATCCGCTGGCGCTGAATAACGTCATGTCAGAGCAGAAAAAGACCTGGGC +GGCTGAAGACCAGCTTCGCGGGAACTGGATGGCAGGCCTGAAGTCCGGCT +GGAGTGAGTGGGAAGAGAGCGCCACGGACAGTATGTCGCAGGTAAAAAGT +GCAGCCACGCAGACCTTTGATGGTATTGCACAGAATATGGCGGCGATGCT +GACCGGCAGTGAGCAGAACTGGCGCAGCTTCACCCGTTCCGTGCTGTCCA +TGATGACAGAAATTCTGCTTAAGCAGGCAATGGTGGGGATTGTCGGGAGT +ATCGGCAGCGCCATTGGCGGGGCTGTTGGTGGCGGCGCATCCGCGTCAGG +CGGTACAGCCATTCAGGCCGCTGCGGCGAAATTCCATTTTGCAACCGGAG +GATTTACGGGAACCGGCGGCAAATATGAGCCAGCGGGGATTGTTCACCGT +GGTGAGTTTGTCTTCACGAAGGAGGCAACCAGCCGGATTGGCGTGGGGAA +TCTTTACCGGCTGATGCGCGGCTATGCCACCGGCGGTTATGTCGGTACAC +CGGGCAGCATGGCAGACAGCCGGTCGCAGGCGTCCGGGACGTTTGAGCAG +AATAACCATGTGGTGATTAACAACGACGGCACGAACGGGCAGATAGGTCC +GGCTGCTCTGAAGGCGGTGTATGACATGGCCCGCAAGGGTGCCCGTGATG +AAATTCAGACACAGATGCGTGATGGTGGCCTGTTCTCCGGAGGTGGACGA +TGAAGACCTTCCGCTGGAAAGTGAAACCCGGTATGGATGTGGCTTCGGTC +CCTTCTGTAAGAAAGGTGCGCTTTGGTGATGGCTATTCTCAGCGAGCGCC +TGCCGGGCTGAATGCCAACCTGAAAACGTACAGCGTGACGCTTTCTGTCC +CCCGTGAGGAGGCCACGGTACTGGAGTCGTTTCTGGAAGAGCACGGGGGC +TGGAAATCCTTTCTGTGGACGCCGCCTTATGAGTGGCGGCAGATAAAGGT +GACCTGCGCAAAATGGTCGTCGCGGGTCAGTATGCTGCGTGTTGAGTTCA +GCGCAGAGTTTGAACAGGTGGTGAACTGATGCAGGATATCCGGCAGGAAA +CACTGAATGAATGCACCCGTGCGGAGCAGTCGGCCAGCGTGGTGCTCTGG +GAAATCGACCTGACAGAGGTCGGTGGAGAACGTTATTTTTTCTGTAATGA +GCAGAACGAAAAAGGTGAGCCGGTCACCTGGCAGGGGCGACAGTATCAGC +CGTATCCCATTCAGGGGAGCGGTTTTGAACTGAATGGCAAAGGCACCAGT +ACGCGCCCCACGCTGACGGTTTCTAACCTGTACGGTATGGTCACCGGGAT +GGCGGAAGATATGCAGAGTCTGGTCGGCGGAACGGTGGTCCGGCGTAAGG +TTTACGCCCGTTTTCTGGATGCGGTGAACTTCGTCAACGGAAACAGTTAC +GCCGATCCGGAGCAGGAGGTGATCAGCCGCTGGCGCATTGAGCAGTGCAG +CGAACTGAGCGCGGTGAGTGCCTCCTTTGTACTGTCCACGCCGACGGAAA +CGGATGGCGCTGTTTTTCCGGGACGTATCATGCTGGCCAACACCTGCACC +TGGACCTATCGCGGTGACGAGTGCGGTTATAGCGGTCCGGCTGTCGCGGA +TGAATATGACCAGCCAACGTCCGATATCACGAAGGATAAATGCAGCAAAT +GCCTGAGCGGTTGTAAGTTCCGCAATAACGTCGGCAACTTTGGCGGCTTC +CTTTCCATTAACAAACTTTCGCAGTAAATCCCATGACACAGACAGAATCA +GCGATTCTGGCGCACGCCCGGCGATGTGCGCCAGCGGAGTCGTGCGGCTT +CGTGGTAAGCACGCCGGAGGGGGAAAGATATTTCCCCTGCGTGAATATCT +CCGGTGAGCCGGAGGCTATTTCCGTATGTCGCCGGAAGACTGGCTGCAGG +CAGAAATGCAGGGTGAGATTGTGGCGCTGGTCCACAGCCACCCCGGTGGT +CTGCCCTGGCTGAGTGAGGCCGACCGGCGGCTGCAGGTGCAGAGTGATTT +GCCGTGGTGGCTGGTCTGCCGGGGGACGATTCATAAGTTCCGCTGTGTGC +CGCATCTCACCGGGCGGCGCTTTGAGCACGGTGTGACGGACTGTTACACA +CTGTTCCGGGATGCTTATCATCTGGCGGGGATTGAGATGCCGGACTTTCA +TCGTGAGGATGACTGGTGGCGTAACGGCCAGAATCTCTATCTGGATAATC +TGGAGGCGACGGGGCTGTATCAGGTGCCGTTGTCAGCGGCACAGCCGGGC +GATGTGCTGCTGTGCTGTTTTGGTTCATCAGTGCCGAATCACGCCGCAAT +TTACTGCGGCGACGGCGAGCTGCTGCACCATATTCCTGAACAACTGAGCA +AACGAGAGAGGTACACCGACAAATGGCAGCGACGCACACACTCCCTCTGG +CGTCACCGGGCATGGCGCGCATCTGCCTTTACGGGGATTTACAACGATTT +GGTCGCCGCATCGACCTTCGTGTGAAAACGGGGGCTGAAGCCATCCGGGC +ACTGGCCACACAGCTCCCGGCGTTTCGTCAGAAACTGAGCGACGGCTGGT +ATCAGGTACGGATTGCCGGGCGGGACGTCAGCACGTCCGGGTTAACGGCG +CAGTTACATGAGACTCTGCCTGATGGCGCTGTAATTCATATTGTTCCCAG +AGTCGCCGGGGCCAAGTCAGGTGGCGTATTCCAGATTGTCCTGGGGGCTG +CCGCCATTGCCGGATCATTCTTTACCGCCGGAGCCACCCTTGCAGCATGG +GGGGCAGCCATTGGGGCCGGTGGTATGACCGGCATCCTGTTTTCTCTCGG +TGCCAGTATGGTGCTCGGTGGTGTGGCGCAGATGCTGGCACCGAAAGCCA +GAACTCCCCGTATACAGACAACGGATAACGGTAAGCAGAACACCTATTTC +TCCTCACTGGATAACATGGTTGCCCAGGGCAATGTTCTGCCTGTTCTGTA +CGGGGAAATGCGCGTGGGGTCACGCGTGGTTTCTCAGGAGATCAGCACGG +CAGACGAAGGGGACGGTGGTCAGGTTGTGGTGATTGGTCGCTGATGCAAA +ATGTTTTATGTGAAACCGCCTGCGGGCGGTTTTGTCATTTATGGAGCGTG +AGGAATGGGTAAAGGAAGCAGTAAGGGGCATACCCCGCGCGAAGCGAAGG +ACAACCTGAAGTCCACGCAGTTGCTGAGTGTGATCGATGCCATCAGCGAA +GGGCCGATTGAAGGTCCGGTGGATGGCTTAAAAAGCGTGCTGCTGAACAG +TACGCCGGTGCTGGACACTGAGGGGAATACCAACATATCCGGTGTCACGG +TGGTGTTCCGGGCTGGTGAGCAGGAGCAGACTCCGCCGGAGGGATTTGAA +TCCTCCGGCTCCGAGACGGTGCTGGGTACGGAAGTGAAATATGACACGCC +GATCACCCGCACCATTACGTCTGCAAACATCGACCGTCTGCGCTTTACCT +TCGGTGTACAGGCACTGGTGGAAACCACCTCAAAGGGTGACAGGAATCCG +TCGGAAGTCCGCCTGCTGGTTCAGATACAACGTAACGGTGGCTGGGTGAC +GGAAAAAGACATCACCATTAAGGGCAAAACCACCTCGCAGTATCTGGCCT +CGGTGGTGATGGGTAACCTGCCGCCGCGCCCGTTTAATATCCGGATGCGC +AGGATGACGCCGGACAGCACCACAGACCAGCTGCAGAACAAAACGCTCTG +GTCGTCATACACTGAAATCATCGATGTGAAACAGTGCTACCCGAACACGG +CACTGGTCGGCGTGCAGGTGGACTCGGAGCAGTTCGGCAGCCAGCAGGTG +AGCCGTAATTATCATCTGCGCGGGCGTATTCTGCAGGTGCCGTCGAACTA +TAACCCGCAGACGCGGCAATACAGCGGTATCTGGGACGGAACGTTTAAAC +CGGCATACAGCAACAACATGGCCTGGTGTCTGTGGGATATGCTGACCCAT +CCGCGCTACGGCATGGGGAAACGTCTTGGTGCGGCGGATGTGGATAAATG +GGCGCTGTATGTCATCGGCCAGTACTGCGACCAGTCAGTGCCGGACGGCT +TTGGCGGCACGGAGCCGCGCATCACCTGTAATGCGTACCTGACCACACAG +CGTAAGGCGTGGGATGTGCTCAGCGATTTCTGCTCGGCGATGCGCTGTAT +GCCGGTATGGAACGGGCAGACGCTGACGTTCGTGCAGGACCGACCGTCGG +ATAAGACGTGGACCTATAACCGCAGTAATGTGGTGATGCCGGATGATGGC +GCGCCGTTCCGCTACAGCTTCAGCGCCCTGAAGGACCGCCATAATGCCGT +TGAGGTGAACTGGATTGACCCGAACAACGGCTGGGAGACGGCGACAGAGC +TTGTTGAAGATACGCAGGCCATTGCCCGTTACGGTCGTAATGTTACGAAG +ATGGATGCCTTTGGCTGTACCAGCCGGGGGCAGGCACACCGCGCCGGGCT +GTGGCTGATTAAAACAGAACTGCTGGAAACGCAGACCGTGGATTTCAGCG +TCGGCGCAGAAGGGCTTCGCCATGTACCGGGCGATGTTATTGAAATCTGC +GATGATGACTATGCCGGTATCAGCACCGGTGGTCGTGTGCTGGCGGTGAA +CAGCCAGACCCGGACGCTGACGCTCGACCGTGAAATCACGCTGCCATCCT +CCGGTACCGCGCTGATAAGCCTGGTTGACGGAAGTGGCAATCCGGTCAGC +GTGGAGGTTCAGTCCGTCACCGACGGCGTGAAGGTAAAAGTGAGCCGTGT +TCCTGACGGTGTTGCTGAATACAGCGTATGGGAGCTGAAGCTGCCGACGC +TGCGCCAGCGACTGTTCCGCTGCGTGAGTATCCGTGAGAACGACGACGGC +ACGTATGCCATCACCGCCGTGCAGCATGTGCCGGAAAAAGAGGCCATCGT +GGATAACGGGGCGCACTTTGACGGCGAACAGAGTGGCACGGTGAATGGTG +TCACGCCGCCAGCGGTGCAGCACCTGACCGCAGAAGTCACTGCAGACAGC +GGGGAATATCAGGTGCTGGCGCGATGGGACACACCGAAGGTGGTGAAGGG +CGTGAGTTTCCTGCTCCGTCTGACCGTAACAGCGGACGACGGCAGTGAGC +GGCTGGTCAGCACGGCCCGGACGACGGAAACCACATACCGCTTCACGCAA +CTGGCGCTGGGGAACTACAGGCTGACAGTCCGGGCGGTAAATGCGTGGGG +GCAGCAGGGCGATCCGGCGTCGGTATCGTTCCGGATTGCCGCACCGGCAG +CACCGTCGAGGATTGAGCTGACGCCGGGCTATTTTCAGATAACCGCCACG +CCGCATCTTGCCGTTTATGACCCGACGGTACAGTTTGAGTTCTGGTTCTC +GGAAAAGCAGATTGCGGATATCAGACAGGTTGAAACCAGCACGCGTTATC +TTGGTACGGCGCTGTACTGGATAGCCGCCAGTATCAATATCAAACCGGGC +CATGATTATTACTTTTATATCCGCAGTGTGAACACCGTTGGCAAATCGGC +ATTCGTGGAGGCCGTCGGTCGGGCGAGCGATGATGCGGAAGGTTACCTGG +ATTTTTTCAAAGGCAAGATAACCGAATCCCATCTCGGCAAGGAGCTGCTG +GAAAAAGTCGAGCTGACGGAGGATAACGCCAGCAGACTGGAGGAGTTTTC +GAAAGAGTGGAAGGATGCCAGTGATAAGTGGAATGCCATGTGGGCTGTCA +AAATTGAGCAGACCAAAGACGGCAAACATTATGTCGCGGGTATTGGCCTC +AGCATGGAGGACACGGAGGAAGGCAAACTGAGCCAGTTTCTGGTTGCCGC +CAATCGTATCGCATTTATTGACCCGGCAAACGGGAATGAAACGCCGATGT +TTGTGGCGCAGGGCAACCAGATATTCATGAACGACGTGTTCCTGAAGCGC +CTGACGGCCCCCACCATTACCAGCGGCGGCAATCCTCCGGCCTTTTCCCT +GACACCGGACGGAAAGCTGACCGCTAAAAATGCGGATATCAGTGGCAGTG +TGAATGCGAACTCCGGGACGCTCAGTAATGTGACGATAGCTGAAAACTGT +ACGATAAACGGTACGCTGAGGGCGGAAAAAATCGTCGGGGACATTGTAAA +GGCGGCGAGCGCGGCTTTTCCGCGCCAGCGTGAAAGCAGTGTGGACTGGC +CGTCAGGTACCCGTACTGTCACCGTGACCGATGACCATCCTTTTGATCGC +CAGATAGTGGTGCTTCCGCTGACGTTTCGCGGAAGTAAGCGTACTGTCAG +CGGCAGGACAACGTATTCGATGTGTTATCTGAAAGTACTGATGAACGGTG +CGGTGATTTATGATGGCGCGGCGAACGAGGCGGTACAGGTGTTCTCCCGT +ATTGTTGACATGCCAGCGGGTCGGGGAAACGTGATCCTGACGTTCACGCT +TACGTCCACACGGCATTCGGCAGATATTCCGCCGTATACGTTTGCCAGCG +ATGTGCAGGTTATGGTGATTAAGAAACAGGCGCTGGGCATCAGCGTGGTC +TGAGTGTGTTACAGAGGTTCGTCCGGGAACGGGCGTTTTATTATAAAACA +GTGAGAGGTGAACGATGCGTAATGTGTGTATTGCCGTTGCTGTCTTTGCC +GCACTTGCGGTGACAGTCACTCCGGCCCGTGCGGAAGGTGGACATGGTAC +GTTTACGGTGGGCTATTTTCAAGTGAAACCGGGTACATTGCCGTCGTTGT +CGGGCGGGGATACCGGTGTGAGTCATCTGAAAGGGATTAACGTGAAGTAC +CGTTATGAGCTGACGGACAGTGTGGGGGTGATGGCTTCCCTGGGGTTCGC +CGCGTCGAAAAAGAGCAGCACAGTGATGACCGGGGAGGATACGTTTCACT +ATGAGAGCCTGCGTGGACGTTATGTGAGCGTGATGGCCGGACCGGTTTTA +CAAATCAGTAAGCAGGTCAGTGCGTACGCCATGGCCGGAGTGGCTCACAG +TCGGTGGTCCGGCAGTACAATGGATTACCGTAAGACGGAAATCACTCCCG +GGTATATGAAAGAGACGACCACTGCCAGGGACGAAAGTGCAATGCGGCAT +ACCTCAGTGGCGTGGAGTGCAGGTATACAGATTAATCCGGCAGCGTCCGT +CGTTGTTGATATTGCTTATGAAGGCTCCGGCAGTGGCGACTGGCGTACTG +ACGGATTCATCGTTGGGGTCGGTTATAAATTCTGATTAGCCAGGTAACAC +AGTGTTATGACAGCCCGCCGGAACCGGTGGGCTTTTTTGTGGGGTGAATA +TGGCAGTAAAGATTTCAGGAGTCCTGAAAGACGGCACAGGAAAACCGGTA +CAGAACTGCACCATTCAGCTGAAAGCCAGACGTAACAGCACCACGGTGGT +GGTGAACACGGTGGGCTCAGAGAATCCGGATGAAGCCGGGCGTTACAGCA +TGGATGTGGAGTACGGTCAGTACAGTGTCATCCTGCAGGTTGACGGTTTT +CCACCATCGCACGCCGGGACCATCACCGTGTATGAAGATTCACAACCGGG +GACGCTGAATGATTTTCTCTGTGCCATGACGGAGGATGATGCCCGGCCGG +AGGTGCTGCGTCGTCTTGAACTGATGGTGGAAGAGGTGGCGCGTAACGCG +TCCGTGGTGGCACAGAGTACGGCAGACGCGAAGAAATCAGCCGGCGATGC +CAGTGCATCAGCTGCTCAGGTCGCGGCCCTTGTGACTGATGCAACTGACT +CAGCACGCGCCGCCAGCACGTCCGCCGGACAGGCTGCATCGTCAGCTCAG +GAAGCGTCCTCCGGCGCAGAAGCGGCATCAGCAAAGGCCACTGAAGCGGA +AAAAAGTGCCGCAGCCGCAGAGTCCTCAAAAAACGCGGCGGCCACCAGTG +CCGGTGCGGCGAAAACGTCAGAAACGAATGCTGCAGCGTCACAACAATCA +GCCGCCACGTCTGCCTCCACCGCGGCCACGAAAGCGTCAGAGGCCGCCAC +TTCAGCACGAGATGCGGTGGCCTCAAAAGAGGCAGCAAAATCATCAGAAA +CGAACGCATCATCAAGTGCCGGTCGTGCAGCTTCCTCGGCAACGGCGGCA +GAAAATTCTGCCAGGGCGGCAAAAACGTCCGAGACGAATGCCAGGTCATC +TGAAACAGCAGCGGAACGGAGCGCCTCTGCCGCGGCAGACGCAAAAACAG +CGGCGGCGGGGAGTGCGTCAACGGCATCCACGAAGGCGACAGAGGCTGCG +GGAAGTGCGGTATCAGCATCGCAGAGCAAAAGTGCGGCAGAAGCGGCGGC +AATACGTGCAAAAAATTCGGCAAAACGTGCAGAAGATATAGCTTCAGCTG +TCGCGCTTGAGGATGCGGACACAACGAGAAAGGGGATAGTGCAGCTCAGC +AGTGCAACCAACAGCACGTCTGAAACGCTTGCTGCAACGCCAAAGGCGGT +TAAGGTGGTAATGGATGAAACGAACAGAAAAGCCCACTGGACAGTCCGGC +ACTGACCGGAACGCCAACAGCACCAACCGCGCTCAGGGGAACAAACAATA +CCCAGATTGCGAACACCGCTTTTGTACTGGCCGCGATTGCAGATGTTATC +GACGCGTCACCTGACGCACTGAATACGCTGAATGAACTGGCCGCAGCGCT +CGGGAATGATCCAGATTTTGCTACCACCATGACTAACGCGCTTGCGGGTA +AACAACCGAAGAATGCGACACTGACGGCGCTGGCAGGGCTTTCCACGGCG +AAAAATAAATTACCGTATTTTGCGGAAAATGATGCCGCCAGCCTGACTGA +ACTGACTCAGGTTGGCAGGGATATTCTGGCAAAAAATTCCGTTGCAGATG +TTCTTGAATACCTTGGGGCCGGTGAGAATTCGGCCTTTCCGGCAGGTGCG +CCGATCCCGTGGCCATCAGATATCGTTCCGTCTGGCTACGTCCTGATGCA +GGGGCAGGCGTTTGACAAATCAGCCTACCCAAAACTTGCTGTCGCGTATC +CATCGGGTGTGCTTCCTGATATGCGAGGCTGGACAATCAAGGGGAAACCC +GCCAGCGGTCGTGCTGTATTGTCTCAGGAACAGGATGGAATTAAGTCGCA +CACCCACAGTGCCAGTGCATCCGGTACGGATTTGGGGACGAAAACCACAT +CGTCGTTTGATTACGGGACGAAAACAACAGGCAGTTTCGATTACGGCACC +AAATCGACGAATAACACGGGGGCTCATGCTCACAGTCTGAGCGGTTCAAC +AGGGGCCGCGGGTGCTCATGCCCACACAAGTGGTTTAAGGATGAACAGTT +CTGGCTGGAGTCAGTATGGAACAGCAACCATTACAGGAAGTTTATCCACA +GTTAAAGGAACCAGCACACAGGGTATTGCTTATTTATCGAAAACGGACAG +TCAGGGCAGCCACAGTCACTCATTGTCCGGTACAGCCGTGAGTGCCGGTG +CACATGCGCATACAGTTGGTATTGGTGCGCACCAGCATCCGGTTGTTATC +GGTGCTCATGCCCATTCTTTCAGTATTGGTTCACACGGACACACCATCAC +CGTTAACGCTGCGGGTAACGCGGAAAACACCGTCAAAAACATTGCATTTA +ACTATATTGTGAGGCTTGCATAATGGCATTCAGAATGAGTGAACAACCAC +GGACCATAAAAATTTATAATCTGCTGGCCGGAACTAATGAATTTATTGGT +GAAGGTGACGCATATATTCCGCCTCATACCGGTCTGCCTGCAAACAGTAC +CGATATTGCACCGCCAGATATTCCGGCTGGCTTTGTGGCTGTTTTCAACA +GTGATGAGGCATCGTGGCATCTCGTTGAAGACCATCGGGGTAAAACCGTC +TATGACGTGGCTTCCGGCGACGCGTTATTTATTTCTGAACTCGGTCCGTT +ACCGGAAAATTTTACCTGGTTATCGCCGGGAGGGGAATATCAGAAGTGGA +ACGGCACAGCCTGGGTGAAGGATACGGAAGCAGAAAAACTGTTCCGGATC +CGGGAGGCGGAAGAAACAAAAAAAAGCCTGATGCAGGTAGCCAGTGAGCA +TATTGCGCCGCTTCAGGATGCTGCAGATCTGGAAATTGCAACGAAGGAAG +AAACCTCGTTGCTGGAAGCCTGGAAGAAGTATCGGGTGTTGCTGAACCGT +GTTGATACATCAACTGCACCTGATATTGAGTGGCCTGCTGTCCCTGTTAT +GGAGTAATCGTTTTGTGATATGCCGCAGAAACGTTGTATGAAATAACGTT +CTGCGGTTAGTTAGTATATTGTAAAGCTGAGTATTGGTTTATTTGGCGAT +TATTATCTTCAGGAGAATAATGGAAGTTCTATGACTCAATTGTTCATAGT +GTTTACATCACCGCCAATTGCTTTTAAGACTGAACGCATGAAATATGGTT +TTTCGTCATGTTTTGAGTCTGCTGTTGATATTTCTAAAGTCGGTTTTTTT +TCTTCGTTTTCTCTAACTATTTTCCATGAAATACATTTTTGATTATTATT +TGAATCAATTCCAATTACCTGAAGTCTTTCATCTATAATTGGCATTGTAT +GTATTGGTTTATTGGAGTAGATGCTTGCTTTTCTGAGCCATAGCTCTGAT +ATCCAAATGAAGCCATAGGCATTTGTTATTTTGGCTCTGTCAGCTGCATA +ACGCCAAAAAATATATTTATCTGCTTGATCTTCAAATGTTGTATTGATTA +AATCAATTGGATGGAATTGTTTATCATAAAAAATTAATGTTTGAATGTGA +TAACCGTCCTTTAAAAAAGTCGTTTCTGCAAGCTTGGCTGTATAGTCAAC +TAACTCTTCTGTCGAAGTGATATTTTTAGGCTTATCTACCAGTTTTAGAC +GCTCTTTAATATCTTCAGGAATTATTTTATTGTCATATTGTATCATGCTA +AATGACAATTTGCTTATGGAGTAATCTTTTAATTTTAAATAAGTTATTCT +CCTGGCTTCATCAAATAAAGAGTCGAATGATGTTGGCGAAATCACATCGT +CACCCATTGGATTGTTTATTTGTATGCCAAGAGAGTTACAGCAGTTATAC +ATTCTGCCATAGATTATAGCTAAGGCATGTAATAATTCGTAATCTTTTAG +CGTATTAGCGACCCATCGTCTTTCTGATTTAATAATAGATGATTCAGTTA +AATATGAAGGTAATTTCTTTTGTGCAAGTCTGACTAACTTTTTTATACCA +ATGTTTAACATACTTTCATTTGTAATAAACTCAATGTCATTTTCTTCAAT +GTAAGATGAAATAAGAGTAGCCTTTGCCTCGCTATACATTTCTAAATCGC +CTTGTTTTTCTATCGTATTGCGAGAATTTTTAGCCCAAGCCATTAATGGA +TCATTTTTCCATTTTTCAATAACATTATTGTTATACCAAATGTCATATCC +TATAATCTGGTTTTTGTTTTTTTGAATAATAAATGTTACTGTTCTTGCGG +TTTGGAGGAATTGATTCAAATTCAAGCGAAATAATTCAGGGTCAAAATAT +GTATCAATGCAGCATTTGAGCAAGTGCGATAAATCTTTAAGTCTTCTTTC +CCATGGTTTTTTAGTCATAAAACTCTCCATTTTGATAGGTTGCATGCTAG +ATGCTGATATATTTTAGAGGTGATAAAATTAACTGCTTAACTGTCAATGT +AATACAAGTTGTTTGATCTTTGCAATGATTCTTATCAGAAACCATATAGT +AAATTAGTTACACAGGAAATTTTTAATATTATTATTATCATTCATTATGT +ATTAAAATTAGAGTTGTGGCTTGGCTCTGCTAACACGTTGCTCATAGGAG +ATATGGTAGAGCCGCAGACACGTCGTATGCAGGAACGTGCTGCGGCTGGC +TGGTGAACTTCCGATAGTGCGGGTGTTGAATGATTTCCAGTTGCTACCGA +TTTTACATATTTTTTGCATGAGAGAATTTGTACCACCTCCCACCGACCAT +CTATGACTGTACGCCACTGTCCCTAGGACTGCTATGTGCCGGAGCGGACA +TTACAAACGTCCTTCTCGGTGCATGCCACTGTTGCCAATGACCTGCCTAG +GAATTGGTTAGCAAGTTACTACCGGATTTTGTAAAAACAGCCCTCCTCAT +ATAAAAAGTATTCGTTCACTTCCGATAAGCGTCGTAATTTTCTATCTTTC +ATCATATTCTAGATCCCTCTGAAAAAATCTTCCGAGTTTGCTAGGCACTG +ATACATAACTCTTTTCCAATAATTGGGGAAGTCATTCAAATCTATAATAG +GTTTCAGATTTGCTTCAATAAATTCTGACTGTAGCTGCTGAAACGTTGCG +GTTGAACTATATTTCCTTATAACTTTTACGAAAGAGTTTCTTTGAGTAAT +CACTTCACTCAAGTGCTTCCCTGCCTCCAAACGATACCTGTTAGCAATAT +TTAATAGCTTGAAATGATGAAGAGCTCTGTGTTTGTCTTCCTGCCTCCAG +TTCGCCGGGCATTCAACATAAAAACTGATAGCACCCGGAGTTCCGGAAAC +GAAATTTGCATATACCCATTGCTCACGAAAAAAAATGTCCTTGTCGATAT +AGGGATGAATCGCTTGGTGTACCTCATCTACTGCGAAAACTTGACCTTTC +TCTCCCATATTGCAGTCGCGGCACGATGGAACTAAATTAATAGGCATCAC +CGAAAATTCAGGATAATGTGCAATAGGAAGAAAATGATCTATATTTTTTG +TCTGTCCTATATCACCACAAAATGGACATTTTTCACCTGATGAAACAAGC +ATGTCATCGTAATATGTTCTAGCGGGTTTGTTTTTATCTCGGAGATTATT +TTCATAAAGCTTTTCTAATTTAACCTTTGTCAGGTTACCAACTACTAAGG +TTGTAGGCTCAAGAGGGTGTGTCCTGTCGTAGGTAAATAACTGACCTGTC +GAGCTTAATATTCTATATTGTTGTTCTTTCTGCAAAAAAGTGGGGAAGTG +AGTAATGAAATTATTTCTAACATTTATCTGCATCATACCTTCCGAGCATT +TATTAAGCATTTCGCTATAAGTTCTCGCTGGAAGAGGTAGTTTTTTCATT +GTACTTTACCTTCATCTCTGTTCATTATCATCGCTTTTAAAACGGTTCGA +CCTTCTAATCCTATCTGACCATTATAATTTTTTAGAATGGTTTCATAAGA +AAGCTCTGAATCAACGGACTGCGATAATAAGTGGTGGTATCCAGAATTTG +TCACTTCAAGTAAAAACACCTCACGAGTTAAAACACCTAAGTTCTCACCG +AATGTCTCAATATCCGGACGGATAATATTTATTGCTTCTCTTGACCGTAG +GACTTTCCACATGCAGGATTTTGGAACCTCTTGCAGTACTACTGGGGAAT +GAGTTGCAATTATTGCTACACCATTGCGTGCATCGAGTAAGTCGCTTAAT +GTTCGTAAAAAAGCAGAGAGCAAAGGTGGATGCAGATGAACCTCTGGTTC +ATCGAATAAAACTAATGACTTTTCGCCAACGACATCTACTAATCTTGTGA +TAGTAAATAAAACAATTGCATGTCCAGAGCTCATTCGAAGCAGATATTTC +TGGATATTGTCATAAAACAATTTAGTGAATTTATCATCGTCCACTTGAAT +CTGTGGTTCATTACGTCTTAACTCTTCATATTTAGAAATGAGGCTGATGA +GTTCCATATTTGAAAAGTTTTCATCACTACTTAGTTTTTTGATAGCTTCA +AGCCAGAGTTGTCTTTTTCTATCTACTCTCATACAACCAATAAATGCTGA +AATGAATTCTAAGCGGAGATCGCCTAGTGATTTTAAACTATTGCTGGCAG +CATTCTTGAGTCCAATATAAAAGTATTGTGTACCTTTTGCTGGGTCAGGT +TGTTCTTTAGGAGGAGTAAAAGGATCAAATGCACTAAACGAAACTGAAAC +AAGCGATCGAAAATATCCCTTTGGGATTCTTGACTCGATAAGTCTATTAT +TTTCAGAGAAAAAATATTCATTGTTTTCTGGGTTGGTGATTGCACCAATC +ATTCCATTCAAAATTGTTGTTTTACCACACCCATTCCGCCCGATAAAAGC +ATGAATGTTCGTGCTGGGCATAGAATTAACCGTCACCTCAAAAGGTATAG +TTAAATCACTGAATCCGGGAGCACTTTTTCTATTAAATGAAAAGTGGAAA +TCTGACAATTCTGGCAAACCATTTAACACACGTGCGAACTGTCCATGAAT +TTCTGAAAGAGTTACCCCTCTAAGTAATGAGGTGTTAAGGACGCTTTCAT +TTTCAATGTCGGCTAATCGATTTGGCCATACTACTAAATCCTGAATAGCT +TTAAGAAGGTTATGTTTAAAACCATCGCTTAATTTGCTGAGATTAACATA +GTAGTCAATGCTTTCACCTAAGGAAAAAAACATTTCAGGGAGTTGACTGA +ATTTTTTATCTATTAATGAATAAGTGCTTACTTCTTCTTTTTGACCTACA +AAACCAATTTTAACATTTCCGATATCGCATTTTTCACCATGCTCATCAAA +GACAGTAAGATAAAACATTGTAACAAAGGAATAGTCATTCCAACCATCTG +CTCGTAGGAATGCCTTATTTTTTTCTACTGCAGGAATATACCCGCCTCTT +TCAATAACACTAAACTCCAACATATAGTAACCCTTAATTTTATTAAAATA +ACCGCAATTTATTTGGCGGCAACACAGGATCTCTCTTTTAAGTTACTCTC +TATTACATACGTTTTCCATCTAAAAATTAGTAGTATTGAACTTAACGGGG +CATCGTATTGTAGTTTTCCATATTTAGCTTTCTGCTTCCTTTTGGATAAC +CCACTGTTATTCATGTTGCATGGTGCACTGTTTATACCAACGATATAGTC +TATTAATGCATATATAGTATCGCCGAACGATTAGCTCTTCAGGCTTCTGA +AGAAGCGTTTCAAGTACTAATAAGCCGATAGATAGCCACGGACTTCGTAG +CCATTTTTCATAAGTGTTAACTTCCGCTCCTCGCTCATAACAGACATTCA +CTACAGTTATGGCGGAAAGGTATGCATGCTGGGTGTGGGGAAGTCGTGAA +AGAAAAGAAGTCAGCTGCGTCGTTTGACATCACTGCTATCTTCTTACTGG +TTATGCAGGTCGTAGTGGGTGGCACACAAAGCTTTGCACTGGATTGCGAG +GCTTTGTGCTTCTCTGGAGTGCGACAGGTTTGATGACAAAAAATTAGCGC +AAGAAGACAAAAATCACCTTGCGCTAATGCTCTGTTACAGGTCACTAATA +CCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTGTGTTTTACAGTA +TTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTA +TATCATTTTACGTTTCTCGTTCAGCTTTTTTATACTAAGTTGGCATTATA +AAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCA +AAATAAAATCATTATTTGATTTCAATTTTGTCCCACTCCCTGCCTCTGTC +ATCACGATACTGTGATGCCATGGTGTCCGACTTATGCCCGAGAAGATGTT +GAGCAAACTTATCGCTTATCTGCTTCTCATAGAGTCTTGCAGACAAACTG +CGCAACTCGTGAAAGGTAGGCGGATCCCCTTCGAAGGAAAGACCTGATGC +TTTTCGTGCGCGCATAAAATACCTTGATACTGTGCCGGATGAAAGCGGTT +CGCGACGAGTAGATGCAATTATGGTTTCTCCGCCAAGAATCTCTTTGCAT +TTATCAAGTGTTTCCTTCATTGATATTCCGAGAGCATCAATATGCAATGC +TGTTGGGATGGCAATTTTTACGCCTGTTTTGCTTTGCTCGACATAAAGAT +ATCCATCTACGATATCAGACCACTTCATTTCGCATAAATCACCAACTCGT +TGCCCGGTAACAACAGCCAGTTCCATTGCAAGTCTGAGCCAACATGGTGA +TGATTCTGCTGCTTGATAAATTTTCAGGTATTCGTCAGCCGTAAGTCTTG +ATCTCCTTACCTCTGATTTTGCTGCGCGAGTGGCAGCGACATGGTTTGTT +GTTATATGGCCTTCAGCTATTGCCTCTCGGAATGCATCGCTCAGTGTTGA +TCTGATTAACTTGGCTGACGCCGCCTTGCCCTCGTCTATGTATCCATTGA +GCATTGCCGCAATTTCTTTTGTGGTGATGTCTTCAAGTGGAGCATCAGGC +AGACCCCTCCTTATTGCTTTAATTTTGCTCATGTAATTTATGAGTGTCTT +CTGCTTGATTCCTCTGCTGGCCAGGATTTTTTCGTAGCGATCAAGCCATG +AATGTAACGTAACGGAATTATCACTGTTGATTCTCGCTGTCAGAGGCTTG +TGTTTGTGTCCTGAAAATAACTCAATGTTGGCCTGTATAGCTTCAGTGAT +TGCGATTCGCCTGTCTCTGCCTAATCCAAACTCTTTACCCGTCCTTGGGT +CCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTTAGGGGGTAAA +TCCCGGCGCTCATGACTTCGCCTTCTTCCCATTTCTGATCCTCTTCAAAA +GGCCACCTGTTACTGGTCGATTTAAGTCAACCTTTACCGCTGATTCGTGG +AACAGATACTCTCTTCCATCCTTAACCGGAGGTGGGAATATCCTGCATTC +CCGAACCCATCGACGAACTGTTTCAAGGCTTCTTGGACGTCGCTGGCGTG +CGTTCCACTCCTGAAGTGTCAAGTACATCGCAAAGTCTCCGCAATTACAC +GCAAGAAAAAACCGCCATCAGGCGGCTTGGTGTTCTTTCAGTTCTTCAAT +TCGAATATTGGTTACGTCTGCATGTGCTATCTGCGCCCATATCATCCAGT +GGTCGTAGCAGTCGTTGATGTTCTCCGCTTCGATAACTCTGTTGAATGGC +TCTCCATTCCATTCTCCTGTGACTCGGAAGTGCATTTATCATCTCCATAA +AACAAAACCCGCCGTAGCGAGTTCAGATAAAATAAATCCCCGCGAGTGCG +AGGATTGTTATGTAATATTGGGTTTAATCATCTATATGTTTTGTACAGAG +AGGGCAAGTATCGTTTCCACCGTACTCGTGATAATAATTTTGCACGGTAT +CAGTCATTTCTCGCACATTGCAGAATGGGGATTTGTCTTCATTAGACTTA +TAAACCTTCATGGAATATTTGTATGCCGACTCTATATCTATACCTTCATC +TACATAAACACCTTCGTGATGTCTGCATGGAGACAAGACACCGGATCTGC +ACAACATTGATAACGCCCAATCTTTTTGCTCAGACTCTAACTCATTGATA +CTCATTTATAAACTCCTTGCAATGTATGTCGTTTCAGCTAAACGGTATCA +GCAATGTTTATGTAAAGAAACAGTAAGATAATACTCAACCCGATGTTTGA +GTACGGTCATCATCTGACACTACAGACTCTGGCATCGCTGTGAAGACGAC +GCGAAATTCAGCATTTTCACAAGCGTTATCTTTTACAAAACCGATCTCAC +TCTCCTTTGATGCGAATGCCAGCGTCAGACATCATATGCAGATACTCACC +TGCATCCTGAACCCATTGACCTCCAACCCCGTAATAGCGATGCGTAATGA +TGTCGATAGTTACTAACGGGTCTTGTTCGATTAACTGCCGCAGAAACTCT +TCCAGGTCACCAGTGCAGTGCTTGATAACAGGAGTCTTCCCAGGATGGCG +AACAACAAGAAACTGGTTTCCGTCTTCACGGACTTCGTTGCTTTCCAGTT +TAGCAATACGCTTACTCCCATCCGAGATAACACCTTCGTAATACTCACGC +TGCTCGTTGAGTTTTGATTTTGCTGTTTCAAGCTCAACACGCAGTTTCCC +TACTGTTAGCGCAATATCCTCGTTCTCCTGGTCGCGGCGTTTGATGTATT +GCTGGTTTCTTTCCCGTTCATCCAGCAGTTCCAGCACAATCGATGGTGTT +ACCAATTCATGGAAAAGGTCTGCGTCAAATCCCCAGTCGTCATGCATTGC +CTGCTCTGCCGCTTCACGCAGTGCCTGAGAGTTAATTTCGCTCACTTCGA +ACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAG +TCCGACAACCCTGAACGACCAGGCGTCTTCGTTCATCTATCGGATCGCCA +CACTCACAACAATGAGTGGCAGATATAGCCTGGTGGTTCAGGCGGCGCAT +TTTTATTGCTGTGTTGCGCTGTAATTCTTCTATTTCTGATGCTGAATCAA +TGATGTCTGCCATCTTTCATTAATCCCTGAACTGTTGGTTAATACGCTTG +AGGGTGAATGCGAATAATAAAAAAGGAGCCTGTAGCTCCCTGATGATTTT +GCTTTTCATGTTCATCGTTCCTTAAAGACGCCGTTTAACATGCCGATTGC +CAGGCTTAAATGAGTCGGTGTGAATCCCATCAGCGTTACCGTTTCGCGGT +GCTTCTTCAGTACGCTACGGCAAATGTCATCGACGTTTTTATCCGGAAAC +TGCTGTCTGGCTTTTTTTGATTTCAGAATTAGCCTGACGGGCAATGCTGC +GAAGGGCGTTTTCCTGCTGAGGTGTCATTGAACAAGTCCCATGTCGGCAA +GCATAAGCACACAGAATATGAAGCCCGCTGCCAGAAAAATGCATTCCGTG +GTTGTCATACCTGGTTTCTCTCATCTGCTTCTGCTTTCGCCACCATCATT +TCCAGCTTTTGTGAAAGGGATGCGGCTAACGTATGAAATTCTTCGTCTGT +TTCTACTGGTATTGGCACAAACCTGATTCCAATTTGAGCAAGGCTATGTG +CCATCTCGATACTCGTTCTTAACTCAACAGAAGATGCTTTGTGCATACAG +CCCCTCGTTTATTATTTATCTCCTCAGCCAGCCGCTGTGCTTTCAGTGGA +TTTCGGATAACAGAAAGGCCGGGAAATACCCAGCCTCGCTTTGTAACGGA +GTAGACGAAAGTGATTGCGCCTACCCGGATATTATCGTGAGGATGCGTCA +TCGCCATTGCTCCCCAAATACAAAACCAATTTCAGCCAGTGCCTCGTCCA +TTTTTTCGATGAACTCCGGCACGATCTCGTCAAAACTCGCCATGTACTTT +TCATCCCGCTCAATCACGACATAATGCAGGCCTTCACGCTTCATACGCGG +GTCATAGTTGGCAAAGTACCAGGCATTTTTTCGCGTCACCCACATGCTGT +ACTGCACCTGGGCCATGTAAGCTGACTTTATGGCCTCGAAACCACCGAGC +CGGAACTTCATGAAATCCCGGGAGGTAAACGGGCATTTCAGTTCAAGGCC +GTTGCCGTCACTGCATAAACCATCGGGAGAGCAGGCGGTACGCATACTTT +CGTCGCGATAGATGATCGGGGATTCAGTAACATTCACGCCGGAAGTGAAT +TCAAACAGGGTTCTGGCGTCGTTCTCGTACTGTTTTCCCCAGGCCAGTGC +TTTAGCGTTAACTTCCGGAGCCACACCGGTGCAAACCTCAGCAAGCAGGG +TGTGGAAGTAGGACATTTTCATGTCAGGCCACTTCTTTCCGGAGCGGGGT +TTTGCTATCACGTTGTGAACTTCTGAAGCGGTGATGACGCCGAGCCGTAA +TTTGTGCCACGCATCATCCCCCTGTTCGACAGCTCTCACATCGATCCCGG +TACGCTGCAGGATAATGTCCGGTGTCATGCTGCCACCTTCTGCTCTGCGG +CTTTCTGTTTCAGGAATCCAAGAGCTTTTACTGCTTCGGCCTGTGTCAGT +TCTGACGATGCACGAATGTCGCGGCGAAATATCTGGGAACAGAGCGGCAA +TAAGTCGTCATCCCATGTTTTATCCAGGGCGATCAGCAGAGTGTTAATCT +CCTGCATGGTTTCATCGTTAACCGGAGTGATGTCGCGTTCCGGCTGACGT +TCTGCAGTGTATGCAGTATTTTCGACAATGCGCTCGGCTTCATCCTTGTC +ATAGATACCAGCAAATCCGAAGGCCAGACGGGCACACTGAATCATGGCTT +TATGACGTAACATCCGTTTGGGATGCGACTGCCACGGCCCCGTGATTTCT +CTGCCTTCGCGAGTTTTGAATGGTTCGCGGCGGCATTCATCCATCCATTC +GGTAACGCAGATCGGATGATTACGGTCCTTGCGGTAAATCCGGCATGTAC +AGGATTCATTGTCCTGCTCAAAGTCCATGCCATCAAACTGCTGGTTTTCA +TTGATGATGCGGGACCAGCCATCAACGCCCACCACCGGAACGATGCCATT +CTGCTTATCAGGAAAGGCGTAAATTTCTTTCGTCCACGGATTAAGGCCGT +ACTGGTTGGCAACGATCAGTAATGCGATGAACTGCGCATCGCTGGCATCA +CCTTTAAATGCCGTCTGGCGAAGAGTGGTGATCAGTTCCTGTGGGTCGAC +AGAATCCATGCCGACACGTTCAGCCAGCTTCCCAGCCAGCGTTGCGAGTG +CAGTACTCATTCGTTTTATACCTCTGAATCAATATCAACCTGGTGGTGAG +CAATGGTTTCAACCATGTACCGGATGTGTTCTGCCATGCGCTCCTGAAAC +TCAACATCGTCATCAAACGCACGGGTAATGGATTTTTTGCTGGCCCCGTG +GCGTTGCAAATGATCGATGCATAGCGATTCAAACAGGTGCTGGGGCAGGC +CTTTTTCCATGTCGTCTGCCAGTTCTGCCTCTTTCTCTTCACGGGCGAGC +TGCTGGTAGTGACGCGCCCAGCTCTGAGCCTCAAGACGATCCTGAATGTA +ATAAGCGTTCATGGCTGAACTCCTGAAATAGCTGTGAAAATATCGCCCGC +GAAATGCCGGGCTGATTAGGAAAACAGGAAAGGGGGTTAGTGAATGCTTT +TGCTTGATCTCAGTTTCAGTATTAATATCCATTTTTTATAAGCGTCGACG +GCTTCACGAAACATCTTTTCATCGCCAATAAAAGTGGCGATAGTGAATTT +AGTCTGGATAGCCATAAGTGTTTGATCCATTCTTTGGGACTCCTGGCTGA +TTAAGTATGTCGATAAGGCGTTTCCATCCGTCACGTAATTTACGGGTGAT +TCGTTCAAGTAAAGATTCGGAAGGGCAGCCAGCAACAGGCCACCCTGCAA +TGGCATATTGCATGGTGTGCTCCTTATTTATACATAACGAAAAACGCCTC +GAGTGAAGCGTTATTGGTATGCGGTAAAACCGCACTCAGGCGGCCTTGAT +AGTCATATCATCTGAATCAAATATTCCTGATGTATCGATATCGGTAATTC +TTATTCCTTCGCTACCATCCATTGGAGGCCATCCTTCCTGACCATTTCCA +TCATTCCAGTCGAACTCACACACAACACCATATGCATTTAAGTCGCTTGA +AATTGCTATAAGCAGAGCATGTTGCGCCAGCATGATTAATACAGCATTTA +ATACAGAGCCGTGTTTATTGAGTCGGTATTCAGAGTCTGACCAGAAATTA +TTAATCTGGTGAAGTTTTTCCTCTGTCATTACGTCATGGTCGATTTCAAT +TTCTATTGATGCTTTCCAGTCGTAATCAATGATGTATTTTTTGATGTTTG +ACATCTGTTCATATCCTCACAGATAAAAAATCGCCCTCACACTGGAGGGC +AAAGAAGATTTCCAATAATCAGAACAAGTCGGCTCCTGTTTAGTTACGAG +CGACATTGCTCCGTGTATTCACTCGTTGGAATGAATACACAGTGCAGTGT +TTATTCTGTTATTTATGCCAAAAATAAAGGCCACTATCAGGCAGCTTTGT +TGTTCTGTTTACCAAGTTCTCTGGCAATCATTGCCGTCGTTCGTATTGCC +CATTTATCGACATATTTCCCATCTTCCATTACAGGAAACATTTCTTCAGG +CTTAACCATGCATTCCGATTGCAGCTTGCATCCATTGCATCGCTTGAATT +GTCCACACCATTGATTTTTATCAATAGTCGTAGTCATACGGATAGTCCTG +GTATTGTTCCATCACATCCTGAGGATGCTCTTCGAACTCTTCAAATTCTT +CTTCCATATATCACCTTAAATAGTGGATTGCGGTAGTAAAGATTGTGCCT +GTCTTTTAACCACATCAGGCTCGGTGGTTCTCGTGTACCCCTACAGCGAG +AAATCGGATAAACTATTACAACCCCTACAGTTTGATGAGTATAGAAATGG +ATCCACTCGTTATTCTCGGACGAGTGTTCAGTAATGAACCTCTGGAGAGA +ACCATGTATATGATCGTTATCTGGGTTGGACTTCTGCTTTTAAGCCCAGA +TAACTGGCCTGAATATGTTAATGAGAGAATCGGTATTCCTCATGTGTGGC +ATGTTTTCGTCTTTGCTCTTGCATTTTCGCTAGCAATTAATGTGCATCGA +TTATCAGCTATTGCCAGCGCCAGATATAAGCGATTTAAGCTAAGAAAACG +CATTAAGATGCAAAACGATAAAGTGCGATCAGTAATTCAAAACCTTACAG +AAGAGCAATCTATGGTTTTGTGCGCAGCCCTTAATGAAGGCAGGAAGTAT +GTGGTTACATCAAAACAATTCCCATACATTAGTGAGTTGATTGAGCTTGG +TGTGTTGAACAAAACTTTTTCCCGATGGAATGGAAAGCATATATTATTCC +CTATTGAGGATATTTACTGGACTGAATTAGTTGCCAGCTATGATCCATAT +AATATTGAGATAAAGCCAAGGCCAATATCTAAGTAACTAGATAAGAGGAA +TCGATTTTCCCTTAATTTTCTGGCGTCCACTGCATGTTATGCCGCGTTCG +CCAGGCTTGCTGTACCATGTGCGCTGATTCTTGCGCTCAATACGTTGCAG +GTTGCTTTCAATCTGTTTGTGGTATTCAGCCAGCACTGTAAGGTCTATCG +GATTTAGTGCGCTTTCTACTCGTGATTTCGGTTTGCGATTCAGCGAGAGA +ATAGGGCGGTTAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGC +TGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTT +GTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAG +TTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATC +CGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAG +CCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGT +CACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCC +AGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGT +TATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTT +GGTAGGTGAGAGATCTGAATTGCTATGTTTAGTGAGTTGTATCTATTTAT +TTTTCAATAAATACAATTGGTTATGTGTTTTGGGGGCGATCGTGAGGCAA +AGAAAACCCGGCGCTGAGGCCGGGTTATTCTTGTTCTCTGGTCAAATTAT +ATAGTTGGAAAACAAGGATGCATATATGAATGAACGATGCAGAGGCAATG +CCGATGGCGATAGTGGGTATCATGTAGCCGCTTATGCTGGAAAGAAGCAA +TAACCCGCAGAAAAACAAAGCTCCAAGCTCAACAAAACTAAGGGCATAGA +CAATAACTACCGATGTCATATACCCATACTCTCTAATCTTGGCCAGTCGG +CGCGTTCTGCTTCCGATTAGAAACGTCAAGGCAGCAATCAGGATTGCAAT +CATGGTTCCTGCATATGATGACAATGTCGCCCCAAGACCATCTCTATGAG +CTGAAAAAGAAACACCAGGAATGTAGTGGCGGAAAAGGAGATAGCAAATG +CTTACGATAACGTAAGGAATTATTACTATGTAAACACCAGGCATGATTCT +GTTCCGCATAATTACTCCTGATAATTAATCCTTAACTTTGCCCACCTGCC +TTTTAAAACATTCCAGTATATCACTTTTCATTCTTGCGTAGCAATATGCC +ATCTCTTCAGCTATCTCAGCATTGGTGACCTTGTTCAGAGGCGCTGAGAG +ATGGCCTTTTTCTGATAGATAATGTTCTGTTAAAATATCTCCGGCCTCAT +CTTTTGCCCGCAGGCTAATGTCTGAAAATTGAGGTGACGGGTTAAAAATA +ATATCCTTGGCAACCTTTTTTATATCCCTTTTAAATTTTGGCTTAATGAC +TATATCCAATGAGTCAAAAAGCTCCCCTTCAATATCTGTTGCCCCTAAGA +CCTTTAATATATCGCCAAATACAGGTAGCTTGGCTTCTACCTTCACCGTT +GTTCGGCCGATGAAATGCATATGCATAACATCGTCTTTGGTGGTTCCCCT +CATCAGTGGCTCTATCTGAACGCGCTCTCCACTGCTTAATGACATTCCTT +TCCCGATTAAAAAATCTGTCAGATCGGATGTGGTCGGCCCGAAAACAGTT +CTGGCAAAACCAATGGTGTCGCCTTCAACAAACAAAAAAGATGGGAATCC +CAATGATTCGTCATCTGCGAGGCTGTTCTTAATATCTTCAACTGAAGCTT +TAGAGCGATTTATCTTCTGAACCAGACTCTTGTCATTTGTTTTGGTAAAG +AGAAAAGTTTTTCCATCGATTTTATGAATATACAAATAATTGGAGCCAAC +CTGCAGGTGATGATTATCAGCCAGCAGAGAATTAAGGAAAACAGACAGGT +TTATTGAGCGCTTATCTTTCCCTTTATTTTTGCTGCGGTAAGTCGCATAA +AAACCATTCTTCATAATTCAATCCATTTACTATGTTATGTTCTGAGGGGA +GTGAAAATTCCCCTAATTCGATGAAGATTCTTGCTCAATTGTTATCAGCT +ATGCGCCGACCAGAACACCTTGCCGATCAGCCAAACGTCTCTTCAGGCCA +CTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTGCATGGGA +TCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATCC +CTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCA +GAAATCACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTAACATTC +CGTCAGGAAAGCTTGGCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCT +TCAACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGCTTAC +CCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTCAGGTGAGAACA +TCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGT +GACGGCTGCATACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGAT +TGAAGGGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGCAAGCAATG +CGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAACG +CCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGCC +AAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCT +CAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGCTCATACGTTAAATC +TATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTAC +CTCTGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTGTATGGAACAAC +GCATAACCCTGAAAGATTATGCAATGCGCTTTGGGCAAACCAAGACAGCT +AAAGATCTCGGCGTATATCAAAGCGCGATCAACAAGGCCATTCATGCAGG +CCGAAAGATTTTTTTAACTATAAACGCTGATGGAAGCGTTTATGCGGAAG +AGGTAAAGCCCTTCCCGAGTAACAAAAAAACAACAGCATAAATAACCCCG +CTCTTACACATTCCAGCCCTGAAAAAGGGCATCAAATTAAACCACACCTA +TGGTGTATGCATTTATTTGCATACATTCAATCAATTGTTATCTAAGGAAA +TACTTACATATGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGA +GAGTGCGTTGCTTAACAAAATCGCAATGCTTGGAACTGAGAAGACAGCGG +AAGCTGTGGGCGTTGATAAGTCGCAGATCAGCAGGTGGAAGAGGGACTGG +ATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTCGTTGA +CGACGACATGGCTCGATTGGCGCGACAAGTTGCTGCGATTCTCACCAATA +AAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTC +TGAGGTCATTACTGGATCTATCAACAGGAGTCATTATGACAAATACAGCA +AAAATACTCAACTTCGGCAGAGGTAACTTTGCCGGACAGGAGCGTAATGT +GGCAGATCTCGATGATGGTTACGCCAGACTATCAAATATGCTGCTTGAGG +CTTATTCGGGCGCAGATCTGACCAAGCGACAGTTTAAAGTGCTGCTTGCC +ATTCTGCGTAAAACCTATGGGTGGAATAAACCAATGGACAGAATCACCGA +TTCTCAACTTAGCGAGATTACAAAGTTACCTGTCAAACGGTGCAATGAAG +CCAAGTTAGAACTCGTCAGAATGAATATTATCAAGCAGCAAGGCGGCATG +TTTGGACCAAATAAAAACATCTCAGAATGGTGCATCCCTCAAAACGAGGG +AAAATCCCCTAAAACGAGGGATAAAACATCCCTCAAATTGGGGGATTGCT +ATCCCTCAAAACAGGGGGACACAAAAGACACTATTACAAAAGAAAAAAGA +AAAGATTATTCGTCAGAGAATTCTGGCGAATCCTCTGACCAGCCAGAAAA +CGACCTTTCTGTGGTGAAACCGGATGCTGCAATTCAGAGCGGCAGCAAGT +GGGGGACAGCAGAAGACCTGACCGCCGCAGAGTGGATGTTTGACATGGTG +AAGACTATCGCACCATCAGCCAGAAAACCGAATTTTGCTGGGTGGGCTAA +CGATATCCGCCTGATGCGTGAACGTGACGGACGTAACCACCGCGACATGT +GTGTGCTGTTCCGCTGGGCATGCCAGGACAACTTCTGGTCCGGTAACGTG +CTGAGCCCGGCCAAACTCCGCGATAAGTGGACCCAACTCGAAATCAACCG +TAACAAGCAACAGGCAGGCGTGACAGCCAGCAAACCAAAACTCGACCTGA +CAAACACAGACTGGATTTACGGGGTGGATCTATGAAAAACATCGCCGCAC +AGATGGTTAACTTTGACCGTGAGCAGATGCGTCGGATCGCCAACAACATG +CCGGAACAGTACGACGAAAAGCCGCAGGTACAGCAGGTAGCGCAGATCAT +CAACGGTGTGTTCAGCCAGTTACTGGCAACTTTCCCGGCGAGCCTGGCTA +ACCGTGACCAGAACGAAGTGAACGAAATCCGTCGCCAGTGGGTTCTGGCT +TTTCGGGAAAACGGGATCACCACGATGGAACAGGTTAACGCAGGAATGCG +CGTAGCCCGTCGGCAGAATCGACCATTTCTGCCATCACCCGGGCAGTTTG +TTGCATGGTGCCGGGAAGAAGCATCCGTTACCGCCGGACTGCCAAACGTC +AGCGAGCTGGTTGATATGGTTTACGAGTATTGCCGGAAGCGAGGCCTGTA +TCCGGATGCGGAGTCTTATCCGTGGAAATCAAACGCGCACTACTGGCTGG +TTACCAACCTGTATCAGAACATGCGGGCCAATGCGCTTACTGATGCGGAA +TTACGCCGTAAGGCCGCAGATGAGCTTGTCCATATGACTGCGAGAATTAA +CCGTGGTGAGGCGATCCCTGAACCAGTAAAACAACTTCCTGTCATGGGCG +GTAGACCTCTAAATCGTGCACAGGCTCTGGCGAAGATCGCAGAAATCAAA +GCTAAGTTCGGACTGAAAGGAGCAAGTGTATGACGGGCAAAGAGGCAATT +ATTCATTACCTGGGGACGCATAATAGCTTCTGTGCGCCGGACGTTGCCGC +GCTAACAGGCGCAACAGTAACCAGCATAAATCAGGCCGCGGCTAAAATGG +CACGGGCAGGTCTTCTGGTTATCGAAGGTAAGGTCTGGCGAACGGTGTAT +TACCGGTTTGCTACCAGGGAAGAACGGGAAGGAAAGATGAGCACGAACCT +GGTTTTTAAGGAGTGTCGCCAGAGTGCCGCGATGAAACGGGTATTGGCGG +TATATGGAGTTAAAAGATGACCATCTACATTACTGAGCTAATAACAGGCC +TGCTGGTAATCGCAGGCCTTTTTATTTGGGGGAGAGGGAAGTCATGAAAA +AACTAACCTTTGAAATTCGATCTCCAGCACATCAGCAAAACGCTATTCAC +GCAGTACAGCAAATCCTTCCAGACCCAACCAAACCAATCGTAGTAACCAT +TCAGGAACGCAACCGCAGCTTAGACCAAAACAGGAAGCTATGGGCCTGCT +TAGGTGACGTCTCTCGTCAGGTTGAATGGCATGGTCGCTGGCTGGATGCA +GAAAGCTGGAAGTGTGTGTTTACCGCAGCATTAAAGCAGCAGGATGTTGT +TCCTAACCTTGCCGGGAATGGCTTTGTGGTAATAGGCCAGTCAACCAGCA +GGATGCGTGTAGGCGAATTTGCGGAGCTATTAGAGCTTATACAGGCATTC +GGTACAGAGCGTGGCGTTAAGTGGTCAGACGAAGCGAGACTGGCTCTGGA +GTGGAAAGCGAGATGGGGAGACAGGGCTGCATGATAAATGTCGTTAGTTT +CTCCGGTGGCAGGACGTCAGCATATTTGCTCTGGCTAATGGAGCAAAAGC +GACGGGCAGGTAAAGACGTGCATTACGTTTTCATGGATACAGGTTGTGAA +CATCCAATGACATATCGGTTTGTCAGGGAAGTTGTGAAGTTCTGGGATAT +ACCGCTCACCGTATTGCAGGTTGATATCAACCCGGAGCTTGGACAGCCAA +ATGGTTATACGGTATGGGAACCAAAGGATATTCAGACGCGAATGCCTGTT +CTGAAGCCATTTATCGATATGGTAAAGAAATATGGCACTCCATACGTCGG +CGGCGCGTTCTGCACTGACAGATTAAAACTCGTTCCCTTCACCAAATACT +GTGATGACCATTTCGGGCGAGGGAATTACACCACGTGGATTGGCATCAGA +GCTGATGAACCGAAGCGGCTAAAGCCAAAGCCTGGAATCAGATATCTTGC +TGAACTGTCAGACTTTGAGAAGGAAGATATCCTCGCATGGTGGAAGCAAC +AACCATTCGATTTGCAAATACCGGAACATCTCGGTAACTGCATATTCTGC +ATTAAAAAATCAACGCAAAAAATCGGACTTGCCTGCAAAGATGAGGAGGG +ATTGCAGCGTGTTTTTAATGAGGTCATCACGGGATCCCATGTGCGTGACG +GACATCGGGAAACGCCAAAGGAGATTATGTACCGAGGAAGAATGTCGCTG +GACGGTATCGCGAAAATGTATTCAGAAAATGATTATCAAGCCCTGTATCA +GGACATGGTACGAGCTAAAAGATTCGATACCGGCTCTTGTTCTGAGTCAT +GCGAAATATTTGGAGGGCAGCTTGATTTCGACTTCGGGAGGGAAGCTGCA +TGATGCGATGTTATCGGTGCGGTGAATGCAAAGAAGATAACCGCTTCCGA +CCAAATCAACCTTACTGGAATCGATGGTGTCTCCGGTGTGAAAGAACACC +AACAGGGGTGTTACCACTACCGCAGGAAAAGGAGGACGTGTGGCGAGACA +GCGACGAAGTATCACCGACATAATCTGCGAAAACTGCAAATACCTTCCAA +CGAAACGCACCAGAAATAAACCCAAGCCAATCCCAAAAGAATCTGACGTA +AAAACCTTCAACTACACGGCTCACCTGTGGGATATCCGGTGGCTAAGACG +TCGTGCGAGGAAAACAAGGTGATTGACCAAAATCGAAGTTACGAACAAGA +AAGCGTCGAGCGAGCTTTAACGTGCGCTAACTGCGGTCAGAAGCTGCATG +TGCTGGAAGTTCACGTGTGTGAGCACTGCTGCGCAGAACTGATGAGCGAT +CCGAATAGCTCGATGCACGAGGAAGAAGATGATGGCTAAACCAGCGCGAA +GACGATGTAAAAACGATGAATGCCGGGAATGGTTTCACCCTGCATTCGCT +AATCAGTGGTGGTGCTCTCCAGAGTGTGGAACCAAGATAGCACTCGAACG +ACGAAGTAAAGAACGCGAAAAAGCGGAAAAAGCAGCAGAGAAGAAACGAC +GACGAGAGGAGCAGAAACAGAAAGATAAACTTAAGATTCGAAAACTCGCC +TTAAAGCCCCGCAGTTACTGGATTAAACAAGCCCAACAAGCCGTAAACGC +CTTCATCAGAGAAAGAGACCGCGACTTACCATGTATCTCGTGCGGAACGC +TCACGTCTGCTCAGTGGGATGCCGGACATTACCGGACAACTGCTGCGGCA +CCTCAACTCCGATTTAATGAACGCAATATTCACAAGCAATGCGTGGTGTG +CAACCAGCACAAAAGCGGAAATCTCGTTCCGTATCGCGTCGAACTGATTA +GCCGCATCGGGCAGGAAGCAGTAGACGAAATCGAATCAAACCATAACCGC +CATCGCTGGACTATCGAAGAGTGCAAGGCGATCAAGGCAGAGTACCAACA +GAAACTCAAAGACCTGCGAAATAGCAGAAGTGAGGCCGCATGACGTTCTC +AGTAAAAACCATTCCAGACATGCTCGTTGAAGCATACGGAAATCAGACAG +AAGTAGCACGCAGACTGAAATGTAGTCGCGGTACGGTCAGAAAATACGTT +GATGATAAAGACGGGAAAATGCACGCCATCGTCAACGACGTTCTCATGGT +TCATCGCGGATGGAGTGAAAGAGATGCGCTATTACGAAAAAATTGATGGC +AGCAAATACCGAAATATTTGGGTAGTTGGCGATCTGCACGGATGCTACAC +GAACCTGATGAACAAACTGGATACGATTGGATTCGACAACAAAAAAGACC +TGCTTATCTCGGTGGGCGATTTGGTTGATCGTGGTGCAGAGAACGTTGAA +TGCCTGGAATTAATCACATTCCCCTGGTTCAGAGCTGTACGTGGAAACCA +TGAGCAAATGATGATTGATGGCTTATCAGAGCGTGGAAACGTTAATCACT +GGCTGCTTAATGGCGGTGGCTGGTTCTTTAATCTCGATTACGACAAAGAA +ATTCTGGCTAAAGCTCTTGCCCATAAAGCAGATGAACTTCCGTTAATCAT +CGAACTGGTGAGCAAAGATAAAAAATATGTTATCTGCCACGCCGATTATC +CCTTTGACGAATACGAGTTTGGAAAGCCAGTTGATCATCAGCAGGTAATC +TGGAACCGCGAACGAATCAGCAACTCACAAAACGGGATCGTGAAAGAAAT +CAAAGGCGCGGACACGTTCATCTTTGGTCATACGCCAGCAGTGAAACCAC +TCAAGTTTGCCAACCAAATGTATATCGATACCGGCGCAGTGTTCTGCGGA +AACCTAACATTGATTCAGGTACAGGGAGAAGGCGCATGAGACTCGAAAGC +GTAGCTAAATTTCATTCGCCAAAAAGCCCGATGATGAGCGACTCACCACG +GGCCACGGCTTCTGACTCTCTTTCCGGTACTGATGTGATGGCTGCTATGG +GGATGGCGCAATCACAAGCCGGATTCGGTATGGCTGCATTCTGCGGTAAG +CACGAACTCAGCCAGAACGACAAACAAAAGGCTATCAACTATCTGATGCA +ATTTGCACACAAGGTATCGGGGAAATACCGTGGTGTGGCAAAGCTTGAAG +GAAATACTAAGGCAAAGGTACTGCAAGTGCTCGCAACATTCGCTTATGCG +GATTATTGCCGTAGTGCCGCGACGCCGGGGGCAAGATGCAGAGATTGCCA +TGGTACAGGCCGTGCGGTTGATATTGCCAAAACAGAGCTGTGGGGGAGAG +TTGTCGAGAAAGAGTGCGGAAGATGCAAAGGCGTCGGCTATTCAAGGATG +CCAGCAAGCGCAGCATATCGCGCTGTGACGATGCTAATCCCAAACCTTAC +CCAACCCACCTGGTCACGCACTGTTAAGCCGCTGTATGACGCTCTGGTGG +TGCAATGCCACAAAGAAGAGTCAATCGCAGACAACATTTTGAATGCGGTC +ACACGTTAGCAGCATGATTGCCACGGATGGCAACATATTAACGGCATGAT +ATTGACTTATTGAATAAAATTGGGTAAATTTGACTCAACGATGGGTTAAT +TCGCTCGTTGTGGTAGTGAGATGAAAAGAGGCGGCGCTTACTACCGATTC +CGCCTAGTTGGTCACTTCGACGTATCGTCTGGAACTCCAACCATCGCAGG +CAGAGAGGTCTGCAAAATGCAATCCCGAAACAGTTCGCAGGTAATAGTTA +GAGCCTGCATAACGGTTTCGGGATTTTTTATATCTGCACAACAGGTAAGA +GCATTGAGTCGATAATCGTGAAGAGTCGGCGAGCCTGGTTAGCCAGTGCT +CTTTCCGTTGTGCTGAATTAAGCGAATACCGGAAGCAGAACCGGATCACC +AAATGCGTACAGGCGTCATCGCCGCCCAGCAACAGCACAACCCAAACTGA +GCCGTAGCCACTGTCTGTCCTGAATTCATTAGTAATAGTTACGCTGCGGC +CTTTTACACATGACCTTCGTGAAAGCGGGTGGCAGGAGGTCGCGCTAACA +ACCTCCTGCCGTTTTGCCCGTGCATATCGGTCACGAACAAATCTGATTAC +TAAACACAGTAGCCTGGATTTGTTCTATCAGTAATCGACCTTATTCCTAA +TTAAATAGAGCAAATCCCCTTATTGGGGGTAAGACATGAAGATGCCAGAA +AAACATGACCTGTTGGCCGCCATTCTCGCGGCAAAGGAACAAGGCATCGG +GGCAATCCTTGCGTTTGCAATGGCGTACCTTCGCGGCAGATATAATGGCG +GTGCGTTTACAAAAACAGTAATCGACGCAACGATGTGCGCCATTATCGCC +TGGTTCATTCGTGACCTTCTCGACTTCGCCGGACTAAGTAGCAATCTCGC +TTATATAACGAGCGTGTTTATCGGCTACATCGGTACTGACTCGATTGGTT +CGCTTATCAAACGCTTCGCTGCTAAAAAAGCCGGAGTAGAAGATGGTAGA +AATCAATAATCAACGTAAGGCGTTCCTCGATATGCTGGCGTGGTCGGAGG +GAACTGATAACGGACGTCAGAAAACCAGAAATCATGGTTATGACGTCATT +GTAGGCGGAGAGCTATTTACTGATTACTCCGATCACCCTCGCAAACTTGT +CACGCTAAACCCAAAACTCAAATCAACAGGCGCCGGACGCTACCAGCTTC +TTTCCCGTTGGTGGGATGCCTACCGCAAGCAGCTTGGCCTGAAAGACTTC +TCTCCGAAAAGTCAGGACGCTGTGGCATTGCAGCAGATTAAGGAGCGTGG +CGCTTTACCTATGATTGATCGTGGTGATATCCGTCAGGCAATCGACCGTT +GCAGCAATATCTGGGCTTCACTGCCGGGCGCTGGTTATGGTCAGTTCGAG +CATAAGGCTGACAGCCTGATTGCAAAATTCAAAGAAGCGGGCGGAACGGT +CAGAGAGATTGATGTATGAGCAGAGTCACCGCGATTATCTCCGCTCTGGT +TATCTGCATCATCGTCTGCCTGTCATGGGCTGTTAATCATTACCGTGATA +ACGCCATTACCTACAAAGCCCAGCGCGACAAAAATGCCAGAGAACTGAAG +CTGGCGAACGCGGCAATTACTGACATGCAGATGCGTCAGCGTGATGTTGC +TGCGCTCGATGCAAAATACACGAAGGAGTTAGCTGATGCTAAAGCTGAAA +ATGATGCTCTGCGTGATGATGTTGCCGCTGGTCGTCGTCGGTTGCACATC +AAAGCAGTCTGTCAGTCAGTGCGTGAAGCCACCACCGCCTCCGGCGTGGA +TAATGCAGCCTCCCCCCGACTGGCAGACACCGCTGAACGGGATTATTTCA +CCCTCAGAGAGAGGCTGATCACTATGCAAAAACAACTGGAAGGAACCCAG +AAGTATATTAATGAGCAGTGCAGATAGAGTTGCCCATATCGATGGGCAAC +TCATGCAATTATTGTGAGCAATACACACGCGCTTCCAGCGGAGTATAAAT +GCCTAAAGTAATAAAACCGAGCAATCCATTTACGAATGTTTGCTGGGTTT +CTGTTTTAACAACATTTTCTGCGCCGCCACAAATTTTGGCTGCATCGACA +GTTTTCTTCTGCCCAATTCCAGAAACGAAGAAATGATGGGTGATGGTTTC +CTTTGGTGCTACTGCTGCCGGTTTGTTTTGAACAGTAAACGTCTGTTGAG +CACATCCTGTAATAAGCAGGGCCAGCGCAGTAGCGAGTAGCATTTTTTTC +ATGGTGTTATTCCCGATGCTTTTTGAAGTTCGCAGAATCGTATGTGTAGA +AAATTAAACAAACCCTAAACAATGAGTTGAAATTTCATATTGTTAATATT +TATTAATGTATGTCAGGTGCGATGAATCGTCATTGTATTCCCGGATTAAC +TATGTCCACAGCCCTGACGGGGAACTTCTCTGCGGGAGTGTCCGGGAATA +ATTAAAACGATGCACACAGGGTTTAGCGCGTACACGTATTGCATTATGCC +AACGCCCCGGTGCTGACACGGAAGAAACCGGACGTTATGATTTAGCGTGG +AAAGATTTGTGTAGTGTTCTGAATGCTCTCAGTAAATAGTAATGAATTAT +CAAAGGTATAGTAATATCTTTTATGTTCATGGATATTTGTAACCCATCGG +AAAACTCCTGCTTTAGCAAGATTTTCCCTGTATTGCTGAAATGTGATTTC +TCTTGATTTCAACCTATCATAGGACGTTTCTATAAGATGCGTGTTTCTTG +AGAATTTAACATTTACAACCTTTTTAAGTCCTTTTATTAACACGGTGTTA +TCGTTTTCTAACACGATGTGAATATTATCTGTGGCTAGATAGTAAATATA +ATGTGAGACGTTGTGACGTTTTAGTTCAGAATAAAACAATTCACAGTCTA +AATCTTTTCGCACTTGATCGAATATTTCTTTAAAAATGGCAACCTGAGCC +ATTGGTAAAACCTTCCATGTGATACGAGGGCGCGTAGTTTGCATTATCGT +TTTTATCGTTTCAATCTGGTCTGACCTCCTTGTGTTTTGTTGATGATTTA +TGTCAAATATTAGGAATGTTTTCACTTAATAGTATTGGTTGCGTAACAAA +GTGCGGTCCTGCTGGCATTCTGGAGGGAAATACAACCGACAGATGTATGT +AAGGCCAACGTGCTCAAATCTTCATACAGAAAGATTTGAAGTAATATTTT +AACCGCTAGATGAAGAGCAAGCGCATGGAGCGACAAAATGAATAAAGAAC +AATCTGCTGATGATCCCTCCGTGGATCTGATTCGTGTAAAAAATATGCTT +AATAGCACCATTTCTATGAGTTACCCTGATGTTGTAATTGCATGTATAGA +ACATAAGGTGTCTCTGGAAGCATTCAGAGCAATTGAGGCAGCGTTGGTGA +AGCACGATAATAATATGAAGGATTATTCCCTGGTGGTTGACTGATCACCA +TAACTGCTAATCATTCAAACTATTTAGTCTGTGACAGAGCCAACACGCAG +TCTGTCACTGTCAGGAAAGTGGTAAAACTGCAACTCAATTACTGCAATGC +CCTCGTAATTAAGTGAATTTACAATATCGTCCTGTTCGGAGGGAAGAACG +CGGGATGTTCATTCTTCATCACTTTTAATTGATGTATATGCTCTCTTTTC +TGACGTTAGTCTCCGACGGCAGGCTTCAATGACCCAGGCTGAGAAATTCC +CGGACCCTTTTTGCTCAAGAGCGATGTTAATTTGTTCAATCATTTGGTTA +GGAAAGCGGATGTTGCGGGTTGTTGTTCTGCGGGTTCTGTTCTTCGTTGA +CATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTG +TTTTTACGTTAAGTTGATGCAGATCAATTAATACGATACCTGCGTCATAA +TTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTGTGATATGTAG +ATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTA +CG diff --git a/samples.map.template b/samples.map.template new file mode 100644 index 0000000..ef69558 --- /dev/null +++ b/samples.map.template @@ -0,0 +1,19 @@ +#Group SampleID fq1 fq2 +GroupA GroupA1 GroupA1.1.fq.gz GroupA1.2.fq.gz +GroupA GroupA2 GroupA2.1.fq.gz GroupA2.2.fq.gz +GroupA GroupA3 GroupA3.1.fq.gz GroupA3.2.fq.gz +GroupA GroupA4 GroupA4.1.fq.gz GroupA4.2.fq.gz +GroupA GroupA5 GroupA5.1.fq.gz GroupA5.2.fq.gz +GroupA GroupA6 GroupA6.1.fq.gz GroupA6.2.fq.gz +GroupB GroupB1 GroupB1.1.fq.gz GroupB1.2.fq.gz +GroupB GroupB2 GroupB2.1.fq.gz GroupB2.2.fq.gz +GroupB GroupB3 GroupB3.1.fq.gz GroupB3.2.fq.gz +GroupB GroupB4 GroupB4.1.fq.gz GroupB4.2.fq.gz +GroupB GroupB5 GroupB5.1.fq.gz GroupB5.2.fq.gz +GroupB GroupB6 GroupB6.1.fq.gz GroupB6.2.fq.gz +GroupC GroupC1 GroupC1.1.fq.gz GroupC1.2.fq.gz +GroupC GroupC2 GroupC2.1.fq.gz GroupC2.2.fq.gz +GroupC GroupC3 GroupC3.1.fq.gz GroupC3.2.fq.gz +GroupC GroupC4 GroupC4.1.fq.gz GroupC4.2.fq.gz +GroupC GroupC5 GroupC5.1.fq.gz GroupC5.2.fq.gz +GroupC GroupC6 GroupC6.1.fq.gz GroupC6.2.fq.gz diff --git a/script/00_FQ_trim.2.pl b/script/00_FQ_trim.2.pl new file mode 100644 index 0000000..4e0803d --- /dev/null +++ b/script/00_FQ_trim.2.pl @@ -0,0 +1,44 @@ +#!/usr/bin/perl +#=============================================================================== +# FILE: fastq_clean.pl +# USAGE: perl fastq_clean.pl +# DESCRIPTION: +# OPTIONS: --- +# REQUIREMENTS: --- +# BUGS: --- +# NOTES: --- +# AUTHOR: luhanlin@genomics.org.cn +# COMPANY: BGI +# VERSION: 1.0 +# CREATED: 05/06/2010 05:17:07 PM +#=============================================================================== +use strict; +use warnings; +use PerlIO::gzip; +die "Usage: perl $0 in.fq [1|2] out.fq \n" unless @ARGV==3; +my ($fq, $pe, $out) = @ARGV; +if ($fq=~/\.gz$/){ + open IN,"zcat $fq |" or die "$! --> $fq\n"; +} +else{ + open IN,"<$fq" or die "$! --> $fq\n"; +} +if ($out=~/\.gz$/){ + open OUT, ">:gzip", "$out" or die $!; +} +else{ + open OUT, ">$out" or die $!; +} +########## +my $tt = 0; +while(my $line1 = ){ + $tt += 1; + my $num = sprintf("%09d",$tt); + $line1 = "\@R$num:$pe\n"; + my $line2 = ; + my $line3 = ; + my $line4 = ; + print OUT $line1.$line2."+\n".$line4; +} +close IN; +close OUT; diff --git a/script/01_FQ_pas.pl b/script/01_FQ_pas.pl new file mode 100644 index 0000000..d1332de --- /dev/null +++ b/script/01_FQ_pas.pl @@ -0,0 +1,62 @@ +#!/usr/bin/perl +#=============================================================================== +# FILE: fastq_clean.pl +# USAGE: perl fastq_clean.pl +# DESCRIPTION: +# OPTIONS: --- +# REQUIREMENTS: --- +# BUGS: --- +# NOTES: --- +# AUTHOR: luhanlin@genomics.org.cn +# COMPANY: BGI +# VERSION: 1.0 +# CREATED: 05/06/2010 05:17:07 PM +#=============================================================================== +use strict; +use warnings; +use PerlIO::gzip; +die "Usage: perl $0 1.fq 2.fq out.fq \n" unless @ARGV==3; +my ($pe1, $pe2, $out) = @ARGV; +###### +if ($pe1=~/\.gz$/){ + open IA,"<:gzip","$pe1" or die "$! --> $pe1\n"; +} +else{ + open IA,"<$pe1" or die "$! --> $pe1\n"; +} +###### +if ($pe2=~/\.gz$/){ + open IB,"<:gzip","$pe2" or die "$! --> $pe2\n"; +} +else{ + open IB,"<$pe2" or die "$! --> $pe2\n"; +} +###### +if ($out=~/\.gz$/){ + open OUT, ">:gzip", "$out" or die $!; +} +else{ + open OUT, ">$out" or die $!; +} +########## +my $tt = 0; +my $rr = 0; +while(my $a1 = ){ + $rr ++; + my $a2 = ; + my $a3 = ; + my $a4 = ; + my $b1 = ; + my $b2 = ; + my $b3 = ; + my $b4 = ; + my $id1 = substr($a1, 2, 9); + my $id2 = substr($b1, 2, 9); + $tt ++ if $id1 eq $id2; + print OUT $a1.$a2.$a3.$a4; + print OUT $b1.$b2.$b3.$b4; +} +print STDERR "Total $rr reads in PE1..\nTotal $tt pairs of reads..\n"; +close IA; +close IB; +close OUT; diff --git a/script/02_FQ_clean.2.pl b/script/02_FQ_clean.2.pl new file mode 100644 index 0000000..235d18c --- /dev/null +++ b/script/02_FQ_clean.2.pl @@ -0,0 +1,138 @@ +#!/usr/bin/perl +# AUTHOR: luhanlin@genomics.org.cn +# COMPANY: BGI +# VERSION: 1.0 +# CREATED: 05/06/2010 05:17:07 PM +use strict; +use warnings; +use PerlIO::gzip; +die "Usage: perl $0 in.fq out.prefix \n" unless @ARGV==2; + +my ($fq, $out) = @ARGV; +my $in; +if ($fq=~/\.gz$/){ + open $in, "<:gzip","$fq" or die "$! --> $fq\n"; +} +else{ + open $in, "<$fq" or die "$! --> $fq\n"; +} + +open OA, ">:gzip", "$out.1.fq.gz" or die $!; +open OB, ">:gzip", "$out.2.fq.gz" or die $!; +open OC, ">:gzip", "$out.single.fq.gz" or die $!; +########## + +my $flag = 0; +my @pair = (); +my ($f1, $f2, $psum, $ssum); +print STDERR "Check and Clean begin..\n"; +while(1){ + if($flag==0){ + $pair[0] = readin($in); + $pair[1] = readin($in); + $flag = 2; + } + elsif($flag==1){ + $pair[1] = readin($in); + $flag = 2; + } + last unless $pair[0] && $pair[1]; + + ###print STDERR $pair[0] . $pair[1]; + if( paired(\@pair) ){ + $f1 = check($pair[0]); + $f2 = check($pair[1]); + $flag = 0; + if($f1 && $f2){ + $psum += 1; + print OA $pair[0]; + print OB $pair[1]; + } + elsif($f1){ + $ssum += 1; + print OC $pair[0]; + } + elsif($f2){ + $ssum += 1; + print OC $pair[1]; + } + } + else{ + $flag = 1; + $f1 = check($pair[0]); + if($f1){ + $ssum += 1; + print OC $pair[0]; + } + shift @pair; + } +} +if($pair[0]){ + $f1 = check($pair[0]); + print OC $pair[0] if $f1; +} +print STDERR "Check and Clean done..\n"; +print STDERR "Paired reads(p)\tsingle(s)\n"; +print STDERR "$psum * 2\t$ssum\n"; + +close $in; +close OA; +close OB; +close OC; + + +sub paired{ + my $arr = shift; + my $id1 = ( split(/\n/, $$arr[0]) )[0]; + my $id2 = ( split(/\n/, $$arr[1]) )[0]; + my $r1 = substr($id1, 2, 9); + my $r2 = substr($id2, 2, 9); + if($id1=~/:1$/ && $id2=~/:2$/ && $r1 eq $r2){ + return 1; + } + else{ + return 0; + } +} + +sub readin{ + my $fh = shift; + my $id = <$fh>; + if($id){ + my $r = <$fh>; + my $s = <$fh>; + my $q = <$fh>; + return( $id.$r.$s.$q ); + } + else{ + return(""); + } +} + +sub check{ + my $l = shift; + my ($line1, $line2, $line3, $line4) = split(/\n/, $l); + + my @arr = split(//, $line2); + my $i=0; + for(my $j=0; $j<@arr; $j++){ + if($arr[$j] eq 'N'){ + $i = $i+1; + } + } + my $percent1 = $i/@arr; + my @arr1 = split(//, $line4); + my $k=0; + for(my $m=0; $m<@arr1; $m++){ + if( ord($arr1[$m])-33 <= 5 ){ + $k = $k+1; + } + } + my $percent2 = $k/@arr1; + if($percent1 >= 0.1 || $percent2 >= 0.5){ + return 0; + } + else{ + return 1; + } +} diff --git a/script/02_FQ_stat.pl b/script/02_FQ_stat.pl new file mode 100644 index 0000000..2283d2f --- /dev/null +++ b/script/02_FQ_stat.pl @@ -0,0 +1,49 @@ +#!/usr/bin/perl +# AUTHOR: luhanlin@genomics.org.cn +# COMPANY: BGI +# VERSION: 1.0 +# CREATED: 05/06/2010 05:17:07 PM +use strict; +use warnings; +use PerlIO::gzip; +die "Usage: perl $0 fq_list out.prefix \n" unless @ARGV>=2; + +my @file = @ARGV; +my $stat = pop( @file ); +my $reads; +my $base; +my $q20; +my $q30; +my $Q = 33; + +print STDERR "Begin..\n"; +foreach my $fq (@file){ + my $in; + print STDERR "Read $fq..\n"; + if ($fq=~/\.gz$/){ + open $in, "<:gzip","$fq" or die "$! --> $fq\n"; + }else{ + open $in, "<$fq" or die "$! --> $fq\n"; + } + while(<$in>){ + $reads += 1; + my $seq = <$in>; + chomp $seq; + $base += length($seq); + <$in>; + my $qual = <$in>; chomp $qual; + foreach ( split(//,$qual) ){ + $q20 += 1 if ord($_) - $Q >= 20; + $q30 += 1 if ord($_) - $Q >= 30; + } + } + close $in; +} + +open OUT, ">$stat", or die $!; +print OUT "Reads\tBase\tQ20\tQ20_r\tQ30\tQ30_r\n"; +print OUT join("\t",$reads, $base, $q20, sprintf("%.6f", $q20/$base), $q30, sprintf("%.6f", $q30/$base), ) . "\n"; +close OUT; +print STDERR "Done..\n"; +########## + diff --git a/script/BS_CleanFastqc_batch.v1.0.pl b/script/BS_CleanFastqc_batch.v1.0.pl new file mode 100644 index 0000000..c79aff2 --- /dev/null +++ b/script/BS_CleanFastqc_batch.v1.0.pl @@ -0,0 +1,79 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_CleanFastqc_batch + + It is designed for running BS cleandata fastqc in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p fastq files folder path; + -o results file path; + -t Specifies the number of files which can be processed simultaneously. + Each thread will be allocated 250MB of memory so you shouldn't run more + threads than your available memory will cope with, and not more than 6 threads on a 32 bit machine + -fastqc fastqc program; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my ($file,$outfile,$fqpath,$threads,$fastqc); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "t=s" => \$threads, + "fastqc=s" => \$fastqc, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $fqpath; +die $usage unless $fastqc; +$threads=1 unless $threads; +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($file)); + $outfile=abs_path($file); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile; + my $path1=$fqpath."/".$sampleinfo[1]."/".$sampleinfo[1].'.clean.1.fq.gz'; + my $path2=$fqpath."/".$sampleinfo[1]."/".$sampleinfo[1].'.clean.2.fq.gz'; + my $totalpath.=$path1." ".$path2; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "$fastqc -t $threads -o $opath $totalpath\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=$threads $signalpbs\n"; + print SGEall "qsub -cwd -l vf=1G -q all.q $signalpbs\n"; + } +close FILE; +close PBSall; +close SGEall; +########################################################################################################################### diff --git a/script/BS_DMRannotate.v1.1.pl b/script/BS_DMRannotate.v1.1.pl new file mode 100644 index 0000000..336ed04 --- /dev/null +++ b/script/BS_DMRannotate.v1.1.pl @@ -0,0 +1,141 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin $Script); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_DMRannotate + + It is designed for DMR annotation of BS-seq. + version:1.1 + usage: perl $0 + -i "*.dmr.filter_qval.0.05.out" file from DMR detection; + + -a assign a annotation list file; or do not set this by use the script to generate a default annotation list; + the format of annotation list is like(default): + + up2k \$Pj_path/04_global_methy/03_element_methy/element/genome.up2k.bed + down2k \$Pj_path/04_global_methy/03_element_methy/element/genome.down2k.bed + promoter \$Pj_path/04_global_methy/03_element_methy/element/genome.promoter.bed + genebody \$Pj_path/04_global_methy/03_element_methy/element/genome.genebody.bed + intron \$Pj_path/04_global_methy/03_element_methy/element/genome.intron.bed + exon \$Pj_path/04_global_methy/03_element_methy/element/genome.exon.bed + cgi \$Pj_path/04_global_methy/03_element_methy/element/genome.cgi.bed + + format of .bed files: + chr1 start end ID annotation + + + -p project path; + -o annotation results file path; + -ele the annotated element gene list you want; default: "promoter,genebody"; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my $p1="$Bin/p02.DMR-annotator.pl"; + + +my ($file,$outdir,$annolist,$element,$pro); +my $help; +GetOptions("i=s" => \$file, + "a=s" => \$annolist, + "o=s" => \$outdir, + "ele=s" => \$element, + "p=s" => \$pro, + "h=s" => \$help + ); + +die $usage if $help; +die $usage unless $file; +die $usage unless $outdir; +#die $usage if(!($annolist && $pro)); +print("You are using a default annotation list file, which will annotate DMRs to the following elements: +######################## +##### upstream2k ##### +##### downstream2k ##### +##### promoter ##### +##### exon ##### +##### intron ##### +##### genebody ##### +##### cgi ##### +######################## +") unless $annolist; + + +system("mkdir -p $outdir"); +my @dir=split(/\//,$file); +my $out="$outdir\/$dir[-1].annotate.xls"; + +if($annolist){ + open ANNO, $annolist or die "can not open $annolist:$!"; + while(){ + chomp $_; + my @e=split(/\t/,$_); + print "You will annotate DMRs to\: ###### $e[0] ######\n"; + } + close ANNO; + print "|------DMR annotation start...\n"; + system("perl $p1 -DMR $file -annot $annolist \> $out"); +}else{ + my $default_annolist ="$outdir\/default.annolist"; + open DE, ">$default_annolist" or die "can not open $default_annolist:$!"; + print DE "up2k $pro/03_global_methy/03_element_methy/element/genome.up2k.bed\n"; + print DE "down2k $pro/03_global_methy/03_element_methy/element/genome.down2k.bed\n"; + print DE "promoter $pro/03_global_methy/03_element_methy/element/genome.promoter.bed\n"; + print DE "genebody $pro/03_global_methy/03_element_methy/element/genome.genebody.bed\n"; + print DE "intron $pro/03_global_methy/03_element_methy/element/genome.intron.bed\n"; + print DE "exon $pro/03_global_methy/03_element_methy/element/genome.exon.bed\n"; + print DE "cgi $pro/03_global_methy/03_element_methy/element/genome.cgi.bed\n"; + close DE; + print "|------DMR annotation start...\n"; + system("perl $p1 -DMR $file -annot $default_annolist \> $out"); +} + +my @elelist=split(/\,/,$element); +foreach my $e(@elelist){ + print "|------Output $e DMR gene list..\n"; + my $genelist = "$out.$e.genelist.txt"; + open AN, $out or die "can not open $out:$!"; + open LIST, ">$genelist" or die "can not open $genelist:$!"; + my $n; + my $DMR_num=0; + my $DMG_num=0; + my %list; + while(){ + chomp $_; + my @F=split(/\t/,$_); + if($.==1){ + while (my($id, $val) = each @F){ + if($val eq $e){ + $n = $id; + } + } + print "======= In the $n th colum in annotate result file ============\n"; + } + my @a=split(/\,/,$F[$n]);# PARD3|NM_001184785,PARD3|NM_001184792 + foreach my $i (@a){ + my @b=split(/\|/, $i); # PARD3|NM_001184785 + if($b[0] ne "--" && $b[0] ne $e ){ + #print LIST "$b[0]\n"; + #print "$i\n" unless $b[0] eq "--"; + $DMR_num++; + $list{$b[0]}=1; + } + } + } + # print uniq gene list + while (my($key, $val) = each %list){ + print LIST "$key\n"; + $DMG_num++; + } + + print "|------Total annotate $DMR_num DMR to $e region of $DMG_num genes...\n"; + close LIST; + close AN; +} +print "|------Congratulations! DMR annotation finished!\n"; + + + diff --git a/script/BS_DMRdetect.v1.1.pl b/script/BS_DMRdetect.v1.1.pl new file mode 100644 index 0000000..df7811a --- /dev/null +++ b/script/BS_DMRdetect.v1.1.pl @@ -0,0 +1,162 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +#use Math::Combinatorics; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: rrbs_DMRdetect + + It is designed for detect DMR among groups of BS-seq. + version:1.1 + usage: perl $0 + -i map file; + -bedgraph .bedgraph file dir; + -o results file path; + -d C site cut depth; select as your process in previous steps. + -t C type: CG,CHG or CHH; Default is CG; + -thread Default 4; + -maxdist max C distance to define a DMR; Default is 300; + -minc min mC sites number in DMR detection; Default is 3; + -diff min methylation difference in DMR detection;Default is 0.1; + -X minimal number of values in group A (default:-1, means at lest 80% of samples in group A); + -Y minimal number of values in group B (default:-1, means at lest 80% of samples in group B); + -p use p-value to filtering DMRs in DMR detection. you should not assign -q if this parameters is assigned ; Default is 0.05; + -q use q-value to filtering DMRs in DMR detection; Default is 0.05; + -pairwise pairwise group comparison mode list like: "CON-CASE1,CON-CASE2,..."; + -ele "global" for genome-wide DMR detection; provide a bed-file for pre-defined regions DMR detection; Default is "global". + -elename region name for pre-defined regions DMR detection in "-ele" parameter; + -metilene metilene program; + -bedtools bedtools excutable file; + -Rscript Rscript program; + -h help; + author: zhangdu + date: 2018-06-20 +USAGE + +my $p1="$Bin/p01.DMR-detector.v1.2.pl"; + + +my ($file,$outfile,$type,$x,$y,$diff,$bedgraph,$p_value,$q_value,$maxDistance,$mincpg,$thread,$element,$elename,$bedtools,$depth,$pairwise,$Rscript,$metilene); +my $help; +GetOptions("i=s" => \$file, + "b=s" => \$bedgraph, + "d=i" => \$depth, + "t=s" => \$type, + "thread=i" => \$thread, + "ele=s" => \$element, + "elename=s" => \$elename, + "minc=i" => \$mincpg, + "pairwise=s" => \$pairwise, + "maxdist=i" => \$maxDistance, + "diff=f" => \$diff, + "p=f" => \$p_value, + "q=f" => \$q_value, + "X=i" => \$x, + "Y=i" => \$y, + "metilene=s" => \$metilene, + "bedtools=s" => \$bedtools, + "Rscript=s" => \$Rscript, + "o=s" => \$outfile, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $outfile; +die $usage unless $bedgraph; +die $usage unless $depth; +die $usage unless $pairwise; +die $usage unless $metilene; +die $usage unless $bedtools; +die $usage unless $Rscript; +die $usage if(defined $p_value && defined $q_value); +if(!defined $p_value && !defined $q_value){$q_value = 0.05}; +die $usage if(defined $elename && !defined $element); + +$thread ||= 4; +$element ||= "global"; +$diff ||=0.1; +$mincpg ||= 3; +$maxDistance ||= 300; +$x ||= -1; +$y ||= -1; +$type ||= "CG"; + +my %groupindex; +my $group; +print "|-----Reading map file...\n"; +open FILE, $file or die "can not open $file:$!"; #打开文件 +my $group="$outfile\/$type.sample_group_bedgraph.txt"; +open GROUP, ">$group" or die "can not open $group:$!"; + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + #$groupindex{$sampleinfo[0]}=$sampleinfo[1]; + #my $opath="$outfile\/$sampleinfo[1]\/"; + my $in="$bedgraph\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."$type\.bedgraph"; + if(!-e $in){print "INPUT FATAL ERRO: erro depth parameters, please check your input of \"-d\"!!!!!!!\n";die}; + print GROUP "$sampleinfo[1]\t$sampleinfo[0]\t$in\n"; + } +close GROUP; +close FILE; + +##########Combinatorics +#my @grouparry; +#while(my($key, $value) = each(%groupindex)) { +# push @grouparry, $key; +#} + +#my @tmpitem=split(/\n/,join("\n", map { join ",", @$_ } combine(2,@grouparry)),"\n"); +#for(my $i=0;$i<=$#tmpitem;$i++){ +# $groupindex{$tmpitem[$i]}='Y'; +#} + +my @pairwiselist=split(/\,/,$pairwise); + +my $n=0; +my $typedir="$outfile\/m$type\_DMR"; +#while(my($key, $value) = each(%groupindex)){ +foreach my $cmp(@pairwiselist){ + $n++; + my @g =split(/\-/,$cmp); + my $cmpdir="$typedir\/$cmp"; + system("mkdir -p $cmpdir"); + my $subgroup="$cmpdir\/$cmp.$type.compare.map"; + print "=========================================================================\n"; + print "|-----$type DMR detection between groups: $cmp ...\n"; + print "=========================================================================\n"; + system("date"); + system("perl -alne 'print if(\$F[1] eq $g[0] \|\| \$F[1] eq $g[1])' $group > $subgroup"); + if($p_value){ + # use p-value to filter DMR + if($element eq "global"){ + my $spedir="$cmpdir\/global\-$depth"."x"."\-$mincpg$type\-dist$maxDistance\-D$diff\-P$p_value"; # file name: global-5x-3CG-dist300-D0.1-Q0.05/ + system("mkdir -p $spedir"); + system("perl $p1 -list $subgroup -outdir $spedir -name $cmp -max $maxDistance -min $mincpg -t $thread -f 1 -p $p_value -c $mincpg -r $diff -X $x -Y $y -metilene $metilene -bedtools $bedtools -Rscript $Rscript"); + }else{ + my $spedir="$cmpdir\/$elename\-$depth"."x"."\-$mincpg$type\-dist$maxDistance\-D$diff\-P$p_value"; + system("perl $p1 -list $subgroup -outdir $spedir -name $cmp -max $maxDistance -min $mincpg -t $thread -f 2 -p $p_value -c $mincpg -r $diff -X $x -Y $y -metilene $metilene -bedtools $bedtools -B $element -Rscript $Rscript"); + } + + }elsif($q_value){ + # use q-value to filter DMR + if($element eq "global"){ + my $spedir="$cmpdir\/global\-$depth"."x"."\-$mincpg$type\-dist$maxDistance\-D$diff\-Q$q_value"; # file name: global-5x-3CG-dist300-D0.1-Q0.05/ + system("mkdir -p $spedir"); + system("perl $p1 -list $subgroup -outdir $spedir -name $cmp -max $maxDistance -min $mincpg -t $thread -f 1 -q $q_value -c $mincpg -r $diff -X $x -Y $y -metilene $metilene -bedtools $bedtools -Rscript $Rscript"); + }else{ + my $spedir="$cmpdir\/$elename\-$depth"."x"."\-$mincpg$type\-dist$maxDistance\-D$diff\-Q$q_value"; + system("perl $p1 -list $subgroup -outdir $spedir -name $cmp -max $maxDistance -min $mincpg -t $thread -f 2 -q $q_value -c $mincpg -r $diff -X $x -Y $y -metilene $metilene -bedtools $bedtools -B $element -Rscript $Rscript"); + } + + } + print "|-----DMR detection between groups finished: $cmp\n"; + system("date"); + print "\n\n"; + +} + + diff --git a/script/BS_ElementMethy_batch.v1.1.pl b/script/BS_ElementMethy_batch.v1.1.pl new file mode 100644 index 0000000..e250c1e --- /dev/null +++ b/script/BS_ElementMethy_batch.v1.1.pl @@ -0,0 +1,121 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin $Script); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: rrbs_ElementMethy_batch + + It is designed for running gene element methylation level of BS-seq in batch model. + version:1.0 + usage: perl $0 + -i map file; + -c bsmap cout file; + -o results file path; + -db UCSC or NCBI or ensembl; + -r reference genome; + -anno gene annotation file; + --UCSC: refGene.txt.gz + --NCBI: gff or gff + --ensembl: gff3 + -cgi 1.if it is a mammal, species.cpgIslandExt.txt.gz download from UCSC; + 2.if it is a mammal, without UCSC cgi files: "build"; + 3.other species has no CGI definition,just do not assign this parameter; + -Rscript Rscript program; + -h help; + author: zhangdu + date: 2018-05-29 +USAGE + +my $p1="$Bin/parse_UCSC_refGene.sh"; +my $p2="$Bin/parse_NCBI_gff3.sh"; +my $p3="$Bin/CGI.element_finder.v1.0.sh"; +my $p4="$Bin/CGI.element_finder_forUCSC.v1.0.sh"; +my $p5="$Bin/z04.elements_methyl.sh"; + +my ($ref,$file,$outfile,$coutdir,$db,$anno,$cgi,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "c=s" => \$coutdir, + "o=s" => \$outfile, + "db=s" => \$db, + "r=s" => \$ref, + "anno=s" => \$anno, + "cgi=s" => \$cgi, + "Rscript=s" => \$Rscript, + "h=s" => \$help +); + +die $usage if $help; +die $usage unless $file; +die $usage unless $coutdir; +die $usage unless $outfile; +die $usage unless $db; +die $usage unless $ref; +die $usage unless $anno; +die $usage unless $Rscript; +$cgi ||=""; + +$coutdir=~s/\/\//\//; + +#gene element extraction +print "|--------gene element extracting from $anno...\n"; +system("date"); +system("mkdir -p $outfile/element"); +if($db eq "UCSC"){ + system("cd $outfile/element \&\& sh $p1 $ref $anno $outfile/element genome"); +}elsif($db eq "NCBI"){ + system("cd $outfile/element \&\& sh $p2 $ref $anno $outfile/element genome"); +} +print "|--------gene element extraction finished...\n"; +system("date"); + +#CG Island and CG Island shore extraction +print "|--------cgi,cgi shore and shelf extracting from start...\n"; +system("date"); +if($cgi ne ""){ + if($cgi eq "build"){ + print "|--------Building cgi.bed of $ref by Emboss cpgplot tools...\n"; + system("sh $p3 $ref $outfile/element genome"); + }else{ + print "|--------cgi and cgi shore extracting from $cgi...\n"; + system("sh $p4 $ref $cgi $outfile/element genome"); + } +} +print "|--------cgi,cgi shore and shelf extracting finished...\n"; +system("date"); + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $cout=$coutdir."/".$sampleinfo[1]."/".$sampleinfo[1].'.cout.gz'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "sh $p5 $outfile/element $cout $opath CG $sampleinfo[1] $Rscript cgi\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=2G -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/BS_ElementMethy_batch.v1.2.pl b/script/BS_ElementMethy_batch.v1.2.pl new file mode 100644 index 0000000..6859763 --- /dev/null +++ b/script/BS_ElementMethy_batch.v1.2.pl @@ -0,0 +1,132 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin $Script); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: rrbs_ElementMethy_batch + + It is designed for running gene element methylation level of BS-seq in batch model. + version:1.0 + usage: perl $0 + -i map file; + -c bsmap cout file; + -o results file path; + -db UCSC or NCBI or ensembl; + -r reference genome; + -anno gene annotation file; + --UCSC: refGene.txt.gz + --NCBI: gff or gff + --ensembl: gff3 + -cgi 1.if it is a mammal, species.cpgIslandExt.txt.gz download from UCSC; + 2.if it is a mammal, without UCSC cgi files: "build"; + 3.other species has no CGI definition,just do not assign this parameter; + -repeat UCSC repeats annotation gz file; + -Rscript Rscript program; + -h help; + author: zhangdu + date: 2018-05-29 +USAGE + +my $p1="$Bin/parse_UCSC_refGene.sh"; +my $p2="$Bin/parse_NCBI_gff3.sh"; +my $p3="$Bin/CGI.element_finder.v1.0.sh"; +my $p4="$Bin/CGI.element_finder_forUCSC.v1.0.sh"; +my $p5="$Bin/z04.elements_methyl.v1.1.sh"; +my $p6="$Bin/parse_UCSC_repeats.sh"; + +my ($ref,$file,$outfile,$coutdir,$db,$anno,$cgi,$repeats,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "c=s" => \$coutdir, + "o=s" => \$outfile, + "db=s" => \$db, + "r=s" => \$ref, + "anno=s" => \$anno, + "cgi=s" => \$cgi, + "repeat=s" => \$repeats, + "Rscript=s" => \$Rscript, + "h=s" => \$help +); + +die $usage if $help; +die $usage unless $file; +die $usage unless $coutdir; +die $usage unless $outfile; +die $usage unless $db; +die $usage unless $ref; +die $usage unless $anno; +die $usage unless $repeats; +die $usage unless $Rscript; +$cgi ||=""; + +$coutdir=~s/\/\//\//; + +#gene element extraction +print "|--------gene element extracting from $anno...\n"; +system("date"); +system("mkdir -p $outfile/element"); +if($db eq "UCSC"){ + system("cd $outfile/element \&\& sh $p1 $ref $anno $outfile/element genome"); +}elsif($db eq "NCBI"){ + system("cd $outfile/element \&\& sh $p2 $ref $anno $outfile/element genome"); +} +print "|--------gene element extraction finished...\n"; +system("date"); + +#CG Island and CG Island shore extraction +print "|--------cgi,cgi shore and shelf extracting from start...\n"; +system("date"); +if($cgi ne ""){ + if($cgi eq "build"){ + print "|--------Building cgi.bed of $ref by Emboss cpgplot tools...\n"; + system("sh $p3 $ref $outfile/element genome"); + }else{ + print "|--------cgi and cgi shore extracting from $cgi...\n"; + system("sh $p4 $ref $cgi $outfile/element genome"); + } +} +print "|--------cgi,cgi shore and shelf extracting finished...\n"; +system("date"); + +#genome repeat elements extraction +print "|--------Repeat element extracting from $repeats...\n"; +system("date"); +system("sh $p6 $repeats $outfile/element"); +print "|--------Repeat element extraction finished...\n"; +system("date"); + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $cout=$coutdir."/".$sampleinfo[1]."/".$sampleinfo[1].'.cout.gz'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "sh $p5 $outfile/element $cout $opath CG $sampleinfo[1] $Rscript cgi\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=2G -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/BS_GlobalMethy_batch.v1.0.pl b/script/BS_GlobalMethy_batch.v1.0.pl new file mode 100644 index 0000000..706e50e --- /dev/null +++ b/script/BS_GlobalMethy_batch.v1.0.pl @@ -0,0 +1,79 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_GlobalMethy_batch + + It is designed for running Global Methylation profile of BS-seq in batch model. + version:1.0 + usage: perl $0 + -i map file; + -c bsmap cout file; + -o results file path; + -d max plot depth;default is 30x; + -r reference genome fa; + -Rscript Rscript program; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my $p1="$Bin/z01.dep_coverage.pl"; +my $p2="$Bin/z02.mC_seqlogo.sh"; + +my ($file,$outfile,$coutdir,$group,$ref,$maxplotdepth,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "c=s" => \$coutdir, + "o=s" => \$outfile, + "d=i" => \$maxplotdepth, + "r=s" => \$ref, + "Rscript=s" => \$Rscript, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $coutdir; +die $usage unless $ref; +die $usage unless $Rscript; +$maxplotdepth ||= 30; + +$coutdir=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $cout=$coutdir."/".$sampleinfo[1]."/".$sampleinfo[1].'.cout.gz'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "perl $p1 -i $cout -o $opath -n $sampleinfo[1] -d $maxplotdepth -r $Rscript + sh $p2 $sampleinfo[1] $cout $opath $ref $Rscript\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=2G -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/BS_Globalpattern_batch.v1.0.pl b/script/BS_Globalpattern_batch.v1.0.pl new file mode 100644 index 0000000..2082853 --- /dev/null +++ b/script/BS_Globalpattern_batch.v1.0.pl @@ -0,0 +1,108 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_Globalpattern_batch + + It is designed for plot chr global methy pattern of BS-seq in batch model. + version:1.0 + usage: perl $0 + -i map file; + -c bsmap cout file; + -o results file path; + -r reference genome; + -m min chr to plot; default is 400000; + -Rscript Rscript program; + -h help; + author: zhangdu + date: 2018-05-29 +USAGE + +my $p1="$Bin/stat-fa_GC_detect.pl"; +my $p2="$Bin/z03.global_pattern.pl"; + +my ($file,$outfile,$coutdir,$ref,$minChrLen,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "c=s" => \$coutdir, + "o=s" => \$outfile, + "r=s" => \$ref, + "m=i" => \$minChrLen, + "Rscript=s" => \$Rscript, + "h=s" => \$help +); + +die $usage if $help; +die $usage unless $file; +die $usage unless $coutdir; +die $usage unless $outfile; +die $usage unless $ref; +die $usage unless $Rscript; +$minChrLen ||=400000; + +$coutdir=~s/\/\//\//; + + +# genome stat +print "|--------Making genome GC stat...\n"; +system("date"); +my $fa_stat="$outfile/genome.GC_stat"; +my $chr_list="$outfile/genome.$minChrLen.chr_list"; +system("perl $p1 $ref \> $fa_stat"); +print "|--------Genome GC stat finished...\n"; +system("date"); + +# select >= 400000 bp chr to plot +print "|--------> $minChrLen chr filtering...\n"; +system("perl -alne 'if(\$F[1]>$minChrLen){print \"\$F[0]\\t\$F[1]\"}' $fa_stat \> $chr_list"); + +my @chr; +my $n=0; +open CHR, $chr_list or die "$!\n"; +while(){ + chomp; + my @a=split; + push @chr,$a[0]; + $n++; +} +print "|--------> Total $n chr methylation pattern to plot...\n"; +system("date"); + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $cout=$coutdir."/".$sampleinfo[1]."/".$sampleinfo[1].'.cout.gz'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + foreach my $chrm(@chr){ + print SIGNAL "perl $p2 -I $cout -N $chrm -B 10000 -O $opath -R $Rscript && $chrm global methylation pattern analysis finished..\n"; + } + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=2G -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/BS_bedgraph_batch.v1.0.pl b/script/BS_bedgraph_batch.v1.0.pl new file mode 100644 index 0000000..7b9a69e --- /dev/null +++ b/script/BS_bedgraph_batch.v1.0.pl @@ -0,0 +1,80 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_bedgragh_batch + + It is designed for generate .bedgraph from cout of BS-seq in batch model. + version:1.0 + usage: perl $0 + -i map file; + -c bsmap cout file; + -o results file path; + -d >= dx CpG sites with be keeped; default is 5; + -bedtools bedtools dir; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + + +my ($file,$outfile,$coutdir,$depth,$bedtools); +my $help; +GetOptions("i=s" => \$file, + "c=s" => \$coutdir, + "o=s" => \$outfile, + "d=i" => \$depth, + "bedtools=s" => \$bedtools, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $coutdir; +die $usage unless $bedtools; +$depth ||= 5; + +$coutdir=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath="$outfile\/$sampleinfo[1]\/"; + my $out0="$outfile\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."C\.bedgraph"; + my $out1="$outfile\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."CG\.bedgraph"; + my $out2="$outfile\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."CHG\.bedgraph"; + my $out3="$outfile\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."CHH\.bedgraph"; + system("mkdir -p $opath"); + my $cout="$coutdir\/$sampleinfo[1]\/$sampleinfo[1]\.cout\.gz"; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL " +zcat $cout \| perl -alne 'if(\$F[6]+\$F[7] >= $depth){print join \"\\t\",\$F[0],\$F[1],\$F[1]+1,\$F[5]}' \| $bedtools sort -i \> $out0 && echo -e \">=$depth x C site filtering done..\" +zcat $cout \| perl -alne 'if(\$F[3] eq \"CG\" \&\& \$F[6]+\$F[7] >= $depth){print join \"\\t\",\$F[0],\$F[1],\$F[1]+1,\$F[5]}' \| $bedtools sort -i \> $out1 && echo -e \">=$depth x CG site filtering done..\" +zcat $cout \| perl -alne 'if(\$F[3] eq \"CHG\" \&\& \$F[6]+\$F[7] >= $depth){print join \"\\t\",\$F[0],\$F[1],\$F[1]+1,\$F[5]}' \| $bedtools sort -i \> $out2 \&\& echo -e \">=$depth x CHG site filtering done..\" +zcat $cout \| perl -alne 'if(\$F[3] eq \"CHH\" \&\& \$F[6]+\$F[7] >= $depth){print join \"\\t\",\$F[0],\$F[1],\$F[1]+1,\$F[5]}' \| $bedtools sort -i \> $out3 \&\& echo -e \">=$depth x CHH site filtering done..\"\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=2G -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/BS_makeMatrix.v1.2.pl b/script/BS_makeMatrix.v1.2.pl new file mode 100644 index 0000000..c441eec --- /dev/null +++ b/script/BS_makeMatrix.v1.2.pl @@ -0,0 +1,137 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_makeMatrix + + It is designed for generate standard methylation matrix and do PCA,PCC and Cluster analysis from sample .bedgraph of BS-seq. + version:1.1 + usage: perl $0 + -i map file; + -b .bedgraph file dir; + -t methy type to analysis: C,CG,CHG,CHH or combine of them, "," splited like: CG,CHG,CHH; + -o results file path; + -d the cut depth of C site; + -bedtools bedtools excutable file; + -Rscript Rscript program; + -h help; + author:zhangdu + date: 2018-08-08 +USAGE + +my $p1="$Bin/makeMatrix_merge.pl"; +my $p2="$Bin/makeMatrix_cluster.pl"; + +my ($file,$outfile,$depth,$bed,$bedtools,$type,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "b=s" => \$bed, + "t=s" => \$type, + "d=i" => \$depth, + "bedtools=s" => \$bedtools, + "Rscript=s" => \$Rscript, + "o=s" => \$outfile, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $outfile; +die $usage unless $bed; +die $usage unless $depth; +die $usage unless $Rscript; +die $usage unless $bedtools; +$type ||= "CG"; + +my (@C,@CG,@CHG,@CHH,@sample); +print "Reading map file...\n"; +open FILE, $file + or die "can not open $file:$!"; #打开文件 +my $group="$outfile\/sample_group.txt"; +open GROUP, ">$group" or die "can not open $group:$!"; + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + print GROUP $sampleinfo[0]."\n"; + #my $opath="$outfile\/$sampleinfo[1]\/"; + my $in0="$bed\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."C\.bedgraph"; + my $in1="$bed\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."CG\.bedgraph"; + my $in2="$bed\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."CHG\.bedgraph"; + my $in3="$bed\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."CHH\.bedgraph"; + push @C,$in0; + push @CG,$in1; + push @CHG,$in2; + push @CHH,$in3; + push @sample,$sampleinfo[1]; + } +close GROUP; +close FILE; + +my $c=join(",", @C); +my $cg=join(",", @CG); +my $chg=join(",", @CHG); +my $chh=join(",", @CHH); +my $s=join(",", @sample); + +my @mode=split(/,/,$type); + +foreach my $m(@mode){ +if($m eq "C"){ + # analysis mC + print "Making $depth x C methylation matrix file...\n"; + system("date"); + my $mC="$outfile\/mC"; + system("mkdir -p $mC"); + my $oc="$mC\/$depth"."x"."C\.methy\_Matrix.txt"; + system("perl $p1 --in $c --head $s --out $oc -b $bedtools"); + print "Making $depth x C methylation clustering...\n"; + system("date"); + system("perl $p2 --in $oc --class $group --pca --pcc --clust --out $oc --r $Rscript"); +} + +if($m eq "CG"){ +#analysis mCG + print "Making $depth x CG methylation matrix file...\n"; + system("date"); + my $mCG="$outfile\/mCG"; + system("mkdir -p $mCG"); + my $ocg="$mCG\/$depth"."x"."CG\.methy\_Matrix.txt"; + system("perl $p1 --in $cg --head $s --out $ocg -b $bedtools"); + print "Making $depth x CG methylation clustering...\n"; + system("date"); + system("perl $p2 --in $ocg --class $group --pca --pcc --clust --out $ocg --r $Rscript"); +} + +if($m eq "CHG"){ + #analysis mCHG + print "Making $depth x CHG methylation matrix file...\n"; + system("date"); + my $mCHG="$outfile\/mCHG"; + system("mkdir -p $mCHG"); + my $ochg="$mCHG\/$depth"."x"."CHG\.methy\_Matrix.txt"; + system("perl $p1 --in $chg --head $s --out $ochg -b $bedtools"); + print "Making $depth x CHG methylation clustering...\n"; + system("date"); + system("perl $p2 --in $ochg --class $group --pca --pcc --clust --out $ochg --r $Rscript"); +} + + +if($m eq "CHH"){ + #analysis mCHH + print "Making $depth x CHH methylation matrix file...\n"; + system("date"); + my $mCHH="$outfile\/mCHH"; + system("mkdir -p $mCHH"); + my $ochh="$mCHH\/$depth"."x"."CHH\.methy\_Matrix.txt"; + system("perl $p1 --in $chh --head $s --out $ochh -b $bedtools"); + print "Making $depth x CHH methylation clustering...\n"; + system("date"); + system("perl $p2 --in $ochh --class $group --pca --pcc --clust --out $ochh --r $Rscript"); +} +} + +########################################################################################################################### diff --git a/script/BS_qc_stat.v1.0.pl b/script/BS_qc_stat.v1.0.pl new file mode 100644 index 0000000..44dc986 --- /dev/null +++ b/script/BS_qc_stat.v1.0.pl @@ -0,0 +1,83 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: BS_qc_stat + + It is designed for running bs qc stat in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p fastq files folder path; + -o results file path; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my $p1="$Bin/02_FQ_stat.pl"; + +my ($file,$outfile,$fqpath); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $fqpath; +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($file)); + $outfile=abs_path($file); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +if (-e $jobpath){ + print STDOUT "$jobpath exists!"; +}else{ + system("mkdir -p $jobpath"); +} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + if (/^#Group/){next} + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $rpath1=$fqpath."/".$sampleinfo[1]."/".$sampleinfo[1].'.fq.gz'; + #my $rpath2=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.2.fq.gz'; + my $path1=$fqpath."/".$sampleinfo[1]."/".$sampleinfo[1].'.clean.1.fq.gz'; + my $path2=$fqpath."/".$sampleinfo[1]."/".$sampleinfo[1].'.clean.2.fq.gz'; + my $path4=$fqpath."/".$sampleinfo[1]."/".$sampleinfo[1].'.cut1.fq.gz'; + my $opath=$outfile."/".$sampleinfo[1]; + system("mkdir -p $opath"); + my $stat1=$opath."/".$sampleinfo[1].'.raw.xls'; + my $stat2=$opath."/".$sampleinfo[1].'.noAdapter.xls'; + my $stat3=$opath."/".$sampleinfo[1].'.clean.xls'; + $totalpath.=$path1." ".$path2." "; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL " +perl $p1 $rpath1 $stat1 && echo rawdata stat done.. +perl $p1 $path4 $stat2 && echo adapter free data stat done.. +perl $p1 $path1 $path2 $stat3 && echo cleandata stat done..\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=2G -q all.q $signalpbs\n"; + } +close FILE; +close PBSall; +########################################################################################################################### diff --git a/script/BS_stackbar.v1.0.pl b/script/BS_stackbar.v1.0.pl new file mode 100644 index 0000000..87fb5d2 --- /dev/null +++ b/script/BS_stackbar.v1.0.pl @@ -0,0 +1,84 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin $Script); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + +name: rrbs_stackbar + + It is designed for generate standard methylation matrix and do PCA,PCC and Cluster analysis from sample .bedgraph of BS-seq. + version:1.1 + usage: perl $0 + -i map file; + -b .bedgraph file dir; + -t methy type to analysis: C,CG,CHG,CHH or combine of them, "," splited like: CG,CHG,CHH; + -o results file path; + -d the cut depth of C site; + -bedtools bedtools excutable file; + -Rscript Rscript program; + -h help; + author:zhangdu + date: 2018-08-08 +USAGE + +my $p1="$Bin/makeMatrix_merge.pl"; +my $p2="$Bin/methy_stack_bar.v1.1.R"; + +my ($file,$outfile,$depth,$bed,$bedtools,$type,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "b=s" => \$bed, + "t=s" => \$type, + "d=i" => \$depth, + "bedtools=s" => \$bedtools, + "Rscript=s" => \$Rscript, + "o=s" => \$outfile, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $outfile; +die $usage unless $bed; +die $usage unless $depth; +die $usage unless $Rscript; +die $usage unless $bedtools; +$type ||= "CG"; + +my @mode=split(/\,/,$type); +foreach my $m(@mode){ + my (@bedgraph,@sample,@group); + print "Reading map file...\n"; + open FILE, $file or die "can not open $file:$!"; + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print "$sampleinfo[0]\t$sampleinfo[1]\n"; + my $in="$bed\/$sampleinfo[1]\/$sampleinfo[1]\_$depth"."x"."$m\.bedgraph"; + push @bedgraph,$in; + push @sample,$sampleinfo[1]; + push @group,$sampleinfo[0]; + } + close FILE; + + my $c=join(",", @bedgraph); + my $s=join(",", @sample); + my $s_head=join("\t","Sample",@sample); + my $g_head=join("\t","Group",@group); + + print "Making $depth x $m methylation matrix file...\n"; + system("date"); + my $mC="$outfile\/m$m"; + system("mkdir -p $mC"); + my $oc="$mC\/$depth"."x"."$m\.methy\_Matrix.txt"; + print "[COMMAND]: perl $p1 --in $c --head $s --out $oc -b $bedtools\n"; + system("perl $p1 --in $c --head $s --out $oc -b $bedtools"); + system("date"); + print "$m methylation stacked barplot for all samples...\n"; + my $oc_plot="$mC\/$depth"."x"."$m\.methy\_Matrix.forplot"; + system("perl -alne \'if\(\$.==1\)\{print \"$g_head\\n$s_head\";next\};print\' $oc \> $oc_plot"); + system("$Rscript $p2 $oc_plot 5 $mC/all_samples.$depth\X$m"); +} + +########################################################################################################################### diff --git a/script/CGI.element_finder.v1.0.sh b/script/CGI.element_finder.v1.0.sh new file mode 100644 index 0000000..3ac7d03 --- /dev/null +++ b/script/CGI.element_finder.v1.0.sh @@ -0,0 +1,40 @@ +#Usage: +# CGI.element_finder.v1.0.sh +# +# Note: you have to have emboss,samtools and bedtools installed in your system. +# output: 1.prefix.cgi.bed +# 2.prefix.cgi_shore.bed +# 3.prefix.cgi_shelf.bed + +ref=$1 +outdir=$2 +name=$3 +cd $outdir + +# CG island 预测 +################################## +echo "|--------Building $name.cgi.bed of $ref by Emboss cpgplot tools..." +cpgplot -outfeat emboss.cgIsland.txt -window 100 -minlen 200 -minoe 0.6 -minpc 50 -outfile emboss.cpgplot -graph svg $ref +less -S emboss.cgIsland.txt |grep -v '#' | cut -f1,4,5 | perl -alne '$id="cgi".$.; print join "\t",@F,$id,$id'> $name.cgi.bed + +# CGI shore 预测 +################################## +samtools faidx $ref +# 统计得到基因组åºåˆ—é•¿åº¦ä¿¡æ¯ +cut -f-2 $ref.fai > $ref.length +echo "|--------Building $name.cgi_shore.bed.." +slopBed -i $name.cgi.bed -g $ref.length -b 2000 | mergeBed | subtractBed -a - -b $name.cgi.bed > $name.cgi_shore.bed +# slop: åŒä¾§æ‰©å±•2000bp (cgiåŒä¾§æ‰©å±•2k) +# merge: é‡å çš„åºåˆ—åˆå¹¶ä¸ºä¸€æ¡ï¼ˆæœ‰é‡å çš„cgi shoreåˆå¹¶ï¼‰ +# subtract : a - b 生æˆæ–°åºåˆ—(åˆå¹¶ä¹‹åŽå†å‡åŽ»ä¸Žcgié‡å çš„部分) + +# CGI shelf 预测 +################################## +echo "|--------Building $name.cgi_shelf.bed.." +slopBed -i $name.cgi.bed -g $ref.length -b 4000 | mergeBed | subtractBed -a - -b $name.cgi_shore.bed |subtractBed -a - -b $name.cgi.bed > $name.cgi_shelf.bed + + + + + + diff --git a/script/CGI.element_finder_forUCSC.v1.0.sh b/script/CGI.element_finder_forUCSC.v1.0.sh new file mode 100644 index 0000000..d4a35da --- /dev/null +++ b/script/CGI.element_finder_forUCSC.v1.0.sh @@ -0,0 +1,40 @@ +#Usage: +# CGI.element_finder_forUCSC.v1.0.sh +# +# Note: you have to have samtools,bedtools installed in your system. +# output: 1.prefix.cgi.bed +# 2.prefix.cgi_shore.bed +# 3.prefix.cgi_shelf.bed + +ref=$1 +cgi=$2 +outdir=$3 +name=$4 +cd $outdir + +# CG island 预测 +################################## +echo "|--------cgi and cgi shore extracting from $cgi..."; +less -S $cgi | cut -f2,3,4 | perl -alne '$id="cgi".$.; print join "\t",@F,$id,$id'> $name.cgi.bed + +# CGI shore 预测 +################################## +samtools faidx $ref +# 统计得到基因组åºåˆ—é•¿åº¦ä¿¡æ¯ +cut -f-2 $ref.fai > $ref.length +echo "|--------Building $name.cgi_shore.bed..." +slopBed -i $name.cgi.bed -g $ref.length -b 2000 | mergeBed | subtractBed -a - -b $name.cgi.bed > $name.cgi_shore.bed +# slop: åŒä¾§æ‰©å±•2000bp (cgiåŒä¾§æ‰©å±•2k) +# merge: é‡å çš„åºåˆ—åˆå¹¶ä¸ºä¸€æ¡ï¼ˆæœ‰é‡å çš„cgi shoreåˆå¹¶ï¼‰ +# subtract : a - b 生æˆæ–°åºåˆ—(åˆå¹¶ä¹‹åŽå†å‡åŽ»ä¸Žcgié‡å çš„部分) + +# CGI shelf 预测 +################################## +echo "|--------Building $name.cgi_shelf.bed..." +slopBed -i $name.cgi.bed -g $ref.length -b 4000 | mergeBed | subtractBed -a - -b $name.cgi_shore.bed |subtractBed -a - -b $name.cgi.bed > $name.cgi_shelf.bed + + + + + + diff --git a/script/Draw_venn.pl b/script/Draw_venn.pl new file mode 100644 index 0000000..89c43d1 --- /dev/null +++ b/script/Draw_venn.pl @@ -0,0 +1,264 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use FindBin qw($Bin $Script); +use File::Basename qw(basename dirname); +use Cwd; +use PerlIO::gzip; +#~~~~~~~~ USAGE ~~~~~~~~# +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 + -I <> list1 + or list1,list2 + or list1,list2,list3 + or list1,list2,list3,list4 + or list1,list2,list3,list4,list5 + list[1..5] is a input file containing one column (e.g., integers, chars), + with each component corresponding to a separate circle in the Venn diagram + -N [] name1,name2,..name5 + -Rscript [] Rscript program + -O [] out.pdf +author: luhanlin\@genomics.org.cn +date: +USAGE +#~~~~~~~~ Globle Parameters ~~~~~~~~# +my ($filelist, $namelist, $pdf, $help,$Rscript); +GetOptions( + "help|?|h" => \$help, + "I=s" => \$filelist, + "N:s" => \$namelist, + "Rscript:s" => \$Rscript, + "O:s" => \$pdf, +); +die $usage if $help; +die $usage unless ($filelist); +die $usage unless $Rscript; +my (@flist, @nlist); +my ($readtable, $make_list, $p, $Rcode); +my @lab = qw(A B C D E); + +$pdf ||= "Venn.pdf"; + +if ($pdf=~/(\S+)\.pdf$/){ + $p = $1; +} +else{ + $p = $pdf; +} +#~~~~~~~~~ Main Process ~~~~~~~~# + +@flist = split(/,/, $filelist); + +die "\nNumber of filelist must <= 5 && >=1\n\n$usage" if (@flist>5 || @flist<1); + +foreach (@flist){ + if(!-e $_){ + die "$_ : can not find it, please check it!\n$usage"; + } +} + +if (defined $namelist){ + @nlist = split(/,/, $namelist); + die "# of namelist != # of filelist\n" unless $#nlist == $#flist; +} +else{ + for (my $i=0; $i<=$#flist; $i++){ + push @nlist, $lab[$i]; + } +} + +foreach (my $i=0; $i<@flist; $i++){ + $readtable .= "$lab[$i]<-read.table(\"$flist[$i]\")\n"; + $make_list .= " \"$nlist[$i]\"=as.vector($lab[$i]"."[,1]),"; +} +$make_list=~s/,$//; +$make_list = 'x=list(' . $make_list . '),'; +$Rcode = $readtable; + +if(@flist==1){ + $Rcode .= "Venn( $make_list filename = \"$p.pdf\", col = \"black\", lwd = 8, fontface = \"bold\", fill = \"grey\", alpha = 0.75, cex = 4, cat.cex = 3, cat.fontface = \"bold\", margin = 0.1 );\n"; +} +elsif(@flist==2){ + $Rcode .= "Venn( $make_list filename = \"$p.pdf\", lwd = 4, fill = c(\"cornflowerblue\", \"darkorchid1\"), alpha = 0.75, label.col = \"black\", cex = 3, fontfamily = \"serif\", fontface = \"bold\", cat.col = c(\"cornflowerblue\", \"darkorchid1\"), cat.cex = 2, cat.fontfamily = \"serif\", cat.fontface = \"bold\", cat.dist = c(0.03, 0.03), cat.pos = c(-20, 14), margin = 0.05 );\n"; +} +elsif(@flist==3){ + $Rcode .= "Venn( $make_list filename = \"$p.pdf\", col = \"black\", lwd = 4, fill = c(\"red\", \"blue\", \"green\"), alpha = 0.5, label.col = c(\"darkred\", \"white\", \"darkblue\", \"white\", \"white\", \"white\", \"darkgreen\"), cex = 2.5, fontfamily = \"serif\", fontface = \"bold\", cat.default.pos = \"text\", cat.col = c(\"darkred\", \"darkblue\", \"darkgreen\"), cat.cex = 2.5, cat.fontfamily = \"serif\", cat.dist = c(0.06, 0.06, 0.03), cat.pos = 0, margin = 0.1 );\n"; +} +elsif(@flist==4){ + $Rcode .= "Venn( $make_list filename = \"$p.pdf\", col = \"black\", lwd = 3, fill = c(\"cornflowerblue\", \"green\", \"yellow\", \"darkorchid1\"), alpha = 0.50, label.col = c(\"orange\", \"white\", \"darkorchid4\", \"white\", \"white\", \"white\", \"white\", \"white\", \"darkblue\", \"white\", \"white\" , \"white\", \"white\", \"darkgreen\", \"white\"), cex = 1.5, fontfamily = \"serif\", fontface = \"bold\", cat.col = c(\"darkblue\", \"darkgreen\", \"orange\", \"darkorchid4\"), cat.cex = 1.5, cat.pos = 0, cat.dist = 0.07, cat.fontfamily = \"serif\", rotation.degree = 0, margin = 0.1 );\n"; +} +elsif(@flist==5){ + $Rcode .= "Venn( $make_list filename = \"$p.pdf\", col = \"black\", fill = c(\"dodgerblue\", \"goldenrod1\", \"darkorange1\", \"seagreen3\", \"orchid3\"), alpha = 0.50, cex = c(1.5, 1.5, 1.5, 1.5, 1.5, 1, 0.8, 1, 0.8, 1, 0.8, 1, 0.8, 1, 0.8, 1, 0.55, 1, 0.55, 1, 0.55, 1, 0.55, 1, 0.55, 1, 1, 1, 1, 1, 1.5), cat.col = c(\"dodgerblue\", \"goldenrod1\", \"darkorange1\", \"seagreen3\", \"orchid3\"), cat.cex = 1.5, cat.fontface = \"bold\", margin = 0.05 );\n"; +} + +$Rcode = Rfunc().$Rcode; +open R,">$p.R" or die "$p.R : $!"; +print R $Rcode; +close R; +system ("$Rscript $p.R"); + +sub Rfunc{ + my $Rfunc=< 5) { + stop("Incorrect number of elements.", call. = FALSE) + } + if (1 == length(x)) { + list.names <- category.names + if (is.null(list.names)) { + list.names <- "" + } + grob.list <- VennDiagram::draw.single.venn(area = length(x[[1]]), + category = list.names, ind = FALSE, ...) + } + else if (2 == length(x)) { + grob.list <- VennDiagram::draw.pairwise.venn(area1 = length(x[[1]]), + area2 = length(x[[2]]), cross.area = length(intersect(x[[1]], + x[[2]])), category = category.names, ind = FALSE, + ...) + } + else if (3 == length(x)) { + A <- x[[1]] + B <- x[[2]] + C <- x[[3]] + list.names <- category.names + nab <- intersect(A, B) + nbc <- intersect(B, C) + nac <- intersect(A, C) + nabc <- intersect(nab, C) + grob.list <- VennDiagram::draw.triple.venn(area1 = length(A), + area2 = length(B), area3 = length(C), n12 = length(nab), + n23 = length(nbc), n13 = length(nac), n123 = length(nabc), + category = list.names, ind = FALSE, list.order = 1:3, + ...) + } + else if (4 == length(x)) { + A <- x[[1]] + B <- x[[2]] + C <- x[[3]] + D <- x[[4]] + list.names <- category.names + n12 <- intersect(A, B) + n13 <- intersect(A, C) + n14 <- intersect(A, D) + n23 <- intersect(B, C) + n24 <- intersect(B, D) + n34 <- intersect(C, D) + n123 <- intersect(n12, C) + n124 <- intersect(n12, D) + n134 <- intersect(n13, D) + n234 <- intersect(n23, D) + n1234 <- intersect(n123, D) + grob.list <- VennDiagram::draw.quad.venn(area1 = length(A), + area2 = length(B), area3 = length(C), area4 = length(D), + n12 = length(n12), n13 = length(n13), n14 = length(n14), + n23 = length(n23), n24 = length(n24), n34 = length(n34), + n123 = length(n123), n124 = length(n124), n134 = length(n134), + n234 = length(n234), n1234 = length(n1234), category = list.names, + ind = FALSE, ...) + } + else if (5 == length(x)) { + A <- x[[1]] + B <- x[[2]] + C <- x[[3]] + D <- x[[4]] + E <- x[[5]] + list.names <- category.names + n12 <- intersect(A, B) + n13 <- intersect(A, C) + n14 <- intersect(A, D) + n15 <- intersect(A, E) + n23 <- intersect(B, C) + n24 <- intersect(B, D) + n25 <- intersect(B, E) + n34 <- intersect(C, D) + n35 <- intersect(C, E) + n45 <- intersect(D, E) + n123 <- intersect(n12, C) + n124 <- intersect(n12, D) + n125 <- intersect(n12, E) + n134 <- intersect(n13, D) + n135 <- intersect(n13, E) + n145 <- intersect(n14, E) + n234 <- intersect(n23, D) + n235 <- intersect(n23, E) + n245 <- intersect(n24, E) + n345 <- intersect(n34, E) + n1234 <- intersect(n123, D) + n1235 <- intersect(n123, E) + n1245 <- intersect(n124, E) + n1345 <- intersect(n134, E) + n2345 <- intersect(n234, E) + n12345 <- intersect(n1234, E) + grob.list <- VennDiagram::draw.quintuple.venn(area1 = length(A), + area2 = length(B), area3 = length(C), area4 = length(D), + area5 = length(E), n12 = length(n12), n13 = length(n13), + n14 = length(n14), n15 = length(n15), n23 = length(n23), + n24 = length(n24), n25 = length(n25), n34 = length(n34), + n35 = length(n35), n45 = length(n45), n123 = length(n123), + n124 = length(n124), n125 = length(n125), n134 = length(n134), + n135 = length(n135), n145 = length(n145), n234 = length(n234), + n235 = length(n235), n245 = length(n245), n345 = length(n345), + n1234 = length(n1234), n1235 = length(n1235), n1245 = length(n1245), + n1345 = length(n1345), n2345 = length(n2345), n12345 = length(n12345), + category = list.names, ind = FALSE, ...) + } + else { + stop("Invalid size of input object") + } + if (!is.null(sub)) { + grob.list <- add.title(gList = grob.list, x = sub, pos = sub.pos, + fontface = sub.fontface, fontfamily = sub.fontfamily, + col = sub.col, cex = sub.cex) + } + if (!is.null(main)) { + grob.list <- add.title(gList = grob.list, x = main, pos = main.pos, + fontface = main.fontface, fontfamily = main.fontfamily, + col = main.col, cex = main.cex) + } + if (!is.null(filename)) { + pdf(file = filename, height = height, width = width) + grid.draw(grob.list) + dev.off() + return(1) + } + plot.new() + grid.draw(grob.list) +}\n +Rline + return($Rfunc); +} diff --git a/script/EAGE_function.v1.0.pl b/script/EAGE_function.v1.0.pl new file mode 100644 index 0000000..00cdbd6 --- /dev/null +++ b/script/EAGE_function.v1.0.pl @@ -0,0 +1,70 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: EAGE_function + + It is designed for gene list GO and KEGG function analysis by EAGE program. + version:1.0 + usage: perl $0 + -i input gene symbol list; + -s specify the species names, default is hsa; + optional: + hsa : human + mmu : mouse + ssc : pig + csv : Cucumis sativus (cucumber) + ath : Arabidopsis thaliana (thale cress) + mcc : Macaca mulatta (rhesus monkey) + rro : Rhinopithecus roxellana (golden snub-nosed monkey) + -q q-value cut off; default is 0.05; + -o results file path; + -eage EAGE program abs dir; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my ($infile,$outdir,$species,$q_val,$EAGE); +my $help; +GetOptions("i=s" => \$infile, + "s=s" => \$species, + "o=s" => \$outdir, + "q=s" => \$q_val, + "eage=s" => \$EAGE, + "h=s" => \$help + ); + +die $usage if $help; +die $usage unless $infile; +die $usage unless $outdir; +die $usage unless $species; +die $usage unless $EAGE; +$q_val ||=0.05; + +my @a=split(/\//,$infile); +my $outdir1="$outdir\/Q$q_val"; +system("mkdir -p $outdir1"); +system("cp $infile $outdir"); +my $outp="$outdir1\/$a[-1]"; + +# GO enrichment +print "================ GO enrichment analysis ======================\n"; +print "GO function analysing...\n"; +system("perl $EAGE/EAGE.pl -L $infile -S $species -O $outp.GO -A BH -C $q_val"); + +# KEGG enrichment +print "================ KEGG enrichment analysis ======================\n"; +print "KEGG function analysing...\n"; +system("perl $EAGE/EAGE.pl -L $infile -T kegg -S $species -O $outp.KEGG -A BH -C $q_val"); + +# plot +print "================ Barplot and bubble polt ======================\n"; +print "[run] perl $EAGE/EAGE_plot.pl -i $outp.GO.xls -t GO -n 2 -f 2 -q $q_val -o $outp.GO\n"; +system("perl $EAGE/EAGE_plot.pl -i $outp.GO.xls -t GO -n 2 -f 2 -q $q_val -o $outp.GO"); +print "[run] perl $EAGE/EAGE_plot.pl -i $outp.KEGG.xls -t KEGG -n 2 -f 2 -q $q_val -o $outp.KEGG\n"; +system("perl $EAGE/EAGE_plot.pl -i $outp.KEGG.xls -t KEGG -n 2 -f 2 -q $q_val -o $outp.KEGG"); + +print "================ GO and KEGG enrichment finished ======================\n"; diff --git a/script/HCLUST.R b/script/HCLUST.R new file mode 100644 index 0000000..07ebd66 --- /dev/null +++ b/script/HCLUST.R @@ -0,0 +1,22 @@ +#### usage +# Rscript this.R input.mat(with col.names) prefix.of.output +# pca analysis for each col of input.file +#### + +##### +args <- commandArgs(TRUE) +input.file <- args[1] +prefix <- args[2] + +###### +rt <- read.table(file=input.file, header=T) +rt <- t( as.matrix( rt ) ) + + + +###### +pdf(file= paste(prefix, ".pdf", sep=""), height=6, width=10) + +plot( hclust( dist( rt ) ) ) + +dev.off() diff --git a/script/PCA.R b/script/PCA.R new file mode 100644 index 0000000..e633192 --- /dev/null +++ b/script/PCA.R @@ -0,0 +1,79 @@ +#### usage +# Rscript this.R input.mat(with col.names) class.file prefix.of.output +# pca analysis for each col of input.file +#### + +##### +args <- commandArgs(TRUE) +input.file <- args[1] +class.file <- args[2] +prefix <- args[3] + +###### +rt1 <- read.table(file=input.file, header=T) +rt1 <- t( as.matrix( rt1 ) ) + +rt2 <- read.table(file=class.file) +class <- as.character( rt2[,1] ) + +###### +library(ade4) +pca <- prcomp(rt1) +s <- summary(pca) +write.table(pca$x, paste(prefix,".x.txt",sep=""), quote = F, sep="\t") +write.table(s$importance, paste(prefix,".eig.txt",sep=""), quote = F, sep="\t") + +###### +fac <- as.factor(class) +fac.len <- length(levels(fac)) +colo <- c("red3", "green4","yellow3", + "purple3","blue3","orange3", + "pink3","deepskyblue","black") + +pdf(file= paste(prefix, ".pdf", sep="") ) +par(mfrow=c(2,2)) + +kip <- s$importance + + +plot(pca$x[, 1:2], type="n",xlab="",ylab="") +text(pca$x[, 1:2], rownames(rt1), adj=c(1,1), pos=1, cex=0.6) +title(xlab=paste("PC1(",round(kip[2,1]*100,2),"%)"),ylab=paste("PC2(",round(kip[2,2]*100,2),"%)")) +s.class(pca$x[, 1:2], fac, col=colo[1:fac.len], include.origin=FALSE, cpoint=1, addaxes=TRUE, cstar=1, add.plot = TRUE) + +plot(pca$x[, 2:3], type="n",xlab="",ylab="") +text(pca$x[, 2:3], rownames(rt1), adj=c(1,1), pos=1, cex=0.6) +title(xlab=paste("PC2(",round(kip[2,2]*100,2),"%)"),ylab=paste("PC3(",round(kip[2,3]*100,2),"%)")) +s.class(pca$x[, 2:3], fac, col=colo[1:fac.len], include.origin=FALSE, cpoint=1, addaxes=TRUE, cstar=1, add.plot = TRUE) + +plot(pca$x[, c(1,3)], type="n",xlab="",ylab="") +text(pca$x[, c(1,3)], rownames(rt1), adj=c(1,1), pos=1, cex=0.6) +title(xlab=paste("PC1(",round(kip[2,1]*100,2),"%)"),ylab=paste("PC3(",round(kip[2,3]*100,2),"%)")) +s.class(pca$x[, c(1,3)], fac, col=colo[1:fac.len], include.origin=FALSE, cpoint=1, addaxes=TRUE, cstar=1, add.plot = TRUE) + +dev.off() + +########## +#pca <- dudi.pca(rt1, center =TRUE, scale = TRUE, scannf = FALSE, nf=dim(rt1)[1] - 1) +#write.table(pca$eig, paste(prefix,"_eig.txt",sep=""),quote = F, sep="\t") +#write.table(pca$c1, paste(prefix,"_c1.txt",sep=""), quote = F, sep="\t") +#write.table(pca$li, paste(prefix,"_li.txt",sep=""), quote = F, sep="\t") + + +#kip <- 100 * pca$eig/sum(pca$eig) +#kip +#pdf(file=paste(prefix,"_2.pdf",sep="")) +#plot(pca$c1[, 1:2],ann=FALSE) +#text(pca$c1[, 1:2], colnames(rt1), adj=c(1,1), pos=1, cex=0.5) +#options(digits=2) +#title(xlab=paste("PC1(",round(kip[1],2),"%)"),ylab=paste("PC2(",round(kip[2],2),"%)")) +#s.class(pca$c1[, 1:2], fac, col=colo[1:fac.len], include.origin=FALSE, cpoint=1.2, addaxes=TRUE, cstar=1, add.plot = TRUE) +#barplot(pca$eig) +#dev.off() +#png(file=paste(prefix,"_pca.png",sep=""),width = 1024, height = 1024) +#plot(pca$c1[, 1:2],ann=FALSE) +#text(pca$c1[, 1:2], colnames(rt1), adj=c(1,1), pos=3, cex=1) +#title(xlab=paste("PC1(",round(kip[1],2),"%)"),ylab=paste("PC2(",round(kip[2],2),"%)")) +#s.class(pca$c1[, 1:2], fac, col=colo[1:fac.len], include.origin=FALSE, cpoint=3, addaxes=TRUE, cstar=1, add.plot = TRUE) +#dev.off() + diff --git a/script/PCC.R b/script/PCC.R new file mode 100644 index 0000000..2398f0e --- /dev/null +++ b/script/PCC.R @@ -0,0 +1,48 @@ +#### usage +#### Rscript this.R input.mat prefix.output +#### corrplot for each col of input.mat +########## + + +##### lib +library(corrplot) + +##### +args <- commandArgs(T) +file.input = args[1] +pdf.output = args[2] + +dat <- read.table(file= file.input, header=T) + +corpct <- round( as.matrix( cor( dat )) * 100, 2) + +# diag(corpct) <- min(corpct) + +col1 <- colorRampPalette( c("blue", "cyan", "yellow", "orange", "red") ) + +col2 <- colorRampPalette( c("#7F0000","red","#FF7F00", + "yellow","#7FFF7F", "cyan", + "#007FFF", "blue","#00007F")) + +col3 <- colorRampPalette( c("#67001F", "#B2182B", "#D6604D", + "#F4A582", "#FDDBC7", "#FFFFFF", + "#D1E5F0", "#92C5DE", "#4393C3", + "#2166AC", "#053061") ) + +pdf(file=paste(pdf.output,"pdf",sep="."), pointsize=12) +par( xpd = NA ) +corrplot( corpct, + type= "full",# c("full", "lower", "upper") + order= "original", # c("original", "AOE", "FPC", "hclust", "alphabet") + cl.lim= c(min(corpct), max(corpct) ), + diag= FALSE, # coefficients on the principal diagonal + addCoefasPercent = FALSE, + addCoef.col = NULL, # NULL, add no Conrf + tl.col= "black", + is.corr= F, + method= "circle", # c("circle", "square", "ellipse", "number", "shade", "color", "pie") + mar= c(0,0,4,0), + col= col1(20), + main= "Pairwise Correlation") + +dev.off() diff --git a/script/PVCLUST.R b/script/PVCLUST.R new file mode 100644 index 0000000..fd66fc3 --- /dev/null +++ b/script/PVCLUST.R @@ -0,0 +1,38 @@ +######################################## +### usage Rscript this.R input.mat ouput.pdf +### pvclust for each col of input.mat +######################################## +args <- commandArgs(T) +inputfile <- args[1] +output.pdf <- args[2] + +x <- read.table(file=inputfile, header=T) +x <- as.matrix(x) + +library(pvclust) + +# method.hclust -> "average", "ward", "single", "complete", "mcquitty", "median" or "centroid" +# "ward", "single", "complete", "average", "mcquitty", "median" or "centroid" +# method.dist -> "correlation", "uncentered", "abscor" +# "euclidean", "maximum", "manhattan", "canberra", "binary" or "minkowski" +# use.cor -> "pairwise.complete.obs", "all.obs" or "complete.obs" +## pvclust(data, +# method.hclust="average", +# method.dist="correlation", +# use.cor="pairwise.complete.obs", +# nboot=1000, +# r=seq(.5,1.4,by=.1), +# store=FALSE, +# weight=FALSE) + + +x.pv <- pvclust(x, nboot = 100) + +pdf(file=output.pdf, height=6, width=10) + +plot(x.pv, cex=1, cex.pv=0.8) + +dev.off() + +##### + diff --git a/script/RawFastqc_batch.v1.0.pl b/script/RawFastqc_batch.v1.0.pl new file mode 100644 index 0000000..f7bdc62 --- /dev/null +++ b/script/RawFastqc_batch.v1.0.pl @@ -0,0 +1,78 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: RawFastqc_batch + + It is designed for running fastqc for any rawdata in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p fastq files folder path; + -o results file path; + -t Specifies the number of files which can be processed simultaneously. + Each thread will be allocated 250MB of memory so you shouldn't run more + threads than your available memory will cope with, and not more than 6 threads on a 32 bit machine + -fastqc fastqc program; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my ($file,$outfile,$fqpath,$threads,$fastqc); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "t=s" => \$threads, + "fastqc=s" => \$fastqc, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $fqpath; +$threads=1 unless $threads; +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($file)); + $outfile=abs_path($file); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile; + my $path1=$fqpath."/".$sampleinfo[1].'_R1.fq.gz'; + my $path2=$fqpath."/".$sampleinfo[1].'_R2.fq.gz'; + my $totalpath.=$path1." ".$path2; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "$fastqc -t $threads -o $opath $totalpath\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=$threads $signalpbs\n"; + print SGEall "qsub -cwd -l vf=1G -q all.q $signalpbs"; + } +close FILE; +close PBSall; +close SGEall; +########################################################################################################################### diff --git a/script/RawFastqc_batch.v1.1.pl b/script/RawFastqc_batch.v1.1.pl new file mode 100644 index 0000000..055b2bb --- /dev/null +++ b/script/RawFastqc_batch.v1.1.pl @@ -0,0 +1,78 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: RawFastqc_batch + + It is designed for running fastqc for any rawdata in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p fastq files folder path; + -o results file path; + -t Specifies the number of files which can be processed simultaneously. + Each thread will be allocated 250MB of memory so you shouldn't run more + threads than your available memory will cope with, and not more than 6 threads on a 32 bit machine + -fastqc fastqc program; + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my ($file,$outfile,$fqpath,$threads,$fastqc); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "t=s" => \$threads, + "fastqc=s" => \$fastqc, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $fqpath; +$threads=1 unless $threads; +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($file)); + $outfile=abs_path($file); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile; + my $path1=$fqpath."/".$sampleinfo[2]; + my $path2=$fqpath."/".$sampleinfo[3]; + my $totalpath.=$path1." ".$path2; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "$fastqc -t $threads -o $opath $totalpath\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=$threads $signalpbs\n"; + print SGEall "qsub -cwd -l vf=1G -q all.q $signalpbs\n"; + } +close FILE; +close PBSall; +close SGEall; +########################################################################################################################### diff --git a/script/Venn.pl b/script/Venn.pl new file mode 100644 index 0000000..7a98f77 --- /dev/null +++ b/script/Venn.pl @@ -0,0 +1,170 @@ +#!/usr/bin/perl +use strict; +use warnings; +use File::Basename qw(basename dirname); +use Getopt::Long; + +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Usage + +my $usage=<<"USAGE"; + +name: $0 + A Venn diagram or set diagram is a diagram that shows all + possible logical relations between a finite collection of + different sets. Venn diagrams were conceived around 1880 + by John Venn. + Here, this perl script can show all possible logical + relations of given different sets like Venn diagram. + + +usage: + -I , join file names with ','; + Òª±È½ÏµÄidËùÔڵĸ÷¸öÎļþ£» + + -C [file_a_col_No,file_b_col_No,file_c_col_No,...,file_n_col_NO], join colNO. with ','; + the default is each whole row; + Òª±È½ÏµÄidÔÚ¸÷¸öÎļþÖÐËùÔÚµÄÁеıàºÅ£» + (ÎļþÁÐÊý±àºÅ´Ó0¿ªÊ¼) + + -L Show the classify results only; + ½öչʾ·ÖÀ࣬²»Êä³ö¾ßÌå½á¹û£» +example: + perl $0 -I a,b + perl $0 -I a,b -C 1,1 + perl $0 -I a,b,c -C 1,1,4 -L + perl $0 -I a,b,c,d,e,f -C 1,2,3,4,5,6 -L +author: + luhanlin\@genomics.org.cn + 2012.01.01 +P.S. + ±È½Ï¶à¸öÎļþÖи÷Ö¸¶¨ÁÐÊÇ·ñÓаüº¬¹Øϵ£» + ÓÃÀ´±È½ÏÁ½¸öÎļþ£¬Ö®ºóÊä³öaÎļþÌØÓеģ¬bÎļþÌØÓеĺÍabÁ½¸öÎļþ¹²ÓеÄÐУ» + »òÕßÊäÈëÈý¸öÎļþ£¬Êä³öa,b,cÌØÓеģ¬a,b,c¹²Óеģ¬ÒÔ¼°abÌØÓУ¬bcÌØÓкÍcaÌØÓеÄÐУ» + »òÕßÊäÈë n ¸öÎļþ£¬2^n-1 ÖÖ¹Øϵ...... + [¿ØÖÆÎļþ£¬²»Ò˹ý¶à] + + + +USAGE + +my ($file_in, $cols_in, $list_only, $help); +GetOptions( + "I=s" => \$file_in, + "C=s" => \$cols_in, + "L" => \$list_only, + "h|?|help" => \$help, +); +die $usage if $help; +unless($file_in){ + print "$usage"; + exit 0; +} +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ globle parameters + +my (@files, @f_cols, %ha, %uniq, %typ, @com, %name, %class_2_file); + +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ define the relations + +@files = split(',', $file_in); +for (my $i=0; $i<@files; $i++){ # 1 2 4 8 16 32 64 ... + my $num = 2**$i; + push @com, $num; + my $name = basename( $files[$i] ); + $class_2_file{$num} = $name; +} +for (my $i=1; $i< 2**($#files+1); $i++){ # ÓÐn¸öÎļþ¾ÍÓÐ 2**n-1 ÖÖ¹ØϵÀàÐÍ + my $binary = sprintf( "%b", $i ); + my @dit = reverse split(//, $binary); + my @name_tmp; + for (my $j=0; $j<=$#dit; $j++){ + my $s_bin = $dit[$j] * ( 2 ** $j ); + unless($s_bin == 0){ + push @name_tmp, "$class_2_file{$s_bin}"; + } + } + $name{$i} = join ' ~ ', @name_tmp; +} + +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ reading files + +if (defined $cols_in){ #reading files + @f_cols = split(',', $cols_in); + foreach my $col (@f_cols){ + unless ($col=~/^\d+$/){ + die "$cols_in\nBe Sure your colNO. are all number!\n"; + } + } + unless ($#files == $#f_cols){ + die "files counts != column counts\nBe Sure your files and your designated colNO. are one-to-one!\n" ; + } + for (my $i=0; $i<@files; $i++){ + open IN,"< $files[$i]" or die "can not open file:\t$files[$i]\n"; + %uniq = (); + while (my $row = ){ + chomp $row; + next if ($row=~/^\s*$/); + my $id = (split(/\s+/,$row))[ $f_cols[$i] ]; + next if ($id=~/^\s*$/); + $uniq{$id}++; + } + close IN; + $ha{$_} += $com[$i] foreach (keys %uniq); #¶Ôÿ¸öÔªËØ×ö¹ØϵÀàÐ͵ĹéÊô´¦Àí + } +} +else{ + for (my $i=0;$i<@files;$i++){ + open IN,"< $files[$i]" or die "can not open file:\t$files[$i]\n"; + %uniq = (); + while (my $row = ){ + chomp $row; + next if($row=~/^\s*$/); + $uniq{$row}++; + } + close IN; + $ha{$_} += $com[$i] foreach (keys %uniq); + } +} +%uniq = (); + +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ clustering + +foreach my $uniq_id (keys %ha){ + my $class = $ha{$uniq_id}; + push @{$typ{$class}},$uniq_id; +} +%ha = (); + +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ print the results + +if (defined $list_only){ + foreach my $class_b (sort {$a<=>$b} keys %name){ + if(exists $typ{$class_b}){ + print "$class_b\t$name{$class_b}\t", $#{$typ{$class_b}}+1, "\n"; + } + else{ + print "$class_b\t$name{$class_b}\t0\n"; + } + } +} +else{ + print ">~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~#\n"; + foreach my $class_b (sort {$a<=>$b} keys %name){ + if(exists $typ{$class_b}){ + print ">>> $class_b\t$name{$class_b}\t", $#{$typ{$class_b}}+1, "\n"; + } + else{ + print ">>> $class_b\t$name{$class_b}\t0\n"; + } + } + print ">~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~#\n"; + foreach my $class_b (sort {$a<=>$b} keys %name){ + if(exists $typ{$class_b}){ + print "### $class_b\t$name{$class_b}\t", $#{$typ{$class_b}}+1, "\n"; + foreach (@{$typ{$class_b}}){ + print "$class_b\t$_\n"; + } + #print "\n"; + } + } +} +#~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ END diff --git a/script/averge_methyl.barplot.R b/script/averge_methyl.barplot.R new file mode 100644 index 0000000..9b8dd51 --- /dev/null +++ b/script/averge_methyl.barplot.R @@ -0,0 +1,24 @@ +### This is script for barplot of genome-wide methylation level of C, CG, CHG and CHH #### +### Author: zhangdu +### date: 2018-08-13 +### version:v1.0 + +arg <- commandArgs(T) +if(length(arg) < 2){ + cat("Argument: 04.all.averge_methyl.xls averge_methyl.pdf\n") + quit('no') +} + + +tab<-read.table(arg[1],header=T,skip=1) +library(tidyr) +library(ggplot2) + +tab_long<-gather(tab,Type,Average_Methylation_level,C:CHH) +pdf(file= arg[2],width=8,height=6) +ggplot(data = tab_long, mapping = aes(x = Type, y =Average_Methylation_level , fill = SampleID)) + + geom_bar(stat="identity",position = "dodge",colour="black") + + #geom_text(aes(label=round(Average_Methylation_level,4)), vjust= -1, position = position_dodge(0.9), size=3.5) + + #scale_fill_brewer(palette="Dark2") + + ggtitle("Genome-wide average methylation of total C, CG, CHG and CHH") +dev.off() diff --git a/script/cut_rrbs.pl b/script/cut_rrbs.pl new file mode 100644 index 0000000..50e35cd --- /dev/null +++ b/script/cut_rrbs.pl @@ -0,0 +1,66 @@ +#!/usr/bin/perl +#=============================================================================== +# FILE: fastq_clean.pl +# USAGE: +# DESCRIPTION: This perl is for RRBS, so it will cut the TGA in read1 3', and cut CA in read2 5' +# OPTIONS: --- +# REQUIREMENTS: --- +# BUGS: --- +# NOTES: --- +# AUTHOR: luhanlin@genomics.org.cn +# COMPANY: BGI +# VERSION: 1.0 +# CREATED: 05/06/2010 05:17:07 PM +#=============================================================================== +use strict; +use warnings; +use PerlIO::gzip; +die "Usage: perl $0 in.fq out.fq \n" unless @ARGV==2; +my ($fq, $out) = @ARGV; +my $enzyme = 0; +my $total = 0; +if ($fq=~/\.gz$/){ + open IN,"<:gzip","$fq" or die "$! --> $fq\n"; +} +else{ + open IN,"<$fq" or die "$! --> $fq\n"; +} + +if ($out=~/\.gz$/){ + open OUT, ">:gzip", "$out" or die $!; +} +else{ + open OUT, ">$out" or die $!; +} + +########## +while(my $line1 = ){ + chomp($line1); + my $line2 = ; + chomp $line2; + my $line3 = ; + chomp $line3; + my $line4 = ; + chomp $line4; + if($line1=~/:1$/){ + $total += 1; + $enzyme += 1 if $line2=~/^[NTC]GG/; + if( $line2=~/TG[AN]$/){ + $line2=~s/TG[AN]$//; + $line4=~s/\S{3}$//; + } + }else{ + if( $line2=~/^CA/){ + $line2=~s/^CA//; + $line4=~s/^\S{2}//; + } + } + print OUT "$line1\n$line2\n$line3\n$line4\n"; +} +close IN; +close OUT; + +print STDERR "Total reads pairs -> $total\n"; +print STDERR "Enzymed reads pairs -> $enzyme\n"; + +#### diff --git a/script/cut_rrbs.v1.0.pl b/script/cut_rrbs.v1.0.pl new file mode 100644 index 0000000..cbaabe0 --- /dev/null +++ b/script/cut_rrbs.v1.0.pl @@ -0,0 +1,139 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use PerlIO::gzip; +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 + This perl is for RRBS, For sigle emzyme digest RRBS,it will cut the TGA in read1 3', and cut CA in read2 5'; + For double emzyme digest RRBS,it will cut the TGA or TGWA in read1 3', and cut CA or CWA in read2 5'. + -i in fq[.gz] file; + -o [str] out fq.gz file; + -m mode,0 for RRBS(defualt);1 for DRRBS; + -h help +author: zhangdu +e-mail: zhangducsu\@163.com +date: 2018-10-10 +version: 1.0 +USAGE +my ($fq,$out,$mode,$help); +GetOptions ( + "i=s" => \$fq, + "o=s" => \$out, + "m=i" => \$mode, + "h=s" => \$help + ); + +die $usage if $help; +die $usage unless $fq; +die $usage unless $out; +$mode ||= 0; +my $time=`date`; + +my $enzyme = 0; +my $total = 0; +my $cut3 = 0; +my $cut5 = 0; +if ($fq=~/\.gz$/){ + open IN,"<:gzip","$fq" or die "$! --> $fq\n"; +} +else{ + open IN,"<$fq" or die "$! --> $fq\n"; +} + +if ($out=~/\.gz$/){ + open OUT, ">:gzip", "$out" or die $!; +} +else{ + open OUT, ">$out" or die $!; +} + +########## +if($mode ==0){ + while(my $line1 = ){ + chomp($line1); + my $line2 = ; + chomp $line2; + my $line3 = ; + chomp $line3; + my $line4 = ; + chomp $line4; + if($line1=~/:1$/){ + $total += 1; + $enzyme += 1 if $line2=~/^[NTC]GG/; + if( $line2=~/TG[AN]$/){ + $line2=~s/TG[AN]$//; + $line4=~s/\S{3}$//; + $cut3 +=1; + } + }else{ + if( $line2=~/^CA/){ + $line2=~s/^CA//; + $line4=~s/^\S{2}//; + $cut5 +=1; + } + } + print OUT "$line1\n$line2\n$line3\n$line4\n"; + } + + print STDERR "Total reads pairs -> $total\n"; + print STDERR "Enzymed reads pairs -> $enzyme\n"; + print STDERR "Total cut 3'TG in read1 -> $cut3\n"; + print STDERR "Total cut 5'CA in read2 -> $cut5\n"; +#### +}else{ + while(my $line1 = ){ + chomp($line1); + my $line2 = ; + chomp $line2; + my $line3 = ; + chomp $line3; + my $line4 = ; + chomp $line4; + if($line1=~/:1$/){ + $total += 1; + $enzyme += 1 if($line2=~/^[NTC]GG/ || $line2=~/^[NTC]TG[NTC]/ || $line2=~/^[NTC]AG[NTC]/); + if( $line2=~/TG[AN]$/){ + $line2=~s/TG[AN]$//; + $line4=~s/\S{3}$//; + $cut3 +=1; + }elsif( $line2=~/TAG[AN]$/){ + $line2=~s/TAG[AN]$//; + $line4=~s/\S{4}$//; + $cut3 +=1; + }elsif( $line2=~/TTG[AN]$/){ + $line2=~s/TTG[AN]$//; + $line4=~s/\S{4}$//; + $cut3 +=1; + } + }else{ + if( $line2=~/^CA/){ + $line2=~s/^CA//; + $line4=~s/^\S{2}//; + $cut5 +=1; + }elsif( $line2=~/^CAA/){ + $line2=~s/^CAA//; + $line4=~s/^\S{3}//; + $cut5 +=1; + }elsif( $line2=~/^CTA/){ + $line2=~s/^CTA//; + $line4=~s/^\S{3}//; + $cut5 +=1; + } + } + print OUT "$line1\n$line2\n$line3\n$line4\n"; + } + print STDERR "Total reads pairs -> $total\n"; + print STDERR "Enzymed reads pairs -> $enzyme\n"; + print STDERR "Total cut 3'TG/TWG in read1 -> $cut3\n"; + print STDERR "Total cut 5'CA/CWA in read2 -> $cut5\n"; + +} +close IN; +close OUT; + + + + + diff --git a/script/disposeOverlap.pl b/script/disposeOverlap.pl new file mode 100644 index 0000000..7f05045 --- /dev/null +++ b/script/disposeOverlap.pl @@ -0,0 +1,1503 @@ +#!/usr/bin/perl + +=head1 Name + +disposeOverlap.pl -- dispose overlap relations between one or two block sets + +=head1 Description + +This program is designed to find overlap relations of blocks, the input files +can be one or two tables, just need to point out the column of ID, Stand +and End;This script is modified from fanw's findOverlap.pl + +The algorithm is that: +(1)Order: sort the two block sets based on start positions, seperately; +(2)Traversal: walk along the chromosome, and find out overlap between the twoblock sets. +(3)Output: + (i)if you had chosen "--C" option, the output table would list the regions gone + through combining the overlapping, and use the outstretched one instead of + all the regions each other staying with overlapping; and output all the regions + without redundance + + (ii)if you had chosen "--E" option, the output table would report who and who + overlapped, as well as their regions(start and end postition), their own size + and the overlapped size, it is sourced from fanw's program; + + The output is TAB delimited with each line consisting of + Col 1:ID + COl 2~5:FirstTable: RegionID Start End Length + Col 6:OverlapNumber + Col 7:OverlapSize + Col 8:OverlapRate + Col 9~:SecondTable: RegionID:Start,End,Length,OverlapSize + Col ...:if exists more than one overlapped region the format is the same as Col 4 + + (iii)if you had chosen "--F" option, the output table would list all the regions + which has been filtered out overlapping with others in a certain degree, just left + the relatively uniq regions. + +=head1 Version + + Author: BENM, binxiaofeng@gmail.com + Version: 3.3, Date: 2008-2-25 UpDate: 2009-01-28 + +=head1 Usage + + --i1 input the first table file + --f1 || input the first table format [id-start-end] [id1-id2-start-end] + --i2 input the second table file + --f2 || input the second table format [id-start-end] [id1-id2-start-end] + --OL set the overlapped limited condition, see below "PS", default: 0 + --final Limit the final overlap limited conditions + --mN set the minimuim overlapping number: [0] (only could be used in existing "--E" option + in "--E O" it means that the OverlapNumber should over this setting value(>) + in "--E N" it means that the OverlapNumber should less than this setting value(<=) + --C combine the overlapped regions without redundance and list all the regions + --F <[R|D> filterout the regions with overlap, R: combine two blocks filterout the overlapped regions and renew the table; + D: delete the overlapped blocks from the first table, which these blocks overlap with the second talbe. + --E <[O|N]> enumerate the overlapped blocks(O): overlap rate between two blocks bigger than the rate set by "--OL" + or none(N) Redundance regions: overlap rate between two blocks smaller than the rate set by "--OL" + --X <[O|N]> extract the overlapped segment(O); + extract the none redundance segment(N)(filterout redundance). + --M || <1,str> || <2,str> || <1,str:2,str> only list the region id matching situation if none or all list all:[all] + --interpolation set the interpolation for splitting data columns, + such as [s+,s,t+,t] and some special symbol, default: t (i.e. @col=split/\t/) + --verbose output verbose information to screen + --help output help information to screen + --readme Chinese readme + --version output version information + +PS: +--OL means if there are existing overlapping between two blocks and over this + rate of overlapping in the "first" or "second" section of the tables + (just could be used in "--E" option); or in the "small" size or in the + "big" between two blocks, if the blocks were coincident with this rule, + it will carry out the function work you need, else it will ignore to + compare these two blocks but go on; if the limited condition is set by + "num-bpsize", it will limited the overlapping size bigger than the "num" + bp scale; if the limited condition is set by "numbp", it won't limited + the overlap type, but the Total OverlapSize must bigger than or equal + the "num" bp (>=num bp); Limit-type: "big", "small", "first", "second", + "bpsize" + +=head1 Exmple + + 1.if there are existing overlapping and the size of overlapping is over 50% + more than the small section between two blocks, filter out these two blocks + out of the table + + % perl disposeOverlap.pl --i1 table1.txt --f1 0-2-3 --OL 0.5-small --F >Overlap_result1.txt + + 2.if there are existing overlapping and over 50% more than the small section + between two regions, combine these two regions, meanhile preserve the unique + regions,including the overlap not exceeding 0.5 of the small section, + in order to get rid of the redundance + + % perl disposeOverlap.pl --i1 table1.txt --f1 0-2-3 --i2 table2.txt --f2 0-2-3 --OL 0.5-small --C >Overlap_result2.txt + + 3.list all the none overlapping or total length of overlapped segments less + than the first table regions + + % perl disposeOverlap.pl --i1 table1.txt --f1 0-1-2-3 --i2 table2.txt --f2 0-1-2-3 --OL 0.5-first --mN 2 --E N >Overlap_result3.txt + + 4.list the two table with considerable overlapping(it can be controlled by + the "--mR & mN"), and just list the accurately matching ID of "Variation" + (set by "--M" option) in first table + + % perl disposeOverlap.pl --i1 table1.txt --f1 0-1-2-3 --i2 table2.txt --f2 0-1-2-3 --OL 0.1-small --mN 1 --E O --M 1,"^Variation$" >Overlap_result4.txt + +=cut + +use strict; +use Getopt::Long; +use Data::Dumper; + +my ($input1,$inform1,$input2,$inform2,$OverlapLimited,$Final,$minNum,$Combine,$Filterout,$Enumerate,$Extract,$matchingID,$Interpolation,$Verbose,$Help,$Readme,$Version); +my %opts; +GetOptions( + \%opts, + "i1:s"=>\$input1, + "f1:s"=>\$inform1, + "i2:s"=>\$input2, + "f2:s"=>\$inform2, + "OL:s"=>\$OverlapLimited, + "final:s"=>\$Final, + "mN:i"=>\$minNum, + "C"=>\$Combine, + "F:s"=>\$Filterout, + "E:s"=>\$Enumerate, + "X:s"=>\$Extract, + "M:s"=>\$matchingID, + "interpolation:s"=>\$Interpolation, + "verbose"=>\$Verbose, + "help"=>\$Help, + "readme"=>\$Readme, + "version"=>\$Version +); +if (defined $Readme) +{ + readme(); exit; +} +die " Author: BENM, binxiaofeng\@gmail.com\n Version: 3.3, Date: 2009-01-28\n" if (defined $Version); +die `pod2text $0` if (($Help)||((!defined $Enumerate)&&(!defined $Filterout)&&(!$Combine)&&(!$Extract))); + + + +die(" +Combine Overlap\n +Usage: disposeOverlap.pl --C\n +Options: + --i1 input the first table file [in.table1] + --f1 || input the first table format [id-start-end] or [id1-id2-start-end] + --i2 input the first table file [in.table2] + --f2 || input the first table format [id-start-end] or [id1-id2-start-end] + --OL set the overlapped limited condition [OverlapRate-LimitedType], LimitedType default:0-small + --M || <1,str> || <2,str> || <1,str:2,str> only list the region id matching situation if none or all list all:[Matching ID] + --C combine the overlapped regions without redundance and list all the regions [null]\n +Example:1. perl disposeOverlap.pl --C --i1 table1.txt --f1 0-2-3 --OL 0 > Combine_Overlap_result1.txt + 2. perl disposeOverlap.pl --C --i1 table1.txt --f1 0-2-3 --i2 table2.txt --f2 0-2-3 --OL 0.1-big > Combine_Overlap_result2.txt\n +") if ( ($Combine) && ( (!defined $input1) || (!defined $inform1) ) ); + +die(" +Filterout Overlap\n +Usage: disposeOverlap.pl --F\n +Options: + --i1 input the first table file [in.table1] + --f1 || input the first table format [id-start-end] or [id1-id2-start-end] + --i2 input the first table file [in.table2] + --f2 || input the first table format [id-start-end] or [id1-id2-start-end] + --OL set the overlapped limited condition [OverlapRate-LimitedType], LimitedType default:0-small + --M || <1,str> || <2,str> || <1,str:2,str> only list the region id matching situation if none or all list all:[Matching ID] + --F filterout the regions with overlap [null]\n +Example:1. perl disposeOverlap.pl --F R --i1 table1.txt --f1 0-2-3 --OL 0.5-small > Filter_Overlap_result1.txt + 2. perl disposeOverlap.pl --F D --i1 table1.txt --f1 0-2-3 --i2 table2.txt --f2 0-2-3 --OL 100-bpsize > Filter_Overlap_result2.txt\n +") if ( ($Filterout) && ( ($Filterout ne "R") || ($Filterout ne "D") ) && ( (!defined $input1) || (!defined $inform1) ) ); + +die(" +Enumerate Overlap\n +Usage: disposeOverlap.pl --E \n +Options: + --i1 input the first table file [in.table1] + --f1 || input the first table format [id-start-end] or [id1-id2-start-end] + --i2 input the first table file [in.table2] + --f2 || input the first table format [id-start-end] or [id1-id2-start-end] + --OL set the overlapped limited condition [OverlapRate-LimitedType] LimitedType can be null, default: 0 + --final Limit the final overlap limited conditions + --mN set the minimuim limited overlapping number: [1] for N option it is maxmuim, default: 1 + --M || <1,str> || <2,str> || <1,str:2,str> only list the region id matching situation if none or all list all:[Matching ID] + --E <[O|N]> enumerate the overlapped blocks [null]\n +Example:1. perl disposeOverlap.pl --E O --i1 table1.txt --f1 0-1-2-3 --i2 table2.txt --f2 0-1-2-3 --OL 0.1-first --mN 1 --M Variation > List_Overlap_result.txt + 2. perl disposeOverlap.pl --E N --i1 table1.txt --f1 0-1-2-3 --i2 table2.txt --f2 0-1-2-3 --OL 100bp --mN 10 > None_Overlap_result.txt\n +") if ( ($Enumerate) && ( ($Enumerate ne "O") || ($Enumerate ne "N") ) && ( (!defined $input1) || (!defined $inform1) ) ); + +die(" +eXtract Overlap\n +Usage: disposeOverlap.pl --e \n +Options: + --i1 input the first table file [in.table1] + --f1 || input the first table format [id-start-end] or [id1-id2-start-end] + --i2 input the first table file [in.table2] + --f2 || input the first table format [id-start-end] or [id1-id2-start-end] + --M || <1,str> || <2,str> || <1,str:2,str> only list the region id matching situation if none or all list all:[Matching ID] + --X <[O|N]> extract the overlapped segment(O); extract the none redundance segment(N)(filterout redundance).\n +Example:1. perl disposeOverlap.pl --X O --i1 table1.txt --f1 0-2-3 > Overlap_result1.txt + 2. perl disposeOverlap.pl --X N --i1 table1.txt --f1 0-2-3 > None_Overlap_result1.txt + 3. perl disposeOverlap.pl --X O --i1 table1.txt --f1 0-1-2-3 --i2 table2.txt --f2 0-1-2-3 --M Variation > Overlap_result2.txt + 4. perl disposeOverlap.pl --X N --i1 table1.txt --f1 0-2-3 --i2 table2.txt --f2 0-2-3 > None_Overlap_result2.txt\n +") if ( ($Extract) && ( ($Extract ne "O") || ($Extract ne "N") ) && ( (!defined $input1) || (!defined $inform1) ) ); + +$OverlapLimited = (defined $OverlapLimited) ? $OverlapLimited : 0; +$minNum = (defined $minNum) ? $minNum : 1; +my @orate=(); +my ($minRate,$limitType,$minSize); +if (defined $OverlapLimited) +{ + @orate=split /-/,$OverlapLimited; + $minRate = ($orate[0] =~ m/\d+/) ? $orate[0] : ""; + if ($orate[0] =~ m/bp/) + { + $orate[0] =~ s/bp//; + $minSize = $orate[0]; + $minRate = 0; + } + die "minRate:$minRate is not a available value!" if (($minRate !~ m/^\d+$/)&&($minRate !~ m/^\d+\.\d+$/)&&(($minRate !~ m/^\d+bp$/))); + $limitType = ((defined $orate[1])&&($orate[1] =~ m/\w+/)) ? $orate[1] : ""; +} +my $bpsize = $minRate if ( (defined $limitType) && ($limitType =~ m/bpsize/i) ); +$minNum ||= 0; +$matchingID ||= "all"; +$Interpolation ||="t"; + +chomp $matchingID; +my $matchingT=""; +my ($T1,$matchingT1,$T2,$matchingT2)=("","","",""); +if ($matchingID ne "all") +{ + $matchingID=~s/\s+//g; + if ($matchingID=~m/\:/g) + { + ($matchingT1,$matchingT2)=split/\:/,$matchingID; + ($T1,$matchingT1)=split/\,/,$matchingT1; + ($T2,$matchingT2)=split/\,/,$matchingT2; + } + elsif (($matchingID=~m/\,/)&&($matchingID !~ m/\:/g)) + { + ($matchingT,$matchingID)=split/\,/,$matchingID; + if ($matchingT==1) + { + $T1=1; + $matchingT1=$matchingID; + } + if ($matchingT==2) + { + $T2=2; + $matchingT2=$matchingID; + } + } + elsif (($matchingID !~m/\,/)&&($matchingID !~ m/\:/g)) + { + ($T1,$matchingT1,$T2,$matchingT2)=(1,$matchingID,2,$matchingID); + } +} + +my ($id1,$rd1,$s1,$e1)=("","","",""); +my ($id2,$rd2,$s2,$e2)=("","","",""); +if (defined $inform1) +{ + if (table_col($inform1) == 4) + { + ($id1,$rd1,$s1,$e1)=table_col($inform1); + } + else + { + ($id1,$s1,$e1)=table_col($inform1); + } +} + +if (defined $inform2) +{ + if (table_col($inform2) == 4) + { + ($id2,$rd2,$s2,$e2)=table_col($inform2); + } + else + { + ($id2,$s2,$e2)=table_col($inform2); + } +} + +my %FirstTable=read_table($input1,$id1,$s1,$e1,$rd1,$T1,$matchingT1,1) if (defined $input1); +my %SecondTable=read_table($input2,$id2,$s2,$e2,$rd2,$T2,$matchingT2,2) if (defined $input2); + +if ( (defined $input1) && (!defined $input2) ) +{ + + combine_overlap(\%FirstTable) if (defined $Combine); + + filterout_overlap(\%FirstTable) if ((defined $Filterout)&&($Filterout eq "R")); + + enumerate_overlap(\%FirstTable,\%FirstTable,$Enumerate) if (defined $Enumerate); + + extract_overlap(\%FirstTable) if (defined $Extract); + +} + +if (defined $input2) +{ + enumerate_overlap(\%FirstTable,\%SecondTable,$Enumerate) if (defined $Enumerate); + filterout_overlap(\%FirstTable,\%SecondTable) if ((defined $Filterout)&&($Filterout eq "D")); + + if ((defined $Combine) || (defined $Filterout) ) + { + my %CombineTable; + foreach my $id1 (sort keys %FirstTable) + { + my $table1=$FirstTable{$id1}; + push @{$CombineTable{$id1}},@$table1; + delete $FirstTable{$id1}; + } + delete @FirstTable {keys %FirstTable}; + foreach my $id2 (sort keys %SecondTable) + { + my $table2=$SecondTable{$id2}; + push @{$CombineTable{$id2}},@$table2; + delete $SecondTable{$id2}; + } + delete @SecondTable {keys %SecondTable}; + + combine_overlap(\%CombineTable) if (defined $Combine); + filterout_overlap(\%CombineTable) if ((defined $Filterout)&&($Filterout eq "R")); + extract_overlap(\%CombineTable) if (defined $Extract); + } + +} + +print STDERR "overlap disposing work has completed!\n"; + +#################################################### +################### Sub Routines ################### +#################################################### + +sub table_col +{ + my $string=shift; + chomp $string; + my @table; + if ($string =~ /\-/) + { + @table=split /\-/,$string; + } + elsif ($string =~ /\,/) + { + @table=split /\,/,$string; + } + return @table; +} + +sub read_table +{ + my ($file,$id,$s,$e,$rd,$t,$matching_id,$input_num)=@_; + my %Table=(); + open (IN,$file) || die "can't open $file for reading:$!"; + while () + { + chomp; + my @col; + if ($Interpolation eq "s+") + { + @col=split /\s+/,$_; + } + elsif ($Interpolation eq "s") + { + @col=split /\s/,$_; + } + elsif ($Interpolation eq "t+") + { + @col=split /\t+/,$_; + } + elsif ($Interpolation eq "t") + { + @col=split /\t/,$_; + } + else + { + @col=split/$Interpolation/,$_; + } + + my $ID=$col[$id]; + chomp $ID; + $ID =~ s/[;,:]$//; + next if ((defined $Enumerate)&&($input_num==2)&&(!exists $FirstTable{$ID})); + if ( ($col[$s] =~ /\d+/) && ($col[$e] =~ /\d+/) ) + { + my $Start=$col[$s]; + my $End=$col[$e]; + if ((defined $rd)&&($rd ne "")) + { + my $RegionID=$col[$rd]; + chomp $RegionID; + $RegionID =~ s/[;,:]$//; + push @{$Table{$ID}},[$Start,$End,$RegionID] if ((!defined $t)||($t eq "")||($RegionID =~ m/$matching_id/i)||($matchingID eq "all")); + } + else + { + push @{$Table{$ID}},[$Start,$End]; + } + } + #else + #{ + # if ($rd ne "") + # { + # print STDERR "Warning file$input_num error:$col[$id]\t$col[$s]\t$col[$e]\t$col[$rd]\n" unless ( ($Enumerate) || ($Filterout) ); + # } + # else + # { + # print STDERR "Warning file$input_num error:$col[$id]\t$col[$s]\t$col[$e]\n" unless ( ($Enumerate) || ($Filterout) ); + # } + #} + } + close IN; + + return %Table; +} + +sub combine_overlap +{ + my ($hash_p,$print_mark)=@_; + $limitType ||= "small"; + if ((!defined $print_mark)||($print_mark eq "")) + { + if ($rd1 ne "") + { + print "ID\tStart\tEnd\tCombinedNum\tCombinedTableID...\n"; + } + else + { + print "ID\tStart\tEnd\n"; + } + } + foreach my $table_id (keys %$hash_p) + { + my @array=(); + my @array_order = (exists $hash_p->{$table_id}) ? (sort {$a->[0]<=>$b->[0]} @{$hash_p->{$table_id}}) : (); + print STDERR "combine overlap on $table_id\n" if($Verbose); + if ((defined $array_order[0][2])&&($array_order[0][2] ne "")) + { + push @array,[0,0,""]; + } + else + { + push @array,[0,0]; + } + my $n = scalar @array_order; + my @compare=(); + for (my $j=0;$j<$n;$j++) + { + my ($S1,$E1,$regionID1,$S2,$E2,$regionID2)=("","","","","",""); + + $S1=$array_order[$j][0]; + $E1=$array_order[$j][1]; + $regionID1=$array_order[$j][2] if ($rd1 ne ""); + + if ($j<$n-1) + { + $S2=$array_order[$j+1][0]; + $E2=$array_order[$j+1][1]; + $regionID2=$array_order[$j+1][2] if ($rd2 ne ""); + + if ( ($S1<=$array[-1][1]+1) ) + { + if ($E1>=$array[-1][1]+1) + { + if ( $OverlapLimited ne "" ) + { + @compare=compare_overlap ([$array[-1][0],$array[-1][1]],[$S1,$E1]); + if (scalar (@compare) ==2) + { + if ((defined $array_order[$j][2])&&(defined $array[-1][2])&&($array_order[$j][2] ne "")) + { + $array[-1][1]=$E1; + $array[-1][2].="\t".$regionID1 unless ($array[-1][2] =~ m/$regionID1/g); + } + else + { + $array[-1][1]=$E1; + } + } + else + { + if ((defined $array_order[$j][2])&&($array_order[$j][2] ne "")) + { + push @array,[$S1,$E1,$regionID1]; + } + else + { + push @array,[$S1,$E1]; + } + } + } + else + { + if ((defined $array_order[$j][2])&&($array_order[$j][2] ne "")) + { + $array[-1][1]=$E1; + $array[-1][2].="\t".$regionID1 unless ($array[-1][2] =~ m/$regionID1/g); + } + else + { + $array[-1][1]=$E1; + } + } + } + else + { + if (($rd1 ne "")&&($rd2 ne "")) + { + $array[-1][2].="\t".$regionID1 unless ((defined $array[-1][2])&&($array[-1][2] =~ m/$regionID1/)); + } + } + next; + } + elsif ( ($E1<$S2-1) && ($S1>$array[-1][1]+1) ) + { + if ($rd1 ne "") + { + push @array,[$S1,$E1,$regionID1]; + } + else + { + push @array,[$S1,$E1]; + } + next; + } + elsif ( ($E1>=$E2-1) && ($S1>$array[-1][1])+1 ) + { + if ($OverlapLimited ne "") + { + @compare=compare_overlap ([$S1,$E1],[$S2,$E2]); + if (scalar (@compare) ==2) + { + if ($rd1 ne "") + { + push @array,[$S1,$E1,$regionID1]; + } + else + { + push @array,[$S1,$E1]; + } + } + else + { + if ( ($rd1 ne "") && ($rd2 ne "") ) + { + push @array,[$S1,$E1,$regionID1]; + push @array,[$S2,$E2,$regionID2]; + } + else + { + push @array,[$S1,$E1]; + push @array,[$S2,$E2]; + } + } + } + else + { + if ($rd1 ne "") + { + push @array,[$S1,$E1,$regionID1]; + } + else + { + push @array,[$S1,$E1]; + } + } + next; + } + elsif ( ($E1>=$S2-1) && ($E1<=$E2-1) && ($S1>$array[-1][1]+1) ) + { + if ($OverlapLimited ne "") + { + @compare=compare_overlap ([$S1,$E1],[$S2,$E2]); + if (scalar (@compare) ==2) + { + if ( ($rd1 ne "") && ($rd2 ne "") ) + { + $regionID1.="\t".$regionID2 unless ($regionID1 =~ m/$regionID2/g); + push @array,[$S1,$E2,$regionID1]; + } + else + { + push @array,[$S1,$E2]; + } + } + else + { + if ( ($rd1 ne "") && ($rd2 ne "") ) + { + push @array,[$S1,$E1,$regionID1]; + push @array,[$S2,$E2,$regionID2]; + } + else + { + push @array,[$S1,$E1]; + push @array,[$S2,$E2]; + } + } + } + else + { + if ( ($rd1 ne "") && ($rd2 ne "") ) + { + $regionID1.="\t".$regionID2 unless ($regionID1 =~ m/$regionID2/g); + push @array,[$S1,$E2,$regionID1]; + } + else + { + push @array,[$S1,$E2]; + } + } + next; + } + } + else + { + if ( ($S1<=$array[-1][1]+1) ) + { + if ($E1>=$array[-1][1]+1) + { + if ( $OverlapLimited ne "" ) + { + @compare=compare_overlap ([$array[-1][0],$array[-1][1]],[$S1,$E1]); + if (scalar (@compare) ==2) + { + $array[-1][1]=$E1; + if ($rd1 ne "") + { + $array[-1][2].="\t".$regionID1 unless ($array[-1][2] =~ m/$regionID1/g); + } + } + else + { + if ($array_order[$j][2] ne "") + { + push @array,[$S1,$E1,$regionID1]; + } + else + { + push @array,[$S1,$E1]; + } + } + } + else + { + $array[-1][1]=$E1; + if ($rd1 ne "") + { + $array[-1][2].="\t".$regionID1 unless ($array[-1][2] =~ m/$regionID1/g); + } + } + } + else + { + next; + } + } + else + { + if ($array_order[$j][2] ne "") + { + push @array,[$S1,$E1,$regionID1]; + } + else + { + push @array,[$S1,$E1]; + } + } + } + } + shift @array if ( ($array[0][1]==0) && ($array[0][0] ==0) ); + if ( (defined $print_mark) && (($print_mark eq "null")||($print_mark ne "O")) ) + { + return @array; + } + else + { + foreach my $table (@array) + { + if ( ($rd1 eq "") || ($rd2 eq "") ) + { + print "$table_id\t$table->[0]\t$table->[1]\n"; + } + elsif ( (defined $table->[2]) && ($table->[2] ne "" ) && ($n != 0) ) + { + my @CombineNumber=split/\s+/,$table->[2]; + my $n=scalar (@CombineNumber); + print "$table_id\t$table->[0]\t$table->[1]\t"; + print "$n\t$table->[2]\t" unless (defined $Extract); + print "\n"; + } + } + } + } +} + +sub filterout_overlap +{ + my ($table1_hash,$table2_hash)=@_; + $limitType ||= "small"; + if (!defined $Extract) + { + if ($rd1 ne "") + { + print "ID\tFirstTableID\tStart\tEnd\n"; + } + else + { + print "ID\tStart\tEnd\n"; + } + } + if ((!defined $table2_hash)||(keys %$table2_hash==0)) + { + foreach my $table_id (sort keys %$table1_hash) + { + my @table_order = (exists $table1_hash->{$table_id}) ? (sort {$a->[0]<=>$b->[0]} @{$table1_hash->{$table_id}}) : (); + print STDERR "filterout overlap on $table_id\n" if ($Verbose); + my ($tempS,$tempE,$preS,$preE,$nextS,$nextE) = (0,0,0,0,0,0); + my $n = scalar @table_order; + my @compare1; + my @compare2; + for (my $k=0;$k<$n;$k++) + { + $tempS=$table_order[$k][0]; + $tempE=$table_order[$k][1]; + if ($k>0) + { + if ($compare1[0] ne "") + { + $preS=($compare1[0]<$table_order[$k-1][0]) ? $compare1[0] : $table_order[$k-1][0]; + } + else + { + $preS=$table_order[$k-1][0]; + } + $preE=($compare1[1]>$table_order[$k-1][1]) ? $compare1[1] : $table_order[$k-1][1]; + } + elsif ($k==0) + { + $preS=0; + $preE=0; + } + + if ($k<$n-1) + { + $nextS = $table_order[$k+1][0]; + $nextE = $table_order[$k+1][1]; + if ($OverlapLimited ne "") + { + @compare1=compare_overlap ([$preS,$preE],[$tempS,$tempE]); + @compare2=compare_overlap ([$nextS,$nextE],[$tempS,$tempE]); + if ((@compare1==4) && (@compare2==4) ) + { + if ( (defined $table_order[$k][2])&&($table_order[$k][2] ne "") ) + { + my $regionID=$table_order[$k][2]; + print "$table_id\t$regionID\t$tempS\t$tempE\n"; + } + else + { + print "$table_id\t$tempS\t$tempE\n"; + } + } + } + else + { + if ( ($tempS>$preE+1) && ($tempE<$nextS-1) ) + { + if ( (defined $table_order[$k][2])&&($table_order[$k][2] ne "") ) + { + my $regionID=$table_order[$k][2]; + print "$table_id\t$regionID\t$tempS\t$tempE\n"; + } + else + { + print "$table_id\t$tempS\t$tempE\n"; + } + } + else + { + next; + } + } + } + elsif ($k==$n-1) + { + if ($OverlapLimited ne "") + { + my @compare=compare_overlap ([$preS,$preE],[$tempS,$tempE]); + if (@compare==4) + { + if ( (defined $table_order[$k][2])&&($table_order[$k][2] ne "") ) + { + my $regionID=$table_order[$k][2]; + print "$table_id\t$regionID\t$tempS\t$tempE\n"; + } + else + { + print "$table_id\t$tempS\t$tempE\n"; + } + } + } + else + { + if ($tempS>$preE+1) + { + if ( (defined $table_order[$k][2])&&($table_order[$k][2] ne "") ) + { + my $regionID=$table_order[$k][2]; + print "$table_id\t$regionID\t$tempS\t$tempE\n"; + } + else + { + print "$table_id\t$tempS\t$tempE\n"; + } + } + else + { + next; + } + } + } + } + } + } + else + { + foreach my $table_id (sort keys %$table1_hash) + { + print STDERR "filterout overlap on $table_id\n" if ($Verbose); + my @table1_order = (exists $table1_hash->{$table_id}) ? (sort {$a->[0]<=>$b->[0]} @{$table1_hash->{$table_id}}) : (); + my @table2_order = (exists $table2_hash->{$table_id}) ? (sort {$a->[0]<=>$b->[0]} @{$table2_hash->{$table_id}}) : (); + if (@table2_order<1) + { + for (my $i=0;$i<@table1_order;$i++) + { + my $S=$table1_order[$i][0]; + my $E=$table1_order[$i][1]; + if ($table1_order[$i][2] ne "") + { + my $regionID=$table1_order[$i][2]; + print "$table_id\t"; + print "$regionID\t" unless (defined $Extract); + print "$S\t$E\n"; + } + else + { + print "$table_id\t$S\t$E\n"; + } + } + } + else + { + for (my $j=0;$j<@table1_order;$j++) + { + my $S1=$table1_order[$j][0]; + my $E1=$table1_order[$j][1]; + my $regionID=$table1_order[$j][2] if ( (defined $table1_order[$j][2])&&($table1_order[$j][2] ne "") ); + if ($S1>=$table2_order[-1][1]) + { + $S1 = $S1+1 if ($S1==$table2_order[-1][1]); + if ($table1_order[$j][2] ne "") + { + print "$table_id\t"; + print "$regionID\t" unless (defined $Extract); + print "$S1\t$E1\n"; + } + else + { + print "$table_id\t$S1\t$E1\n"; + } + last; + } + if (scalar @table2_order>0) + { + my $filterID_tmp=""; + for (my $k=0;$k<@table2_order;$k++) + { + my $S2=$table2_order[$k][0]; + my $E2=$table2_order[$k][1]; + my $filterID=$table2_order[$k][2] if ((defined $table2_order[$k][2])&&($table2_order[$k][2] ne "")); + $filterID=$filterID_tmp if ($filterID_tmp ne ""); + next if ($E2<$S1); + if ($S2>=$E1) + { + if ( (defined $table1_order[$j][2])&&($table1_order[$j][2] ne "") ) + { + print "$table_id\t"; + print "$regionID\t" unless (defined $Extract); + print "$S1\t$E1"; + if ((defined $filterID_tmp)&&($filterID_tmp ne "")) + { + print "\t$filterID_tmp" unless (defined $Extract); + } + print "\n"; + } + else + { + print "$table_id\t$S1\t$E1\n"; + } + last; + } + elsif (($S2<$S1)&&($E2>$S1)&&($E2<=$E1)) + { + if ($E1-$E2>1) + { + $S1=$E2+1; + $filterID_tmp=$table2_order[$k][2] if ((defined $table2_order[$k][2])&&($table2_order[$k][2] ne "")); + next; + } + else + { + last; + } + } + elsif (($S2<=$S1)&&($E2>=$E1)) + { + last; + } + elsif (($S2>=$S1)&&($S2<$E1)) + { + if ($S2>$S1+1) + { + if ((defined $table1_order[$j][2])&&($table1_order[$j][2] ne "")) + { + print "$table_id\t"; + print "$regionID\t" unless (defined $Extract); + print "$S1\t".($S2-1)."\t"; + if ((defined $filterID)&&(defined $filterID ne "")) + { + print "$filterID" unless (defined $Extract); + } + print "\n"; + $filterID_tmp=""; + } + else + { + print "$table_id\t$S1\t".($S2-1)."\n"; + } + } + if ($E2<$E1) + { + $S1=$E2+1; + $filterID_tmp=$table2_order[$k][2] if ((defined $table2_order[$k][2])&&($table2_order[$k][2] ne "")); + next; + } + else + { + last; + } + } + } + if ($S1>=$table2_order[-1][1]) + { + if ( (defined $table1_order[$j][2])&&($table1_order[$j][2] ne "") ) + { + print "$table_id\t"; + print "$regionID\t" unless (defined $Extract); + print "$S1\t$E1\t"; + print "$filterID_tmp" unless (defined $Extract); + print "\n"; + } + else + { + print "$table_id\t$S1\t$E1\n"; + } + } + } + } + + } + } + } +} + +sub enumerate_overlap +{ + my ($Fst_table,$Sec_table,$List_form)=@_; + + chomp $List_form; + my $overlap_num=0; + if (($List_form eq "O") || ($List_form eq "N") ) + { + if ( ($rd1 ne "") || ($rd2 ne "") ) + { + print "ID\t"; + print "FirstTableID\t" if ($rd1 ne ""); + print "Start\tEnd\tLength\tOverlapNumber\tOverlapSize\tOverlapRate\t"; + print "SecondTableID:" if ($rd2 ne ""); + print "Start,End,Length,OverlapSize...\n" ; + } + else + { + print "ID\tStart\tEnd\tLength\tOverlapNumber\tOverlapSize\tOverlapRate\tStart,End,Length,OverlapSize...\n" if ($List_form eq "N"); + print "ID\tStart\tEnd\tLength\tOverlapNumber\tOverlapSize\tOverlapRate\tStart,End,Length,OverlapSize...\n" if ($List_form eq "O"); + } + } + foreach my $table_id (sort keys %$Fst_table) + { + my @fst_tab = (exists $Fst_table->{$table_id}) ? (sort {$a->[0] <=> $b->[0]} @{$Fst_table->{$table_id}}) : (); + my @sec_tab = (exists $Sec_table->{$table_id}) ? (sort {$a->[0] <=> $b->[0]} @{$Sec_table->{$table_id}}) : (); + + print STDERR "find overlap on $table_id\n" if($Verbose); + + my $sec_begin=0; + + for (my $i=0; $i<@fst_tab; $i++) + { + my $fst_size = $fst_tab[$i][1] - $fst_tab[$i][0] + 1; + my @overlap=(); + my $total_overlap_size=0; + for (my $j=$sec_begin; $j<@sec_tab; $j++) + { + next if ((!defined $input2) && (!defined $inform2) && + (($rd1 ne "") && ($sec_tab[$j][2] eq $fst_tab[$i][2]) && ($fst_tab[$j][0]==$sec_tab[$j][0]) && ($fst_tab[$j][1]==$sec_tab[$j][1]) ) ); + if ($sec_tab[$j][1] < $fst_tab[$i][0]) + { + next; + } + if ($sec_tab[$j][0] > $fst_tab[$i][1]) + { + last; + } + $sec_begin=$j if (scalar @overlap==0); + + if ($limitType ne "") + { + my @compare=compare_overlap($fst_tab[$i],$sec_tab[$j]); + next if ( ($List_form eq "O") && (@compare==4) ); + last if ( ($List_form eq "N") && (@compare==2) ); + } + + my $sec_size = $sec_tab[$j][1] - $sec_tab[$j][0] + 1; + my $overlap_size = overlap_size($fst_tab[$i],$sec_tab[$j]); + $total_overlap_size += $overlap_size; + + if (($rd2 ne "")&&($sec_tab[$j][2] ne "")) + { + push @overlap,["$sec_tab[$j][2]:$sec_tab[$j][0],$sec_tab[$j][1],$sec_size",$overlap_size]; + } + else + { + push @overlap,["$sec_tab[$j][0],$sec_tab[$j][1],$sec_size",$overlap_size]; + } + } + @overlap=sort{$b->[1]<=>$a->[1]}@overlap; #overlapÁоÙÓÉoverlap size´Ó´óµ½Ð¡ÂÞÁÐ + my $overlap_out=""; + $overlap_num=scalar(@overlap); + if ($overlap_num>0) + { + map{$overlap_out.="\t".(join ",",@$_)}@overlap; + } + my $overlap_rate=$total_overlap_size/$fst_size; + if (($rd1 ne "")&&($fst_tab[$i][2] ne "")) + { + if ( ($List_form eq "O") && ($overlap_num>=$minNum) ) + { + if ((defined $Final)||($limitType eq "")) + { + next if ( (defined $minRate) && ($overlap_rate<$minRate) && (!defined $bpsize) ); + } + next if ( (defined $minSize) && ($total_overlap_size<$minSize)); + } + elsif ( ($List_form eq "N") && ($overlap_num<=$minNum) ) + { + if ((defined $Final)||($limitType eq "")) + { + next unless ( ((!defined $bpsize)&&($total_overlap_size<=$fst_size*$minRate)) || ((!defined $minSize)&&(defined $bpsize)&&($total_overlap_size<=$bpsize)) ); + } + next if ((defined $minSize) && ($total_overlap_size>$minSize)); + } + else + { + next; + } + print ($table_id."\t".$fst_tab[$i][2]."\t".$fst_tab[$i][0]."\t".$fst_tab[$i][1]."\t".$fst_size."\t".$overlap_num."\t".$total_overlap_size."\t".$overlap_rate."$overlap_out\n"); + } + else + { + if ( ($List_form eq "O") && ($overlap_num>=$minNum) ) + { + if ((defined $Final)||($limitType eq "")) + { + next if ((defined $minRate) && ($overlap_rate<$minRate) && (!defined $bpsize) ); + } + next if ((defined $minSize) && ($total_overlap_size<$minSize)); + } + elsif ( ($List_form eq "N") && ($overlap_num<=$minNum) ) + { + if ((defined $Final)||($limitType eq "")) + { + next unless ( ((!defined $bpsize)&&($total_overlap_size<=$fst_size*$minRate)) || ((!defined $minSize)&&(defined $bpsize)&&($total_overlap_size<=$bpsize)) ); + } + next if ((defined $minSize) && ($total_overlap_size>$minSize)); + } + else + { + next; + } + print ($table_id."\t".$fst_tab[$i][0]."\t".$fst_tab[$i][1]."\t".$fst_size."\t".$overlap_num."\t".$total_overlap_size."\t".$overlap_rate."$overlap_out\n"); + } + } + } +} + +sub extract_overlap +{ + my $block_p=shift; + foreach my $table_id (keys %$block_p) + { + my @overlap=(); + my @array_order = (exists $block_p->{$table_id}) ? (sort {$a->[0]<=>$b->[0]} @{$block_p->{$table_id}}) : (); + if (@array_order>1) + { + for (my $i=0;$i<@array_order-1;$i++) + { + my ($S1,$E1)=($array_order[$i][0],$array_order[$i][1]); + for (my $j=$i+1;$j<@array_order;$j++) + { + my ($S2,$E2)=($array_order[$j][0],$array_order[$j][1]); + if ($S2>$E1) + { + last; + } + else + { + my ($S,$E)=find_overlap([$S1,$E1],[$S2,$E2]); + push @overlap,[$S,$E]; + } + } + } + } + else + { + print "$table_id\t${$block_p->{$table_id}}[0][0]\t${$block_p->{$table_id}}[0][1]\n" if ($Extract eq "N"); + } + $limitType = "bpsize"; + $bpsize = 0; + my %tmp_overlap=(); + push @{$tmp_overlap{$table_id}},@overlap; + my @Combine_array = combine_overlap(\%$block_p,"null"); + my @Overlap_array = combine_overlap(\%tmp_overlap,$Extract); + my %Combine=(); + my %Overlap=(); + push @{$Combine{$table_id}},@Combine_array; + push @{$Overlap{$table_id}},@Overlap_array; + filterout_overlap(\%Combine,\%Overlap) if (($Extract eq "N")&&(@{$Combine{$table_id}}>0)&&(@{$Overlap{$table_id}}>0)); + } +} + +sub find_overlap +{ + my ($block1_p,$block2_p) = @_; + my $S = ($block1_p->[0] > $block2_p->[0]) ? $block1_p->[0] : $block2_p->[0]; + my $E = ($block1_p->[1] < $block2_p->[1]) ? $block1_p->[1] : $block2_p->[1]; + return ($S,$E); +} + +sub overlap_size +{ + my ($block1_p,$block2_p) = @_; + + my $combine_start = ($block1_p->[0] < $block2_p->[0]) ? $block1_p->[0] : $block2_p->[0]; + my $combine_end = ($block1_p->[1] > $block2_p->[1]) ? $block1_p->[1] : $block2_p->[1]; + + my $overlap_size = ($block1_p->[1]-$block1_p->[0]+1) + ($block2_p->[1]-$block2_p->[0]+1) - ($combine_end-$combine_start+1); + + return $overlap_size; +} + +sub compare_overlap +{ + my $block1_p = shift; + my $block2_p = shift; + + my $S1=$block1_p->[0]; + my $E1=$block1_p->[1]; + my $S2=$block2_p->[0]; + my $E2=$block2_p->[1]; + my $overlapSize=overlap_size($block1_p,$block2_p); + my $Size1=($E1-$S1+1); + my $Size2=($E2-$S2+1); + my ($big,$small); + my $S=($S1<$S2) ? $S1 : $S2; + my $E=($E1>$E2) ? $E1 : $E2; + if ($Size1>$Size2) + { + $big=$Size1; + $small=$Size2; + } + else + { + $big=$Size2; + $small=$Size1; + } + if ($limitType eq "small") + { + if ($overlapSize<=$small*$minRate) + { + return ($S1,$E1,$S2,$E2); + } + else + { + return ($S,$E); + } + } + elsif ($limitType eq "big") + { + + if ($overlapSize<=$big*$minRate) + { + return ($S1,$E1,$S2,$E2); + } + else + { + return ($S,$E); + } + } + elsif ($limitType eq "first") + { + if ($overlapSize<=$Size1*$minRate) + { + return ($S1,$E1,$S2,$E2); + } + else + { + return ($S,$E); + } + } + elsif($limitType eq "second") + { + if ($overlapSize<=$Size2*$minRate) + { + return ($S1,$E1,$S2,$E2); + } + else + { + return ($S,$E); + } + } + elsif($limitType eq "bpsize") + { + if ($overlapSize<=$bpsize) + { + return ($S1,$E1,$S2,$E2); + } + else + { + return ($S,$E); + } + } + else + { + last; + } +} + +sub readme +{ + my $usage = <=S1) && (S2<=E1) +È¡Smin = (S1E2) ? E1 : E2; +È¥ÈßÓ೤¶È Lc=Emax-Smin+1; +overlap³¤¶È L=L1+L2-Lc=(E1-S1+1)+(E2-S2+1)-(Emax-Smin+1) + +2¡¢´úÂë: +sub overlap_size +{ + my (\$block1_p,\$block2_p) = @_; #([S1,E1],[S2,E2]) + + my \$combine_start = (\$block1_p->[0] < \$block2_p->[0]) ? \$block1_p->[0] : \$block2_p->[0]; + my \$combine_end = (\$block1_p->[1] > \$block2_p->[1]) ? \$block1_p->[1] : \$block2_p->[1]; + + my \$overlap_size = (\$block1_p->[1]-\$block1_p->[0]+1) + (\$block2_p->[1]-\$block2_p->[0]+1) - (\$combine_end-\$combine_start+1); + + return \$overlap_size; +} + +(¶þ£©overlapÖ÷Òª¹Øϵ£¬½éÉÜdisposeOverlap.plµÄÖ÷Òª¹¦ÄÜ +Ò»°ãÈÏΪ¾ß±¸ÈýÖÖ¹¦ÄܾͿÉÒÔ´¦ÀíÖî¶àµÄoverlap¹Øϵ£ºÁоÙ(Enumerate)¡¢ºÏ²¢(Combine)¡¢É¸ÂË(Filterout) +1¡¢Áо٠+ÁоٵÄÒâ˼£¬¼´Èç¹ûÎÒÃÇ´¦ÀíÁ½¸öÎļþ£¬²»·ÁÉèΪaÎļþºÍbÎļþ£¨¼ò³Æa,b)£¬ÕâÁ½¸öÎļþ¶¼ÓÐÒ»¶¨µÄÇøÓòÈç(start,end)×÷Ϊ±È½Ï£» +ÎÒÃÇÒªÕÒbÀïµÄÇøÓòÓжàÉÙÓëaÖеÄÇøÓòÓÐoverlap£¬²¢ÒÔa×÷Ϊ²Î¿¼£¬¼´ÈçaÖÐij¸öÇøÓò¸úbÖжàÉÙµÄÇøÓòÓÐoverlap£¬²¢Áгö¡£ +²»·ÁÉèa£¬bµÄ¸ñʽΪ +Chromosome RegionID Start End 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10-bpsize -final ±È½Ï¶¨Òåoverlapʱͬ--OL 10-bpsize£¬µ«Êä³ö½á¹ûÏÞ¶¨OverlapSize>=10²ÅÊä³ö; +ÕâÀïOverlapRate,OverlapSize,Length¶¼ÊÇtitleµÄÖµ¼´Îª×ܵÄOverlap±ÈÂÊ£¬×ܵÄOverlap´óС£¬ºÍµÚÒ»ÎļþµÄÇøÓòµÄ³¤¶È +×îºóÊä³ö×÷ÁËСÐ޸ģ¬ÒÔºó»áÒÔµÚÒ»¸öÎļþµÄÊ×λÖÃ×÷Ϊ×ù±ê³ß(ÅÅÐò)ÁÐ±í£¬µÚ¶þ¸öÎļþÓëÖ®overlap£¬»á°´overlap´óС´Ó´óµ½Ð¡ÔÚºóÃæÁоٳö¡£ + +´óÄê³õÈýÁ賿2:39am, Happy Å£ Year! +******************************************** + +USAGE + print $usage; +} diff --git a/script/makeMatrix_cluster.pl b/script/makeMatrix_cluster.pl new file mode 100644 index 0000000..af547d0 --- /dev/null +++ b/script/makeMatrix_cluster.pl @@ -0,0 +1,169 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use File::Spec; +use List::Util qw/sum min max/; +use File::Temp qw/tempfile tempdir/; +use File::Basename; +use FindBin qw($Bin $Script); +# ----------------------------------------------------------------------------- +# Variables + +my ($USAGE, $call, $ret, $help); +my ($in1, $class, $out, $pcc, $pca, $clust, $Rscript); +my ($r_pcc, $r_pca, $r_clust1, $r_clust2); +my $SCRIPTNAME = basename($0); + +# ----------------------------------------------------------------------------- +# OPTIONS + + +## R executable! checking of ALL flags + +$USAGE = << "USE"; + + synopsis: + + usage: perl $SCRIPTNAME --in [--out ] --head [-b ] + + [INPUT] --in input file of methyl-matrix file (the output of p01.input.pl); + --class inout file containing subclass identifiers for each sample(one per line), just used in PCA analysis. + --pcc run pearson correlation analysis + --pca run PCA (Principal Component Analysis) analysis + --clust run Clustering Analysis + --out prefix of output + --r path/executable of Rscript executable (default: in PATH) + + + +USE + +if (!@ARGV) { + printf STDERR $USAGE; + exit -1; +} + +unless (GetOptions( + "in=s" => \$in1, + "out=s" => \$out, + "pcc" => \$pcc, + "pca" => \$pca, + "clust" => \$clust, + "class=s" =>\$class, + "r=s" => \$Rscript, + "h|help"=> \$help +)){ + printf STDERR $USAGE; + exit -1; +} + +if (defined $help){ + printf STDERR $USAGE; + exit -1; +} + +# ----------------------------------------------------------------------------- +# MAIN + +my $cur_dir = File::Spec->curdir(); + +############ +## checks ## +############ +print STDERR ("[INFO]" . prettyTime() . "Checking options\n"); + +### input +unless( -e $in1){ + print STDERR "[Err]: the -in $in1 does not exists!\n"; + die $USAGE; +} + +### out +if (defined $out){ + $out = File::Spec->rel2abs($out); +} +else { + $out = ("pattern"); +} + +### script +$r_pcc = File::Spec->rel2abs("$Bin/PCC.R"); +$r_pca = File::Spec->rel2abs("$Bin/PCA.R"); +$r_clust1 = File::Spec->rel2abs("$Bin/HCLUST.R"); +$r_clust2 = File::Spec->rel2abs("$Bin/PVCLUST.R"); + +### $Rscript +if (defined $Rscript){ + $Rscript = File::Spec->rel2abs($Rscript); + if (-e $Rscript){ + unless (-d $Rscript){ + unless (-x $Rscript){ + die "##### AN ERROR has occurred: --r option executable is not executable\n"; + } + } + else{ + die "##### AN ERROR has occurred: --r option executable is directory\n"; + } + } + else{ + die "##### AN ERROR has occurred: --r option executable nonexistent\n"; + } +} +else{ + $Rscript = "Rscript"; +} + +$call = "command -v $Rscript &> /dev/null"; +$ret = system ($call); +if ($ret != 0){ + die "##### AN ERROR has occurred: No bedtools executable found. Please provide path/filename of bedtools executable with --bedtools option\n"; +} + +##################### +## analysi and plot # +##################### +print STDERR ("[INFO]" . prettyTime() . "Begin to analysis ..\n"); + +if(defined $pcc && -e $r_pcc){ + $call = "$Rscript $r_pcc $in1 $out.PCC"; + call($call); + print STDERR ("[INFO]: PCC analysis is done..\n"); +} + +if(defined $pca && -e $r_pca && -e $class){ + my $classfile = File::Spec->rel2abs($class); + $call = "$Rscript $r_pca $in1 $classfile $out.PCA"; + call($call); + print STDERR ("[INFO]: PCA analysis is done..\n"); +} + +if(defined $clust && -e $r_clust1){ + $call = "$Rscript $r_clust1 $in1 $out.hclust \&\& $Rscript $r_clust2 $in1 $out.pvclust.pdf"; + call($call); + print STDERR ("[INFO]: Clustering analysis is done..\n"); +} + + +# ----------------------------------------------------------------------------- +# FUNCTIONS + +sub call{ + my ($sub_call) = @_; + + $ret = system ($sub_call); + + if ($ret != 0){ + die "##### AN ERROR has occurred\n"; + } +} + +sub prettyTime{ + my @months = qw(Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec); + my @weekDays = qw(Sun Mon Tue Wed Thu Fri Sat Sun); + my ($second, $minute, $hour, $dayOfMonth, $month, $yearOffset, $dayOfWeek, $dayOfYear, $daylightSavings) = localtime(); + my $year = 1900 + $yearOffset; + return "\t$weekDays[$dayOfWeek] $months[$month] $dayOfMonth, $hour:$minute:$second, $year\t"; +} + +## END diff --git a/script/makeMatrix_merge.pl b/script/makeMatrix_merge.pl new file mode 100644 index 0000000..9fdb6cb --- /dev/null +++ b/script/makeMatrix_merge.pl @@ -0,0 +1,177 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use File::Spec; +use List::Util qw/sum min max/; +use File::Temp qw/tempfile tempdir/; +use File::Basename; + +# ----------------------------------------------------------------------------- +# Variables + +my ($USAGE, $call, $ret, $help); +my ($in1, $out, $h1, $l, $bedtools); +my ($NA, $out_metilene, $g1_h, $g1_n); +my (@g1, @g1_h); +my $order = ""; +my $SCRIPTNAME = basename($0); + +# ----------------------------------------------------------------------------- +# OPTIONS + + +## R executable! checking of ALL flags + +$USAGE = << "USE"; + + synopsis: Combines multiple bedgraph input files of CpGs methylation levels INTO a single file [methyl matrix]; + + usage: perl $SCRIPTNAME --in [--out ] --head [-b ] + + [INPUT] --in comma-seperated list of sorted (!) bedgraph input files of CpGs methylation levels + --head comma-seperated list of identifier for bedgraph input files (default: numeric) + --out path/file of out file (default: ./methyl_matrix.txt) + -b path/executable of bedtools executable (default: in PATH) + + + +USE + +if (!@ARGV) { + printf STDERR $USAGE; + exit -1; +} + +unless (GetOptions( + "in=s" => \$in1, + "out=s" => \$out, + "head=s" => \$h1, + "b=s" => \$bedtools, + "h|help"=> \$help +)){ + printf STDERR $USAGE; + exit -1; +} + +if (defined $help){ + printf STDERR $USAGE; + exit -1; +} + +# ----------------------------------------------------------------------------- +# MAIN + +my $cur_dir = File::Spec->curdir(); + +############ +## checks ## +############ +print STDERR ("[WARNING]" . prettyTime() . "Input files need to be SORTED, i.e., use \"bedtools sort -i file >file.sorted\"\n"); +print STDERR ("[INFO]" . prettyTime() . "Checking flags\n"); + +$NA = "NA"; + +if (defined $in1){ + @g1 = split(/,/,$in1); + + for (my $i=0; $irel2abs($g1[$i]); + + if ((defined $g1[$i]) && (-e $g1[$i])){ + unless (-r $g1[$i]){ + die "##### AN ERROR has occurred: $g1[$i] (option --g1) not readable\n"; + } + } + else{ + die "##### AN ERROR has occurred: required option --g1 ($g1[$i]) missing or nonexistent\n"; + } + } +} +else { + die "##### AN ERROR has occurred: required option --$in1 missing\n"; +} + +if (defined $h1){ + @g1_h = split(/,/,$h1); + unless(@g1_h == @g1){ + die "##### AN ERROR has occurred: the number of identifier for bedgraph input files is not equal to the number of bedgraph input files!\n"; + } +} +else{ + print STDERR "[Warning]: undefined option --head, automatic numbering will be used..\n"; + for (my $i=0; $irel2abs($out); + $out_metilene = $out; +} +else { + $out_metilene = ("methyl_matrix.txt"); +} + +## bedtools ## +if (defined $bedtools){ + $bedtools = File::Spec->rel2abs($bedtools); + if (-e $bedtools){ + unless (-d $bedtools){ + unless (-x $bedtools){ + die "##### AN ERROR has occurred: --bedtools option executable is not executable\n"; + } + } + else{ + die "##### AN ERROR has occurred: --bedtools option executable is directory\n"; + } + } + else{ + die "##### AN ERROR has occurred: --bedtools option executable nonexistent\n"; + } +} +else{ + $bedtools = "bedtools"; +} +$call = "command -v $bedtools &> /dev/null"; +$ret = system ($call); +if ($ret != 0){ + die "##### AN ERROR has occurred: No bedtools executable found. Please provide path/filename of bedtools executable with --bedtools option\n"; +} + +##################### +## union bedgraph ## +#################### +print STDERR ("[INFO]" . prettyTime() . "Write metilene input to $out_metilene\n"); + +$g1_n = join(" ", @g1); +$g1_h = join(" ", @g1_h); + +$call = "$bedtools unionbedg -header -names $g1_h -filler $NA -i $g1_n | cut -f1,2,4- | sed '/\tNA/d; s/\t/:/' | sed 's/chrom:start//' >$out_metilene"; + +call($call); + +print STDERR ("[NEXT STEP]: The For further parameters: makeMatrix_cluster.pl --help\n"); + +# ----------------------------------------------------------------------------- +# FUNCTIONS + +sub call{ + my ($sub_call) = @_; + + $ret = system ($sub_call); + + if ($ret != 0){ + die "##### AN ERROR has occurred\n"; + } +} + +sub prettyTime{ + my @months = qw(Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec); + my @weekDays = qw(Sun Mon Tue Wed Thu Fri Sat Sun); + my ($second, $minute, $hour, $dayOfMonth, $month, $yearOffset, $dayOfWeek, $dayOfYear, $daylightSavings) = localtime(); + my $year = 1900 + $yearOffset; + return "\t$weekDays[$dayOfWeek] $months[$month] $dayOfMonth, $hour:$minute:$second, $year\t"; +} + +## END diff --git a/script/merge_methy_stat.v1.0.sh b/script/merge_methy_stat.v1.0.sh new file mode 100644 index 0000000..8a937de --- /dev/null +++ b/script/merge_methy_stat.v1.0.sh @@ -0,0 +1,31 @@ +pjdir=$1 +prefix=$2 +Rscript=$3 + +echo -e "Table 5.CG,CHG,CHH sequencing depth and coverrage\nSampleID\ttotal_CG\tmean_depthCG\teffect_depthCG(>=1X)\tcovrageCG\tcoverageCG(d>=5X)\tcoverageCG(d>=10X)\ttotal_CHG\tmean_depthCHG\teffect_depthCHG(>=1X)\tcovrageCHG\tcoverageCHG(d>=5X)\tcoverageCHG(d>=10X)\ttotal_CHH\tmean_depthCHH\teffect_depthCHH(>=1X)\tcovrageCHH\tcoverageCHH(d>=5X)\tcoverageCHH(d>=10X)" > $pjdir/03_global_methy/01_coverage_depth/05.all.coverage_depth.xls +echo -e "Table 4.CG,CHG,CHH average methylation level(>1Xdepth)\nSampleID\tC\tCG\tCHG\tCHH" >$pjdir/03_global_methy/01_coverage_depth/04.all.averge_methyl.xls + +cd $pjdir/03_global_methy/01_coverage_depth +#for sam in `ls -d $prefix*` +for sam in `ls -d *` + do + awk 'NR==2' $sam/$sam.averge_methyl.xls >> $pjdir/03_global_methy/01_coverage_depth/04.all.averge_methyl.xls + awk 'NR==2' $sam/$sam.coverage_depth.xls >> $pjdir/03_global_methy/01_coverage_depth/05.all.coverage_depth.xls + done + +#plot +#get script file dir +getsd() { + oldwd=`pwd` + rw=`dirname $0` + cd $rw + sw=`pwd` + cd $oldwd + echo $sw +} +scriptdir=`getsd` + +$Rscript $scriptdir/averge_methyl.barplot.R $pjdir/03_global_methy/01_coverage_depth/04.all.averge_methyl.xls all.averge_methyl.pdf +$Rscript $scriptdir/methy_type.pieplot.R $pjdir/03_global_methy/01_coverage_depth/05.all.coverage_depth.xls global_methy_type.pieplot.pdf + + diff --git a/script/methy_stack_bar.v1.1.R b/script/methy_stack_bar.v1.1.R new file mode 100644 index 0000000..c699b77 --- /dev/null +++ b/script/methy_stack_bar.v1.1.R @@ -0,0 +1,112 @@ +# A script for stack barplot of methylation data +# zhangdu +# 2018-8-13 +# v1.0 + +# USAGE: +# Rscript methy_stat_bar.R in 5 out_prefix +# "5" is the number of stack blocks for each bar to plot. + +# in file format: +############################ +#Group A A B B +#Sample s1 s2 s3 s4 +#rowname1 0.1 0.1 0.1 0.1 +#rowname2 0.1 0.2 0.1 0.2 +############################ + + + args <- commandArgs(TRUE) +#data <- read.table(args[1],header=T,row.names=1) + data <- read.table(args[1]) +n <- as.numeric(args[2]) + prefix <- args[3] + + grouplabels <- as.character(t(data[1,-1])) +samplelabels <- as.character(t(data[2,-1])) + +#data1 is sample methy + data1<-data[c(-1,-2),-1] +data1 <- matrix(as.numeric(as.matrix(data1)), dim(data1)[1], dim(data1)[2], byrow=F) + rownames(data1)<-data[c(-1,-2),1] + data2<-data1 + colnames(data1)<-samplelabels + +#data3 is group methy + colnames(data2)<-grouplabels +groups<-levels(as.factor(grouplabels)) + if(length(groups) != length(samplelabels)){ + data3<-matrix(nrow=dim(data2)[1],ncol=length(groups)) + for(i in 1:length(groups)){ + data2a<-data2[,colnames(data2)==groups[i]] + data3[,i]<-apply(data2a,1,mean) + } + colnames(data3)<-groups + rownames(data3)<-data[c(-1,-2),1] + grouplabels1<-colnames(data3) + write.table(data3,file=paste(prefix,"group.table",sep="_"),quote=F) + } + +# stat sample methy distribution +mat <- matrix(0,ncol=length(samplelabels),nrow=n) + unit = 1/n + start = 0 +regions <- rep(0,n) + for ( i in 1:n ) {regions[i]=paste( start, start+unit, sep="-"); start = start + unit } + + colnames(mat) <- samplelabels + rownames(mat) <- regions +data1 <- ceiling(data1/unit) + + for (i in 1:length(samplelabels)){ + v = table(data1[,i]) + mat[,i]<-as.numeric(v)[2:(n+1)] + mat[1,i] <- mat[1,i] + as.numeric(v)[1] + } + +base = dim(data1)[1] +mat <- mat/base +write.table(mat,file=paste(prefix, "methy_distribution.xls",sep="_"),quote=F) + +#plot sample methy distribution +colors <- c("red3","green4","yellow3","purple3","blue3","orange3","pink3","deepskyblue","orange","lightcyan2") + + pdf(file=paste(prefix, "stackbar.pdf",sep="_")) +par(mai=c(1.2,1,1.2,1.3),mgp=c(3.5,0.6,0),las=2) + barplot(mat,names.arg=samplelabels,xlab="Sample",ylab="% Cpg coverage",col=colors,cex.names=0.8,cex.axis=0.8) + xy <- par("usr") + legend(x=xy[2]+0.1,y=xy[4]-0.1, regions, cex=0.8, fill=colors,xpd=T) +dev.off() + +# stat group methy distribution + if(length(groups) != length(samplelabels)){ + mat <- matrix(0,ncol=length(grouplabels1),nrow=n) + unit = 1/n + start = 0 + regions <- rep(0,n) + for ( i in 1:n ) {regions[i]=paste( start, start+unit, sep="-"); start = start + unit } + + colnames(mat) <- grouplabels1 + rownames(mat) <- regions + data3 <- ceiling(data3/unit) + + for (i in 1:length(grouplabels1)){ + v = table(data3[,i]) + mat[,i]<-as.numeric(v)[2:(n+1)] + mat[1,i] <- mat[1,i] + as.numeric(v)[1] + } + + base = dim(data3)[1] + mat <- mat/base + write.table(mat,file=paste(prefix, "group_methy_distribution.xls",sep="_"),quote=F) + +#plot group methy distribution + colors <- c("red3","green4","yellow3","purple3","blue3","orange3","pink3","deepskyblue","orange","lightcyan2") + + pdf(file=paste(prefix, "group_stackbar.pdf",sep="_")) + par(mai=c(1.2,1,1.2,1.3),mgp=c(3.5,0.6,0),las=2) + barplot(mat,names.arg=grouplabels1,xlab="Sample",ylab="% Cpg coverage",col=colors,cex.names=0.8,cex.axis=0.8) + xy <- par("usr") + legend(x=xy[2]+0.1,y=xy[4]-0.1, regions, cex=0.8, fill=colors,xpd=T) + dev.off() + } diff --git a/script/methy_type.pieplot.R b/script/methy_type.pieplot.R new file mode 100644 index 0000000..f20a626 --- /dev/null +++ b/script/methy_type.pieplot.R @@ -0,0 +1,39 @@ +### This is script for pieplot of Genome C, CHG and CHH count percentage #### +### Author: zhangdu +### date: 2018-08-13 +### version:v1.0 + +arg <- commandArgs(T) +if(length(arg) < 2){ + cat("Argument: 05.all.coverage_depth.xls methy_type.pieplot.pdf\n") + quit('no') +} + +library(tidyr) +library(ggplot2) +tab<-read.table(arg[1],header=T,skip=1) +tab<-tab[1,] +tab1<-data.frame(CG=tab$total_CG,CHG=tab$total_CHG,CHH=tab$total_CHH) +tab_long<-gather(tab1,Type,Number,CG:CHH) + +tab_long = tab_long[order(tab_long$Number, decreasing = TRUE),] +myLabel = as.vector(tab_long$Type) +tab_long$Number<-as.numeric(tab_long$Number) +myLabel = paste(myLabel, "(",tab_long$Number,",",round(tab_long$Number / sum(tab_long$Number) * 100, 2), "%)", sep = "") + +pdf(arg[2]) + ggplot(tab_long, aes(x = "", y = Number, fill = Type)) + + geom_bar(stat = "identity", color="white", width = 1) + + coord_polar(theta = "y") + + labs(x = "", y = "", title = "") + + theme(axis.ticks = element_blank()) + + theme(legend.title = element_blank(), legend.position = "top") + + scale_fill_discrete(breaks = tab_long$Type, labels = myLabel) + + #scale_fill_brewer(palette="Dark2") + + theme(axis.text.x = element_blank()) + + theme(panel.grid=element_blank()) + ## 去掉白色圆框和中间的å标线 + theme(panel.border=element_blank()) + ## 去掉最外层正方形的框框 + theme(panel.background = element_blank())+ + ggtitle("Genome C, CHG and CHH percentage") + +dev.off() diff --git a/script/metilene_output.v_p-value_filter.R b/script/metilene_output.v_p-value_filter.R new file mode 100644 index 0000000..b59ee35 --- /dev/null +++ b/script/metilene_output.v_p-value_filter.R @@ -0,0 +1,105 @@ +#!/usr/local/bin/Rscript + +######################################### +### This is a p-value fiter version of metilene_output.R +### +### Author: zhangdu +### +### Usage: Rscript metilene_output.v_p-value_filter.R +######################################### + +args <- commandArgs(trailingOnly = TRUE) + + +## Loading packages +library(ggplot2) + +## make nice plots +theme_cfg <- + theme_bw() + + theme( + panel.grid.major = element_blank(), + panel.grid.minor = element_blank(), + panel.border = element_blank(), + + axis.line = element_line(colour = "black"), + + legend.key = element_blank() + ) + +## Read input file +data <- read.table(file=args[1], header=F, col.names=c('chr','start','end','p_val','diff','CpG','group1','group2')) +data$length <- data$end-data$start + +## Plot statistics +#pdf(args[2]) +# 01 difference histogram +pdf(file = paste(args[2],"DMR_difference_histogram.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=diff)) + geom_histogram(binwidth=0.05, fill='steelblue3', color='black') + xlab("Mean methylation difference") + scale_x_continuous(limits=c(-1,1)) + labs(title=paste(args[3],"vs",args[4],"DMR difference histogram",sep=" ")) +# + theme_cfg +dev.off() + +# 02 length distribution CpGs +pdf(file = paste(args[2],"DMR_mC_length_distribution.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=length, fill = 'skyblue3')) + geom_line(stat="Density", size=1) + xlab("DMR length [nt]") + labs(title=paste(args[3],"vs",args[4],"DMR length distribution(mC)",sep=" ")) +#+ theme_cfg +dev.off() + +# 03 length distribution nt +pdf(file = paste(args[2],"DMR_nt_length_distribution.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=CpG)) + geom_line(stat="Density", size=1) + xlab("DMR length [CpG]") + labs(title=paste(args[3],"vs",args[4],"DMR length distribution(nt)",sep=" ")) +#+ theme_cfg +dev.off() + +# 04 p_val vs difference +pdf(file = paste(args[2],"DMR_p_val-difference.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=diff, y=p_val)) + geom_point(alpha=.5) + scale_y_log10() + xlab("Mean methylation difference") + ylab("p-value") + labs(title=paste(args[3],"vs",args[4],"DMR p_val-difference",sep=" ")) +#+ theme_cfg +dev.off() + +# 05 mean1 vs mean2 + +pdf(file = paste(args[2],"DMR_methylation_distribution.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=group1, y=group2)) + geom_point(alpha=.5) + coord_fixed() + xlab(args[3]) + ylab(args[4]) + labs(title=paste(args[3],"vs",args[4],"DMR methylation Level distribution",sep=" ")) +# + theme_cfg +dev.off() + +# 06 nt vs CpGs +pdf(file = paste(args[2],"DMR_nt-mC_number.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=length, y=CpG)) + geom_point(alpha=.5) + xlab("DMR length [nt]") + ylab("DMR length [CpGs]") + labs(title=paste(args[3],"vs",args[4],"DMR nt-mC number",sep=" ")) +#+ theme_cfg +dev.off() + +# 07 DMR scatter plot + pdf(file = paste(args[2],"DMR_scatter_plot.pdf",sep="."),width =5,height=5) +n <- nrow(data) +ggplot(data, aes(x = group1, y = group2)) + +geom_point(alpha=0.5,colour="#0000FF") + +stat_density_2d(aes(alpha=..density..,fill = ..density..), geom = "tile",contour=FALSE)+ +scale_fill_gradientn (colours = c("#FFFFFF", "#0000FF","#00FF00","#FFFF00","#FF0000")) + +labs(title=paste("DMR methylation Level distribution(n=",n,")",sep="")) + xlab(args[3]) + ylab(args[4]) + +theme(plot.title = element_text(hjust = 0.5),panel.background=element_blank(),panel.border=element_rect(fill=NA),legend.position="none") + +scale_y_continuous(limits=c(0,1),expand = c(0,0)) + +scale_x_continuous(limits=c(0,1),expand = c(0,0)) +dev.off() + + +# 08 DMR violin plot +library("vioplot") +### T.test ### +xa<-data$group1 +xb<-data$group2 +ya<-xa[!is.na(xa)] +yb<-xb[!is.na(xb)] +ylim<-max(ya,yb) +maxy=ylim +p=signif(t.test(xa,xb)$p.value,digits=4) +plot.new=TRUE +main=paste("n=",n," P=",p,seq="") +### Plot figure ### + pdf(file = paste(args[2],"DMR_violin_plot.pdf",sep="."),width =5,height=5) +vioplot(xa,xb,names=c(args[3],args[4]),col="gold",ylim=c(0,maxy)) +title(main) +dev.off() + + diff --git a/script/metilene_output.v_p-value_filter.pl b/script/metilene_output.v_p-value_filter.pl new file mode 100644 index 0000000..6a1a7ed --- /dev/null +++ b/script/metilene_output.v_p-value_filter.pl @@ -0,0 +1,201 @@ +#!/usr/bin/perl + +use strict; +use warnings; + +use Getopt::Long; +use File::Spec; +use File::Path qw(make_path remove_tree); +use File::Basename; + +# ----------------------------------------------------------------------------- +# Variables + +my ($USAGE, $help, $ret, $call, $in, $out, $path); +my ($threads, $min_cpgs, $min_diff, $id1, $id2, $p_val, $length); +my ($plot_script, $pval_out, $pval_bed, $pdf,$Rscript); +my $SCRIPTNAME = basename($0); + +# ----------------------------------------------------------------------------- +# OPTIONS + +$USAGE = << "USE"; + + usage: perl $SCRIPTNAME -q [-o ] [-p ] [-c ] [-d ] [-l ] [-a ] [-b ] + + This is a develope version of metilene_output.pl for Mann-Whitney-U test p-value filter instead of adj. p-value filter. + Author: zhangdu + + [INPUT] -i path/filename of metilene DMRs + -o path/prefix of output files (default: input_path/) + -p maximum (<) Mann-Whitney-U test p-value for output of significant DMRs (default: 0.05) + -c minimum (>=) cpgs (default:10) + -d minimum mean methylation difference (>=) (default:0.1) + -l minimum length of DMR [nt] (>=) (post-processing, default: 0) + -a name of group A (default:"g1") + -b name of group B (default:"g2") + -Rscript Rscript program; +USE + +if (!@ARGV) { + printf STDERR $USAGE; + exit -1; +} + +unless (GetOptions( + "i=s" => \$in, + "o=s" => \$out, + "p=s" => \$p_val, + "c=s" => \$min_cpgs, + "d=s" => \$min_diff, + "l=s" => \$length, + "a=s" => \$id1, + "b=s" => \$id2, + "Rscript=s" => \$Rscript, + "h|help" => \$help +)){ + printf STDERR $USAGE; + exit -1; +} +if (defined $help){ + printf STDERR $USAGE; + exit -1; +} +die $USAGE unless $Rscript; + +# ----------------------------------------------------------------------------- +# MAIN + +################# +## check flags ## +################# +print STDERR ("[INFO]" . prettyTime() . "Checking flags\n"); + +## relative to absolut path ## +if (defined $in){$in = File::Spec->rel2abs($in)}; +if (defined $out){$out = File::Spec->rel2abs($out)}; +$plot_script = File::Spec->rel2abs($0); + +## out dir +my ($vol, $dir, $file) = File::Spec->splitpath($in); +if (!defined $out){ + $path = $dir; + $file = "metilene"; +} +else { + ($vol, $dir, $file) = File::Spec->splitpath($out); + $path = $dir; +} +unless (-e $path){ + $ret = make_path($path); + if ($ret != 1){ + die "##### AN ERROR has occurred: Could not make out directory $path\n"; + } + else{ + print STDERR ("[INFO]" . prettyTime() . "Created result-directory $path\n"); + } +} +elsif (-d $path){ + unless (-w $path && -r $path){ + die "##### AN ERROR has occurred: out directory $path not readable or writable\n"; + } +} + +## id group 1 ## +if (!defined $id1){ + $id1 = "g1"; +} + +## id group 2 ## +if (!defined $id2){ + $id2 = "g2"; +} + +## min cpgs ## +if (!defined $min_cpgs){ + $min_cpgs = 10; +} + +## min diff ## +if (!defined $min_diff){ + $min_diff = 0.1; +} + +## pval filter ## +if (!defined $p_val){ + $p_val = 0.05; +} + +## length ## +if (!defined $length){ + $length = 0; +} + +## out file ## +$pval_out = File::Spec->catpath($vol, $path, $file); +$pval_out = ($pval_out . "_pval." . $p_val . ".out"); +$pval_bed = File::Spec->catpath($vol, $path, $file); +$pval_bed = ($pval_bed . "_pval." . $p_val . ".bedgraph"); +$pdf = File::Spec->catpath($vol, $path, $file); +$pdf = ($pdf . "_pval." . $p_val); + +$plot_script =~ s/.pl$/.R/; + +################## +### filter DMRs ## +################## +print STDERR ("[INFO]" . prettyTime() . "Filter DMRs.\n"); + +my $count = 0; +open(OUT, ">$pval_out") or die "##### AN ERROR has occurred: could not write to $pval_out\n"; +open(OUT_B, ">$pval_bed") or die "##### AN ERROR has occurred: could not write to $pval_out\n"; + +open (IN, "$in") || die "cannot open $in\n"; + while () { + chomp; + + my ($chr, $start, $end, $pval, $diff_methyl, $nr_cpgs, $MWU, $KS2D, $mean_g1, $mean_g2) = split(/\t/,$_); + if (($MWU < $p_val) && (abs($diff_methyl) >= $min_diff) && ($nr_cpgs >= $min_cpgs) && (($end-$start) >= $length)) { + print OUT "$chr\t$start\t$end\t$MWU\t$diff_methyl\t$nr_cpgs\t$mean_g1\t$mean_g2\n"; + print OUT_B "$chr\t$start\t$end\t$diff_methyl\n"; + $count++; + } +} +close (IN); +close (OUT); +close (OUT_B); + +print STDERR ("[INFO]" . prettyTime() . "Wrote $count DMRs with Mann-Whitney-U test p-value<$p_val, a minimum absolute difference>=$min_diff, a minimum length [CpG]>=$min_cpgs and a minimum length [nt]>=$length\n"); +print STDERR ("[INFO]" . prettyTime() . "Bedgraph file containing DMR difference: $pval_bed\n"); + + +################# +### plot stats ## +################# +#print "$plot_script $pval_out $pdf\n"; +print STDERR ("[INFO]" . prettyTime() . "Plot DMR statistics.\n"); +$call = ("$Rscript $plot_script $pval_out $pdf $id1 $id2"); +call($call); + +# -----------------------------------------------------------------------------ls +# FUNCTIONS + +sub call{ + my ($sub_call) = @_; + $ret = system ($sub_call); + + if ($ret != 0){ + die "##### AN ERROR has occurred. Occurred at: $sub_call\n"; + } +} + +sub prettyTime{ + my @months = qw(Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec); + my @weekDays = qw(Sun Mon Tue Wed Thu Fri Sat Sun); + my ($second, $minute, $hour, $dayOfMonth, $month, $yearOffset, $dayOfWeek, $dayOfYear, $daylightSavings) = localtime(); + my $year = 1900 + $yearOffset; + return "\t$weekDays[$dayOfWeek] $months[$month] $dayOfMonth, $hour:$minute:$second, $year\t"; +} + + + diff --git a/script/metilene_output.v_q-value_filter.R b/script/metilene_output.v_q-value_filter.R new file mode 100644 index 0000000..952ca3c --- /dev/null +++ b/script/metilene_output.v_q-value_filter.R @@ -0,0 +1,105 @@ +#!/usr/local/bin/Rscript + +######################################### +### This is a q-value fiter version of metilene_output.R +### +### Author: zhangdu +### +### Usage: Rscript metilene_output.v_q-value_filter.R +######################################### + +args <- commandArgs(trailingOnly = TRUE) + + +## Loading packages +library(ggplot2) + +## make nice plots +theme_cfg <- + theme_bw() + + theme( + panel.grid.major = element_blank(), + panel.grid.minor = element_blank(), + panel.border = element_blank(), + + axis.line = element_line(colour = "black"), + + legend.key = element_blank() + ) + +## Read input file +data <- read.table(file=args[1], header=F, col.names=c('chr','start','end','q_val','diff','CpG','group1','group2')) +data$length <- data$end-data$start + +## Plot statistics +#pdf(args[2]) +# 01 difference histogram +pdf(file = paste(args[2],"DMR_difference_histogram.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=diff)) + geom_histogram(binwidth=0.05, fill='steelblue3', color='black') + xlab("Mean methylation difference") + scale_x_continuous(limits=c(-1,1)) + labs(title=paste(args[3],"vs",args[4],"DMR difference histogram",sep=" ")) +# + theme_cfg +dev.off() + +# 02 length distribution CpGs +pdf(file = paste(args[2],"DMR_mC_length_distribution.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=length, fill = 'skyblue3')) + geom_line(stat="Density", size=1) + xlab("DMR length [nt]") + labs(title=paste(args[3],"vs",args[4],"DMR length distribution(mC)",sep=" ")) +#+ theme_cfg +dev.off() + +# 03 length distribution nt +pdf(file = paste(args[2],"DMR_nt_length_distribution.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=CpG)) + geom_line(stat="Density", size=1) + xlab("DMR length [CpG]") + labs(title=paste(args[3],"vs",args[4],"DMR length distribution(nt)",sep=" ")) +#+ theme_cfg +dev.off() + +# 04 q_val vs difference +pdf(file = paste(args[2],"DMR_q_val-difference.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=diff, y=q_val)) + geom_point(alpha=.5) + scale_y_log10() + xlab("Mean methylation difference") + ylab("q-value") + labs(title=paste(args[3],"vs",args[4],"DMR q_val-difference",sep=" ")) +#+ theme_cfg +dev.off() + +# 05 mean1 vs mean2 + +pdf(file = paste(args[2],"DMR_methylation_distribution.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=group1, y=group2)) + geom_point(alpha=.5) + coord_fixed() + xlab(args[3]) + ylab(args[4]) + labs(title=paste(args[3],"vs",args[4],"DMR methylation Level distribution",sep=" ")) +# + theme_cfg +dev.off() + +# 06 nt vs CpGs +pdf(file = paste(args[2],"DMR_nt-mC_number.pdf",sep="."),width =5,height=5) +ggplot(data, aes(x=length, y=CpG)) + geom_point(alpha=.5) + xlab("DMR length [nt]") + ylab("DMR length [CpGs]") + labs(title=paste(args[3],"vs",args[4],"DMR nt-mC number",sep=" ")) +#+ theme_cfg +dev.off() + +# 07 DMR scatter plot + pdf(file = paste(args[2],"DMR_scatter_plot.pdf",sep="."),width =5,height=5) +n <- nrow(data) +ggplot(data, aes(x = group1, y = group2)) + +geom_point(alpha=0.5,colour="#0000FF") + +stat_density_2d(aes(alpha=..density..,fill = ..density..), geom = "tile",contour=FALSE)+ +scale_fill_gradientn (colours = c("#FFFFFF", "#0000FF","#00FF00","#FFFF00","#FF0000")) + +labs(title=paste("DMR methylation Level distribution(n=",n,")",sep="")) + xlab(args[3]) + ylab(args[4]) + +theme(plot.title = element_text(hjust = 0.5),panel.background=element_blank(),panel.border=element_rect(fill=NA),legend.position="none") + +scale_y_continuous(limits=c(0,1),expand = c(0,0)) + +scale_x_continuous(limits=c(0,1),expand = c(0,0)) +dev.off() + + +# 08 DMR violin plot +library("vioplot") +### T.test ### +xa<-data$group1 +xb<-data$group2 +ya<-xa[!is.na(xa)] +yb<-xb[!is.na(xb)] +ylim<-max(ya,yb) +maxy=ylim +p=signif(t.test(xa,xb)$p.value,digits=4) +plot.new=TRUE +main=paste("n=",n," P=",p,seq="") +### Plot figure ### + pdf(file = paste(args[2],"DMR_violin_plot.pdf",sep="."),width =5,height=5) +vioplot(xa,xb,names=c(args[3],args[4]),col="gold",ylim=c(0,maxy)) +title(main) +dev.off() + + diff --git a/script/metilene_output.v_q-value_filter.pl b/script/metilene_output.v_q-value_filter.pl new file mode 100644 index 0000000..c0a5c20 --- /dev/null +++ b/script/metilene_output.v_q-value_filter.pl @@ -0,0 +1,199 @@ +#!/usr/bin/perl + +use strict; +use warnings; + +use Getopt::Long; +use File::Spec; +use File::Path qw(make_path remove_tree); +use File::Basename; + +# ----------------------------------------------------------------------------- +# Variables + +my ($USAGE, $help, $ret, $call, $in, $out, $path); +my ($threads, $min_cpgs, $min_diff, $id1, $id2, $q_val, $length); +my ($plot_script, $qval_out, $qval_bed, $pdf,$Rscript); +my $SCRIPTNAME = basename($0); + +# ----------------------------------------------------------------------------- +# OPTIONS + +$USAGE = << "USE"; + + usage: perl $SCRIPTNAME -q [-o ] [-p ] [-c ] [-d ] [-l ] [-a ] [-b ] + + [INPUT] -i path/filename of metilene DMRs + -o path/prefix of output files (default: input_path/) + -q maximum (<) adj. p-value (q-value) for output of significant DMRs (default: 0.05) + -c minimum (>=) cpgs (default:10) + -d minimum mean methylation difference (>=) (default:0.1) + -l minimum length of DMR [nt] (>=) (post-processing, default: 0) + -a name of group A (default:"g1") + -b name of group B (default:"g2") + -Rscript Rscript program; +USE + +if (!@ARGV) { + printf STDERR $USAGE; + exit -1; +} + +unless (GetOptions( + "i=s" => \$in, + "o=s" => \$out, + "q=s" => \$q_val, + "c=s" => \$min_cpgs, + "d=s" => \$min_diff, + "l=s" => \$length, + "a=s" => \$id1, + "b=s" => \$id2, + "Rscript=s" => \$Rscript, + "h|help" => \$help +)){ + printf STDERR $USAGE; + exit -1; +} +if (defined $help){ + printf STDERR $USAGE; + exit -1; +} + +die $USAGE unless $Rscript; + +# ----------------------------------------------------------------------------- +# MAIN + +################# +## check flags ## +################# +print STDERR ("[INFO]" . prettyTime() . "Checking flags\n"); + +## relative to absolut path ## +if (defined $in){$in = File::Spec->rel2abs($in)}; +if (defined $out){$out = File::Spec->rel2abs($out)}; +$plot_script = File::Spec->rel2abs($0); + +## out dir +my ($vol, $dir, $file) = File::Spec->splitpath($in); +if (!defined $out){ + $path = $dir; + $file = "metilene"; +} +else { + ($vol, $dir, $file) = File::Spec->splitpath($out); + $path = $dir; +} +unless (-e $path){ + $ret = make_path($path); + if ($ret != 1){ + die "##### AN ERROR has occurred: Could not make out directory $path\n"; + } + else{ + print STDERR ("[INFO]" . prettyTime() . "Created result-directory $path\n"); + } +} +elsif (-d $path){ + unless (-w $path && -r $path){ + die "##### AN ERROR has occurred: out directory $path not readable or writable\n"; + } +} + +## id group 1 ## +if (!defined $id1){ + $id1 = "g1"; +} + +## id group 2 ## +if (!defined $id2){ + $id2 = "g2"; +} + +## min cpgs ## +if (!defined $min_cpgs){ + $min_cpgs = 10; +} + +## min diff ## +if (!defined $min_diff){ + $min_diff = 0.1; +} + +## qval filter ## +if (!defined $q_val){ + $q_val = 0.05; +} + +## length ## +if (!defined $length){ + $length = 0; +} + +## out file ## +$qval_out = File::Spec->catpath($vol, $path, $file); +$qval_out = ($qval_out . "_qval." . $q_val . ".out"); +$qval_bed = File::Spec->catpath($vol, $path, $file); +$qval_bed = ($qval_bed . "_qval." . $q_val . ".bedgraph"); +$pdf = File::Spec->catpath($vol, $path, $file); +$pdf = ($pdf . "_qval." . $q_val); + +$plot_script =~ s/.pl$/.R/; + +################## +### filter DMRs ## +################## +print STDERR ("[INFO]" . prettyTime() . "Filter DMRs.\n"); + +my $count = 0; +open(OUT, ">$qval_out") or die "##### AN ERROR has occurred: could not write to $qval_out\n"; +open(OUT_B, ">$qval_bed") or die "##### AN ERROR has occurred: could not write to $qval_out\n"; + +open (IN, "$in") || die "cannot open $in\n"; + while () { + chomp; + + my ($chr, $start, $end, $qval, $diff_methyl, $nr_cpgs, $MWU, $KS2D, $mean_g1, $mean_g2) = split(/\t/,$_); + if (($qval < $q_val) && (abs($diff_methyl) >= $min_diff) && ($nr_cpgs >= $min_cpgs) && (($end-$start) >= $length)) { + print OUT "$chr\t$start\t$end\t$qval\t$diff_methyl\t$nr_cpgs\t$mean_g1\t$mean_g2\n"; + print OUT_B "$chr\t$start\t$end\t$diff_methyl\n"; + $count++; + } +} +close (IN); +close (OUT); +close (OUT_B); + +print STDERR ("[INFO]" . prettyTime() . "Wrote $count DMRs with adj. p-value<$q_val, a minimum absolute difference>=$min_diff, a minimum length [CpG]>=$min_cpgs and a minimum length [nt]>=$length\n"); +print STDERR ("[INFO]" . prettyTime() . "Bedgraph file containing DMR difference: $qval_bed\n"); + + +################# +### plot stats ## +################# +#print "$plot_script $qval_out $pdf\n"; +print STDERR ("[INFO]" . prettyTime() . "Plot DMR statistics.\n"); +$call = ("$Rscript $plot_script $qval_out $pdf $id1 $id2 "); +call($call); + +# -----------------------------------------------------------------------------ls +# FUNCTIONS + +sub call{ + my ($sub_call) = @_; + $ret = system ($sub_call); + + if ($ret != 0){ + die "##### AN ERROR has occurred. Occurred at: $sub_call\n"; + } +} + +sub prettyTime{ + my @months = qw(Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec); + my @weekDays = qw(Sun Mon Tue Wed Thu Fri Sat Sun); + my ($second, $minute, $hour, $dayOfMonth, $month, $yearOffset, $dayOfWeek, $dayOfYear, $daylightSavings) = localtime(); + my $year = 1900 + $yearOffset; + return "\t$weekDays[$dayOfWeek] $months[$month] $dayOfMonth, $hour:$minute:$second, $year\t"; +} + + + diff --git a/script/p01.DMR-detector.v1.2.pl b/script/p01.DMR-detector.v1.2.pl new file mode 100644 index 0000000..b6902b7 --- /dev/null +++ b/script/p01.DMR-detector.v1.2.pl @@ -0,0 +1,200 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use FindBin qw($Bin $Script); +use File::Basename qw(basename dirname); + +my $usage=<<"USAGE"; +name: $0 + +usage: perl $0 + + ############ General parameters ########################## + + -list file containing the sample names and data path you want + this perl to do DMR analysis. Format of this file is : + "samplename\\tgroupname\\tinput.bedgragph"; + -metilene metilene path(metilene_v0.2-6/ abs dir ) + -outdir output dir (default: .) + -name prefix of outputname (default: dmr) + + ############ DMR detection ########################## + + -bedtools the path/executable of bedtools executable + -Rscript Rscript program; + -max maximum distance (default:300) + -min minimum cpgs (default:10) + -d minimum mean methylation difference (default:0.100000) + -t number of threads (default:1) + -f number of method: 1: de-novo, 2: pre-defined regions + -B bed-file for mode 2 containing pre-defined regions; + needs to be SORTED equally to the DataInputFile (default:none) + -groupA name of group A (default: the group name read first time in -list) + -groupB name of group B (default: the group name read second time in -list) + -X minimal number of values in group A (default:-1) + -Y minimal number of values in group B (default:-1) + -v valley filter (0.0 - 1.0) (default:0.700000) + + ########### DMR filtering and stat ################################ + + -p use maximum (<) Mann-Whitney-U test p-value for output of significant DMRs (default: 0.05) + -q use maximum (<) adj. p-value (q-value) for output of significant DMRs (default: 0.05) + -c minimum (>=) cpgs (default:10) + -l minimum length of DMR [nt] (>=) (default: 0, means no filtering) + -r minimum mean methylation difference (>=) (default:0.1) + + -help|?|h + +author: luhanlin.hi\@gmail.com +date: +USAGE + +my ( $LIST, $OUTDIR, $PREFIX, $bedtools, $M, $m, $d, $t, $f, $B, + $groupA, $groupB, $X, $Y, $v, $p, $q, $c, $l, $r, $HELP,$softdir,$Rscript); + +GetOptions( + "list=s" => \$LIST, + "outdir:s" => \$OUTDIR, + "name:s" => \$PREFIX, + "metilene:s" => \$softdir, + "bedtools:s" => \$bedtools, + "Rscript:s" => \$Rscript, + "max:i" => \$M, + "min:i" => \$m, + "d:f" => \$d, + "t:i" => \$t, + "f:i" => \$f, + "B:s" => \$B, + "groupA:s" => \$groupA, + "groupB:s" => \$groupB, + "X:i" => \$X, + "Y:i" => \$Y, + "v:f" => \$v, + "q:f" => \$q, + "p:f" => \$p, + "c:i" => \$c, + "l:i" => \$l, + "r:f" => \$r, + "h|?|help" => \$HELP +); + +die $usage if $HELP; +die $usage unless $LIST; +die $usage unless $bedtools; +die $usage unless $softdir; +die $usage unless $Rscript; + +my $perl01 = "$softdir/metilene_input.pl"; +my $metilene = "$softdir/metilene"; +my $perl02 = "$Bin/metilene_output.v_q-value_filter.pl"; +my $perl03 = "$Bin/metilene_output.v_p-value_filter.pl"; + +$OUTDIR ||= "."; +$PREFIX ||= "dmr"; +$M ||= 300; +$m ||= 10; +$d ||= 0.1; +$t ||= 4; +$f ||= 1; +$B ||= ""; +$X ||= -1; +$Y ||= -1; +$v ||= 0.7; +if(!$p){$q ||=0.05}; +if(!$q){$p ||=0.05}; +$c ||= 10; +$r ||= 0.1; +$l ||= 0; + + +print STDERR "[run]: reading samplelist -> $LIST ..\n"; +my %group = (); +my @group_in_order = (); +open IN, $LIST or die "[err]: can not open file: $LIST\n"; +while(){ + chomp; + next if /^#/; + next if /^\s*$/; + my ($samplename, $groupname, $file) = split; + push @group_in_order, $groupname unless exists $group{$groupname}; + $group{ $groupname }{ $samplename } = $file; +} +close IN; + +foreach (@group_in_order){print "#### -> $_\n";} + +die "[err]:the second group doesn't exists!\n" unless scalar @group_in_order >= 2; +$groupA ||= $group_in_order[0]; +$groupB ||= $group_in_order[1]; + +##### data processing +my @bedA = (); +my @bedB = (); +foreach my $sam (keys %{ $group{ $groupA } }){ + my $bedfile = $group{$groupA}{$sam}; + if(-e $bedfile){ + push @bedA, $bedfile; + }else{ + print STDERR "[err]: file not exists -> $bedfile\n"; + } +} + +foreach my $sam (keys %{ $group{ $groupB } }){ + my $bedfile = $group{$groupB}{$sam}; + if(-e $bedfile){ + push @bedB, $bedfile; + }else{ + print STDERR "[err]: file not exists -> $bedfile\n"; + } +} + +my $bedA_list = join(",", @bedA); +my $bedB_list = join(",", @bedB); +print STDERR "[run]: get metilene input file -> $OUTDIR/$PREFIX.input\n"; +print STDERR "[command]: perl $perl01 --in1 $bedA_list --in2 $bedB_list --out $OUTDIR/$PREFIX.input --h1 $groupA --h2 $groupB -b $bedtools\n"; + +system("perl $perl01 --in1 $bedA_list --in2 $bedB_list --out $OUTDIR/$PREFIX.input --h1 $groupA --h2 $groupB -b $bedtools"); + +print STDERR "[done]: get metilene input file -> $OUTDIR/$PREFIX.input\n"; + +##### DMR detection +print STDERR "[run]: DMR detection between: group $groupA and group $groupB\n"; + +if(-e $B && $f==1){ + print STDERR "[infor]: you have set the -B is $B, and -f is 1, so here -f will be set as 2 !\n"; + $f = 2; +} + +if($f==1){ + print STDERR "[command]: $metilene -M $M -m $m -d $d -t $t -f $f -a $groupA -b $groupB -X $X -Y $Y -v $v $OUTDIR/$PREFIX.input 2> $OUTDIR/$PREFIX.metilene.log |sort -k1,1 -k2,2g > $OUTDIR/$PREFIX.dmr.xls\n"; + system("$metilene -M $M -m $m -d $d -t $t -f $f -a $groupA -b $groupB -X $X -Y $Y -v $v $OUTDIR/$PREFIX.input 2> $OUTDIR/$PREFIX.metilene.log |sort -k1,1 -k2,2g > $OUTDIR/$PREFIX.dmr.xls"); +} +elsif($f==2){ + if(-e $B){ + print STDERR "[command]: $metilene -M $M -m $m -d $d -t $t -f $f -B $B -a $groupA -b $groupB -X $X -Y $Y -v $v $OUTDIR/$PREFIX.input 2> $OUTDIR/$PREFIX.metilene.log |sort -k1,1 -k2,2g > $OUTDIR/$PREFIX.dmr.xls\n"; + system("$metilene -M $M -m $m -d $d -t $t -f $f -B $B -a $groupA -b $groupB -X $X -Y $Y -v $v $OUTDIR/$PREFIX.input 2> $OUTDIR/$PREFIX.metilene.log |sort -k1,1 -k2,2g > $OUTDIR/$PREFIX.dmr.xls"); + } + else{ + die "[err]: if -f is 2, you must default the bed-file for mode 2 containing pre-defined regions\n"; + } +} + +##### filtering +if($p){ + #use p-value to filter + print STDERR "[run]: DMR filtering by p-value..\n [command]: filtering -> perl $perl03 -q $OUTDIR/$PREFIX.dmr.xls -o $OUTDIR/$PREFIX.dmr.filter -p $p -c $c -d $r -l $l -a $groupA -b $groupB -Rscript $Rscript\n"; + system("perl $perl03 -i $OUTDIR/$PREFIX.dmr.xls -o $OUTDIR/$PREFIX.dmr.filter -p $p -c $c -d $r -l $l -a $groupA -b $groupB -Rscript $Rscript"); + system("mv $OUTDIR/$PREFIX.dmr.filter\_pval.$p.out $OUTDIR/$PREFIX.dmr.filter\_pval.$p.out.xls"); + system("sed -i \'1i\#Chr\tStart\tEnd\tP-value\tDifference\t\#CpGs\t$groupA\t$groupB\' $OUTDIR\/$PREFIX.dmr.filter\_pval.$p.out.xls"); +}else{ + #use q-value to filter + print STDERR "[run]: DMR filtering by q-value..\n [command]: filtering -> perl $perl02 -q $OUTDIR/$PREFIX.dmr.xls -o $OUTDIR/$PREFIX.dmr.filter -q $q -c $c -d $r -l $l -a $groupA -b $groupB -Rscript $Rscript\n"; + system("perl $perl02 -i $OUTDIR/$PREFIX.dmr.xls -o $OUTDIR/$PREFIX.dmr.filter -q $q -c $c -d $r -l $l -a $groupA -b $groupB -Rscript $Rscript"); + system("mv $OUTDIR/$PREFIX.dmr.filter\_qval.$q.out $OUTDIR/$PREFIX.dmr.filter\_qval.$q.out.xls"); + system("sed -i \'1i\#Chr\tStart\tEnd\tQ-value\tDifference\t\#CpGs\t$groupA\t$groupB\' $OUTDIR\/$PREFIX.dmr.filter\_qval.$q.out.xls"); +} + +system("sed -i \'1i\#Chr\tStart\tEnd\tQ-value\tDifference\t\#CpGs\tp\(MWU\)\tp\(2D KS\)\t$groupA\t$groupB\' $OUTDIR\/$PREFIX.dmr.xls"); + +print STDERR "[ok] DMR detection is done..\n"; diff --git a/script/p02.DMR-annotator.pl b/script/p02.DMR-annotator.pl new file mode 100644 index 0000000..ed7dd05 --- /dev/null +++ b/script/p02.DMR-annotator.pl @@ -0,0 +1,120 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use FindBin qw($Bin $Script); +use PerlIO::gzip; +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 + + This perl is used to annotate the DMR into genomic elements (including promoter, CGI or any one you gave). + -DMR DMR file (bed format); + chr1 start end ... + -annot list of annotation infomation file (bed format); + #example for list file: + promoter path/promoter.bed + CGI path/CGI.bed + 5utr path/5utr.bed + exon path/exon.bed + #format of promoter.bed : + chr1 start end ID annotation + + -help +example : perl $0 -DMR in.dmr -annot annot.list > in.dmr.annt.txt +author: luhanlin\@genomics.org.cn +date: +USAGE + +my ($dmrfile, $annotlist, $help); + +GetOptions ( + "DMR=s" => \$dmrfile, + "annot=s" => \$annotlist, + "help" => \$help, +); + +die $usage if $help; +die $usage unless $dmrfile && $annotlist; + +my $getoverlap = "$Bin/disposeOverlap.pl"; + +# +print STDERR "|-- reading DMR file: $dmrfile ..\n"; +my $tmp = "tmp.dmr.txt"; +# my @head = (); +open DMR, $dmrfile or die $!; +open OUT, "> $tmp" or die $!; +while(){ + chomp; + if(/^#/){ + next; + } + else{ + my $dmrID = "DMR$."; + print OUT "$dmrID\t$_\n"; + # @head = split(/\t/, $_); + } +} +close DMR; +close OUT; + +# +my %res = (); +my @list = (); +print STDERR "|-- reading annotation file list ..\n"; +open IN, $annotlist or die $!; +while(){ + chomp; + my @aa = split(/\t/, $_); + my $name = $aa[0]; + push @list, $name; + my $file = $aa[1]; + my $output = "tmp.$name.overlap"; + + print STDERR "|-- overlap with ELEMENT: $name, \n\tfile: $file, \n\toutput: $output ..\n"; + system("$getoverlap --i1 $tmp --f1 1-0-2-3 --i2 $file --f2 0-4-1-2 --OL 0.5-small --mN 1 --E O > $output"); + + print STDERR "|-- parse output: $output ..\n"; + open IB, "$output" or die $!; + while(){ + chomp; + next if $.==1; + my @bb = split; + my $dmrid = $bb[1]; + my %u = (); + foreach my $ele (@bb[8..$#bb]){ + my $id = (split(/:/, $ele))[0]; + $u{$id} += 1; + } + my $annot_id = join(",", keys %u); + $res{$dmrid}{$name} = $annot_id; + } + close IB; + + print STDERR "|-- remove output: $output ..\n"; + system("rm -f $output"); + +} +close IN; + +# +print STDERR "|-- write the result ..\n"; +open IA, $tmp or die $!; +print join("\t", "chr", "start", "end", "q-value", "methyl-diff", "CpGs", "methyl_a", "methyl_b", @list), "\n"; +while(){ + chomp; + my @aa = split(/\t/, $_); + my $dmrid = $aa[0]; + my @out = (); + foreach my $name (@list){ + my $o = $res{$dmrid}{$name} ? $res{$dmrid}{$name} : "--"; + push @out, $o; + } + print join("\t", @aa[1..$#aa], @out), "\n"; +} +close IA; + +system("rm -f $tmp"); +print STDERR "|-- program is done .. \n\n\n"; + +# END diff --git a/script/p03.DMR-ploter.pl b/script/p03.DMR-ploter.pl new file mode 100644 index 0000000..c728d05 --- /dev/null +++ b/script/p03.DMR-ploter.pl @@ -0,0 +1,406 @@ +#!/usr/bin/perl +use strict; +use warnings; +use PerlIO::gzip; +use Getopt::Long; + +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 + + plot methylation levels of different samples or groups for given regions. + --I input file containing DMRs;( or output file of p01.DMR-detector.pl ) + ### format + chr1 1 400 ... + chr1 100 500 ... + ... + --E [int] Extend <1000> bp for each region. [1000] + --C matrix of CpG methylation (or input file of p01.DMR-detector.pl) + ### format; + chr pos g1 g1 g2 g2 + chr1 10497 0.950 0.941 0.783 0.930 + chr1 10499 0.000 0.000 0.000 0.000 + chr1 10500 0.000 0.000 0.000 0.000 + ... + --F single sample list or group list you want to plot. + the number means the sample column number(perl colum number, 0 is the firt column) in matrix given -C; + ### file format: (one group per line) + #name list + g1 2,3 + g2 4,5 + --R [str] reference.fa, which will be used to calculate the CG density. + --G [str] bed format file containing genic elements you want to plot;The 3th colum is the gene name; + #format + chr1 1 100 DDX11L1 id + + chr5 29370 30000 WASH7P id - + --O [str] dir of output + --Rscript [str] Rscript program; +author: luhanlin +date: +USAGE + +my $file_dmr; +my $file_matrix; +my $file_format; +my $file_fasta; +my $file_gene; +my $dir_output; +my $extend; +my $Rscript; +my $help; + +GetOptions ( + "I=s" => \$file_dmr, + "C=s" => \$file_matrix, + "F=s" => \$file_format, + "R:s" => \$file_fasta, + "E:i" => \$extend, + "G:s" => \$file_gene, + "O:s" => \$dir_output, + "Rscript:s" => \$Rscript, + "help|?|h" => \$help, +); + +unless( $file_dmr && $file_matrix && $file_format){ + print $usage; + exit 1; +} +if($help){ + print $usage; + exit 0; +} +die $usage unless $Rscript; + +### parameters +$extend ||= 1000; +$dir_output ||= "."; +mkdir $dir_output unless -d $dir_output; + +### read fasta file +print STDERR "|-- read fasta file: $file_fasta ..\n"; +open FA, $file_fasta or die $!; +my %fa; +my $ch; +while () { + chomp; + if (/>(\S+)/){ + $ch = $1; + } + else{ + $fa{$ch} .= $_; + } +} +close FA; +print STDERR " --| read fasta file is done ..\n"; + +### read DMR regions +print STDERR "|-- loading the dmr regions: $file_dmr ..\n"; +my %pos2reg; +my %region; +open DMR, $file_dmr or die $!; +while(){ + chomp; + next if(/^#/); + my @arr = split; + my ($chr, $st, $ed) = @arr[0..2]; + my $n = sprintf("%06d", $.-1); + my $id = "RG$n"; + my $ext_st = $st - $extend > 0 ? $st - $extend : 1; + my $ext_ed = $ed + $extend; + my $cpg = cpg(\%fa, $chr, $ext_st, $ext_ed); + $region{$id}{"chr"} = $chr; + $region{$id}{"st"} = $st; + $region{$id}{"exst"} = $ext_st; + $region{$id}{"ed"} = $ed; + $region{$id}{"exed"} = $ext_ed; + $region{$id}{"cpg"} = $cpg; + foreach my $i ($ext_st..$ext_ed){ + my $pos = "$chr\t$i"; + $pos2reg{$pos}{$id} = 1; + } +} +close DMR; +print STDERR " --| loading dmr regions is done ..\n"; + +### read format file +print STDERR "|-- loading the format list: $file_format ..\n"; +my %g; +open IN, $file_format or die $!; +while(){ + chomp; + my @arr = split; + my $id = $arr[0]; + my $l = $arr[1]; + my @list = split(/,/, $l); + push @{$g{$id}}, @list; +} +close IN; +print STDERR " --| loading the format list is done ..\n"; + +### read the matrix file +print STDERR "|-- loading methyl matrix: $file_matrix ..\n"; +my %dat; +if($file_matrix=~/\.gz/){ + open MAT, "<:gzip", $file_matrix or die $!; +} +else{ + open MAT, $file_matrix or die $!; +} +while(){ + next if $.==1; + chomp; + my @a = split; + my ($chr, $s) = ($a[0], $a[1]); + my $pos = "$chr\t$s"; + if ( exists $pos2reg{$pos} ){ + foreach my $id (keys %{$pos2reg{$pos}}){ + foreach my $gp (keys %g){ + my @b = @{$g{$gp}}; + foreach my $lev (@a[@b]){ + $dat{$id}{$gp}{$pos} .= "$pos\t$lev\n"; + } + } + } + } +} +close MAT; +print STDERR " --| loading methyl matrix is done ..\n"; + +### read the gene structure file +print STDERR "|-- loading gene structure: $file_gene ..\n"; +my %gene; +open BED, $file_gene or die $!; +while(){ + chomp; + my @arr = split; + my ($chr, $st, $ed, $id, $strand) = @arr[0,1,2,4,5]; + foreach my $i ($st..$ed){ + my $pos = "$chr\t$i"; + if(exists $pos2reg{$pos} ){ + foreach my $id (keys %{$pos2reg{$pos}}){ + my $ge = join("\t", @arr[0,1,2,4,5]); + $gene{$id}{$ge} = 1; + } + } + } +} +close BED; +print STDERR " --| loading gene structure is done ..\n"; + + +print STDERR "|-- start to plot ..\n"; +foreach my $id (sort keys %region){ + my $chr = $region{$id}{"chr"}; + my $st = $region{$id}{"st"}; + my $ed = $region{$id}{"ed"}; + my $ext_st = $region{$id}{"exst"}; + my $ext_ed = $region{$id}{"exed"}; + + my $cpgfile = "$dir_output/$id.cpgs.txt"; + print $cpgfile."\n"; + open OA, ">$cpgfile" or die $!; + print OA $region{$id}{"cpg"}; + close OA; + + my $genfile = "$dir_output/$id.gene.txt"; + open OB, ">$genfile" or die $!; + print OB "$chr\t-1\t-1\tNA\t+\n"; + foreach my $gene (keys %{$gene{$id}}){ + print OB "$gene\n"; + } + close OB; + + my @levfiles = (); + my @gpname = (); + foreach my $gp (keys %{$dat{$id}}){ + my $levfile = "$dir_output/$id.level.$gp.txt"; + open OC, ">$levfile" or die $!; + foreach my $pos (keys %{$dat{$id}{$gp}}){ + print OC $dat{$id}{$gp}{$pos}; + } + close OC; + push @levfiles, $levfile; + push @gpname, $gp; + } + + plot($chr, $id, $st, $ed, $ext_st, $ext_ed, $cpgfile, $genfile, \@levfiles, \@gpname, $dir_output); + # system("rm -f $dir_output/$id.*.txt"); + +} +print STDERR " --| plotting is done ..\n\n\n"; + + +######### sub function + +sub cpg{ + my ($h, $chr, $st, $ed) = @_; + my $seq = substr($$h{$chr}, $st-1, $ed-$st+1); + my $out = (); + while($seq =~m/cg/ig){ + my $base = $1; + my $pos = pos($seq) + $st; + $out .= "$chr\t$pos\t1\n"; + $pos = $pos + 1; + $out .= "$chr\t$pos\t1\n"; + } + return $out; +} + + +########### sub function + +sub plot{ + my ($chr, $id, $st, $ed, $ext_st, $ext_ed, $cgf, $genf, $levfs, $names, $dir) = @_; + my $args = join(" ", $cgf, $genf, @$levfs); + my $legendname = join("\",\"", @$names); + $legendname = '"' . $legendname . '"'; + my $c = @$names + 2; + my $Rline=<<"RL"; +######################### +args <- commandArgs(TRUE) +cgf <- read.table(args[1]) +tssf <- read.table(args[2]) + +start <- $ext_st +end <- $ext_ed +dmr_s <- $st +dmr_e <- $ed + + +scol <- c( rgb(0,0,0,1), rgb(1,1,1,1), + rgb(189/255,28/255,44/255,1), rgb(32/255,60/255,153/255,1), + rgb(236/255,99/255,34/255,1), rgb(11/255,161/255,75/255,1), + rgb(127/255,62/255,152/255,1), rgb(252/255,220/255,21/255,1)) + +pcol <- c( rgb(0,0,0,0.7), rgb(1,1,1,0.7), + rgb(189/255,28/255,44/255,0.7), rgb(32/255,60/255,153/255,0.7), + rgb(236/255,99/255,34/255,0.7), rgb(11/255,161/255,75/255,0.7), + rgb(127/255,62/255,152/255,0.7), rgb(252/255,220/255,21/255,0.7)) + +######## +# plot # +######## +pdf( file="$dir/$id.plot.pdf", width=12, height=8) + +nf<-layout(matrix(c(1,2,3), ,1,byrow=TRUE), c(2800), c(250,900,350), TRUE) +#layout.show(nf) + +######## 1 func +sline <- function(Ma){ + a <- subset(Ma,!is.na(Ma[,2])) + a <- a[order(a[,1]),] + c <- unique(a[,1]) + c <- cbind(c,0) + for(k in 1:nrow(c)){ + c[k,2] <- mean( a[which(a[,1]==c[k,1]),2] ) + } + a <- c + r <- nrow(a) + b <- matrix(rep(0,(a[r,1]-a[1,1]+1)*2), , 2, byrow=TRUE) + num <- 1 + b[num,1] <- a[1,1] + b[num,2] <- a[1,2] + for( i in 1:(nrow(a)-1) ){ + for (j in (a[i,1]+1):a[i+1,1]){ + num <- num + 1; + b[num,1] <- j + if(j==a[i+1,1]){ + b[num,2]<-a[i+1,2] + } + else{ + if(a[i+1,1]==a[i,1]){ + b[num,2] <- a[i,1] + } + else{ + b[num,2] <- a[i,2] + ( (a[i+1,2]-a[i,2]) * (j-a[i,1]) / (a[i+1,1]-a[i,1])) + } + } + } + } + b +} + + +######## 2. gene structure +par(mar=c(0,7,4,1), las=1, cex.lab=1.5, cex.axis=1.5) +plot(0, + type="n", + ylim=c(-0.5,1), + xlim=c(start, end), + col=1, + xaxt="n", + yaxt="n", + bty="o", + xlab="", + ylab="") +mtext("$chr", side=2, cex=1.5, line=2) +segments(start, 0.4, end, 0.4, col=1) +for (k in 1:nrow(tssf)){ + text(tssf[k,3], 0, tssf[k,4]) + if(tssf[k,5] == "+"){ + rect(tssf[k,2], 0.2, 0.05*(end-start)+tssf[k,2], 0.6, col=rgb(0,0,0,0.5), border=NA) + segments(tssf[k,2], 0.2, tssf[k,2], 0.8, col=1, lwd=1) + arrows(tssf[k,2], 0.8, 0.07*(end-start)+tssf[k,2], 0.8, angle=15, code=2, length=0.2, lwd=1, col="red") + }else{ + rect(tssf[k,3], 0.2, tssf[k,3]-(0.05*(end-start)), 0.6, col=rgb(0,0,0,0.5), border=NA) + segments(tssf[k,3], 0.2, tssf[k,3], 0.8, col=1, lwd=1) + arrows(tssf[k,3], 0.8, tssf[k,3]-(0.07*(end-start)), 0.8, angle=15, code=2, length=0.2, lwd=1, col="red") + } +} + + +######## 3. plot methy +par(mar=c(0,7,0,1),las=1,cex.lab=2, cex.axis=1.5) +plot(0, + type="n", + xlim=c(start, end), + xaxt="n", + ylim=c(0,1), + ylab="CG\nMethyl Level") + +legend("topleft", + legend=c($legendname), + col=scol[3:$c], + pch=19, + bty="n", + cex=1.5) + + +for (i in 3:length(args)){ + dat <- read.table(args[i]) + points(dat[,2], dat[,3], pch=20, cex=1, col=pcol[i]) + la <- as.matrix(cbind(dat[,2],dat[,3])) + la <- sline(la) + a <- smooth.spline(la[,2], df=25) + points(a\$x+la[1,1]-1, a\$y, type="l", col=pcol[i], lwd=2) +} + +#abline(h=0, lwd=2) +rect(dmr_s, 0, dmr_e, 1, col=rgb(255/255,255/255,0,0.3), border=NA) + +######## 4. GCdensity +par(mar=c(5,7,0,1), las=1, cex.lab=2, cex.axis=1.5) +plot(0, + type="n", + xlim=c(start, end), + ylim=c(-0.05,1), + ylab="CG\ndensity", + xlab="") +#axis(2,at=c(-15,0,15),lab=c(15,0,15)) +#abline(h=0) +points(cgf[,2], rep(-0.05,nrow(cgf)), col=rgb(0.48,0.48,0.48,0.5), type="h", lwd=1, lend=2) + +par(new=T,mar=c(5,7,0,1)) +a <- density(cgf[,2], width=100) +plot(a\$x,a\$y, type="l", lwd=1, xlab="", ylab="", xaxt="n", yaxt="n") +######## +dev.off() +######## + +RL + open RL, ">$dir/$id.plot.R" or die $!; + print RL $Rline; + close RL; + system("$Rscript $dir/$id.plot.R $args"); +} +### diff --git a/script/parse_NCBI_gff3.sh b/script/parse_NCBI_gff3.sh new file mode 100644 index 0000000..c955035 --- /dev/null +++ b/script/parse_NCBI_gff3.sh @@ -0,0 +1,97 @@ +#!/bin/sh +if [ $# != 2 ] +then + echo "----------------------------------------------------------" + echo "Usage: $0 " + echo "Use this perl to get following files from NCBI gff3:" + echo "1. exon.bed" + echo "2. cds.bed" + echo "3. intron.bed" + echo "4. intergenic.bed" + echo "5. upstream.bed" + echo "6. downstream.bed" + echo "7. genebody.bed" + echo "7. promoter.bed" + echo "----------------------------------------------------------" + exit +fi +ref=$1 +prefix=$2 + + +#####1 genebody +echo "|-- $prefix.genebody.bed begin...(TSS~TTS)" +less -S $ref | perl -F"\t" -alne 'if($F[2]=~/gene/){ @a=split(/;/,$F[8]); %h=(); foreach(@a){ @b=split(/=/,$_); $h{$b[0]}=$b[1];} print join "\t", @F[0,3,4], $h{ID}, "$h{Name}|$h{gene_biotype}", $F[6]}' > $prefix.genebody.bed +echo "--| $prefix.genebody.bed is done..." + +##### promoter: -2000bp ~ +500 bp +echo "|-- $prefix.promoter.bed begin...(-2000bp ~ +500 bp)" +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ $start = $F[1]-2000 > 0 ? $F[1]-2000 : 1;$end = $F[1]+500 > $F[2] ? $F[2] : $F[1]+500;print join "\t",$F[0], $start, $end, @F[3,4,5] }else{$end =$F[2]-500 > $F[1] ? $F[2]-500 : $F[1]; print join "\t",$F[0], $end,$F[2]+2000,@F[3,4,5]}' > $prefix.promoter.bed +echo "--| $prefix.promoter.bed is done..." + +##### genebody2 :+500bp ~ TTS +echo "|-- $prefix.genebody2.bed begin...(+500bp ~ TTS)" +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ if($F[1]+500 >= $f[2]){ next}else{print join "\t",$F[0], $F[1]+500,$F[2], @F[3,4,5]}}else{if($F[2]-500 <= $f[1]){ next}else{print join "\t",$F[0], $F[1],$F[2]-500, @F[3,4,5]}}' > $prefix.genebody2.bed +echo "--| $prefix.genebody2.bed is done..." + +####4 up2k +echo "|-- $prefix.up2k.bed begin..." +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ $start = $F[1]-2000 > 0 ? $F[1]-2000 : 1; print join "\t",$F[0], $start, $F[1]-1, @F[3,4,5] }else{print join "\t",$F[0], $F[2]+1, $F[2]+2000,@F[3,4,5]}' > $prefix.upstream2k.bed +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ $start = $F[1]-5000 > 0 ? $F[1]-5000 : 1; print join "\t",$F[0], $start, $F[1]-1, @F[3,4,5] }else{print join "\t",$F[0], $F[2]+1, $F[2]+5000,@F[3,4,5]}' > $prefix.upstream5k.bed +echo "--| $prefix.up2k.bed is done..." + +###5 down2k +echo "|-- $prefix.down2k.bed begin..." +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ print join "\t", $F[0], $F[2]+1, $F[2]+2000, @F[3,4,5]}else{ $start = $F[1]-2000 > 0 ? $F[1]-2000 : 1; print join "\t",$F[0], $start, $F[1]-1,@F[3,4,5]}' > $prefix.downstream2k.bed +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ print join "\t", $F[0], $F[2]+1, $F[2]+5000, @F[3,4,5]}else{ $start = $F[1]-5000 > 0 ? $F[1]-5000 : 1; print join "\t",$F[0], $start, $F[1]-1,@F[3,4,5]}' > $prefix.downstream5k.bed +echo "--| $prefix.down2k.bed is done..." + +####6 exon +echo "|-- $prefix.exon.bed begin..." +less -S $ref | perl -F"\t" -alne 'if($F[2]=~/exon/){ @a=split(/;/,$F[8]); %h=(); foreach(@a){ @b=split(/=/,$_); $h{$b[0]}=$b[1];} print join "\t", @F[0,3,4], $h{ID}, "$h{gene}|$h{transcript_id}|$h{gbkey}", $F[6]}' > $prefix.exon.bed +echo "--| $prefix.exon.bed is done..." + +####7 cds +echo "|-- $prefix.cds.bed begin..." +less -S $ref | perl -F"\t" -alne 'if($F[2]=~/CDS/){ @a=split(/;/,$F[8]); %h=(); foreach(@a){ @b=split(/=/,$_); $h{$b[0]}=$b[1];} print join "\t", @F[0,3,4], $h{ID}, "$h{gene}", $F[6]}' > $prefix.cds.bed +echo "--| $prefix.cds.bed is done..." + +####8 intron +echo "|-- $prefix.intron.bed begin..." +less -S $prefix.exon.bed | perl -alne ' @a=split(/\|/, $F[4]); if($a[1]){ $g{$a[1]}="$F[4]\t$F[5]"; $c{$a[1]}=$F[0]; push @{$h{$a[1]}}, $F[1]; push @{$h{$a[1]}}, $F[2];} +END{ + foreach $i (keys %c){ + $chr = $c{$i}; + $gene = $g{$i}; + if(@{$h{$i}}>2){ + @pos = @{$h{$i}}; + @pos = sort {$a<=>$b} @pos; + for($i=1; $i<$#pos; $i+=2){ + print join "\t", $chr, $pos[$i] + 1, $pos[$i+1] - 1, 0, $gene; + } + } + } +}' | sort -k1,1 -k2,2g | perl -alne 'print join "\t",@F[0,1,2], "intron_$.",@F[4,5]' > $prefix.intron.bed +echo "--| $prefix.intron.bed is done..." + +####9 intergenic +#没有考虑染色体两端的片段,有待改进 +echo "|-- $prefix.intergenic.bed begin..." +sort -k1,1 -k2,2n -u $prefix.genebody.bed | perl -alne ' $g="$F[3]|$F[4]|$F[5]"; $end=$F[2]; if($F[0] eq $tem_chr){ print $F[0],"\t", $tem_end+1, "\t", $F[1]-1, "\t", 0, "\t", "$tem_gene;$g", "\t", "+"; } $tem_gene=$g; $tem_end=$end; $tem_chr=$F[0];' | perl -alne 'print if $F[2]-$F[1] > 200 ' | sort -k1,1 -k2,2g | perl -alne 'print join "\t",@F[0,1,2],"intergenic_$.",@F[4,5]' > $prefix.intergenic.bed +echo "--| $prefix.intergenic.bed is done..." + +###### sort -u +echo "|-- sort ..." +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.genebody.bed $prefix.genebody.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.promoter.bed $prefix.promoter.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.genebody2.bed $prefix.genebody2.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.upstream2k.bed $prefix.upstream2k.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.upstream5k.bed $prefix.upstream5k.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.downstream2k.bed $prefix.downstream2k.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.downstream5k.bed $prefix.downstream5k.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.exon.bed $prefix.exon.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.cds.bed $prefix.cds.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.intron.bed $prefix.intron.bed +sort -k1,1 -k2,2g -k3,3g -u -T . -o $prefix.intergenic.bed $prefix.intergenic.bed +echo "--| sort done ..." +###### diff --git a/script/parse_UCSC_refGene.sh b/script/parse_UCSC_refGene.sh new file mode 100644 index 0000000..b9d0858 --- /dev/null +++ b/script/parse_UCSC_refGene.sh @@ -0,0 +1,240 @@ +#!/bin/sh +if [ $# != 4 ] +then + echo "----------------------------------------------------------" + echo "Usage: $0 " + echo "Use this perl to get following files from UCSC-refGene.txt:" + echo "1. exon.bed" + echo "2. cds.bed" + echo "3. intron.bed" + echo "4. intergenic.bed" + echo "5. 5utr.bed" + echo "6. 3utr.bed" + echo "7. up2k.bed" + echo "8. down2k.bed" + echo "9. TSS.bed" + echo "10. promoter.bed" + echo "11. genebody.bed" + echo "12. up1k.bed" + echo "13. down1k.bed" + echo "14. up5k.bed" + echo "15. down5k.bed" + echo "----------------------------------------------------------" + exit +########################## format of refGene.txt.gz from UCSC (tab splited)###################### +#bin transcriptName chrom strand transcriptStart transcriptEnd cdsStart cdsEnd exonCount exonStarts exonEnds score geneName cdsStartStat cdsEndStat exonFrames +#944 NM_001357077 chr1 + 47140242 47141166 47140242 47140776 2 47140242,47141151, 47140863,47141166, 0 LOC111258524 cmpl cmpl 0,-1, + +#================= perl column number ============ +#0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 + +########################## output bed file format ################### +#chrom Start End transcriptName geneName strand +#chr1 47140242 47141166 NM_001357077 LOC111258524 + + +fi +genome=$1 +ref=$2 +outdir=$3 +prefix=$4 + +##### genome prepare +echo "|-- $genome preparing..." +samtools faidx $genome +cut -f-2 $genome.fai > $genome.length +#slopBed -i $name.cgi.bed -g $ref.length -b 2000 | mergeBed | subtractBed -a - -b $name.cgi.bed > $name.cgi_shore.bed + +#####0 TSS +echo "|-- $prefix.tss.bed begin..." +less -S $ref | perl -alne 'if($F[4]==0){next};if($F[3] eq "+"){ print join "\t", @F[2,4], @F[4,1,12,3]; }else{print join "\t", @F[2,5], @F[5,1,12,3];}' | sort -k1,1 -k2,2n > $outdir/$prefix.tss.bed +echo "--| $prefix.tss.bed is done..." + +##### promoter (-2k~+500) +echo "|-- $prefix.promoter.bed begin..." +slopBed -i $outdir/$prefix.tss.bed -g $genome.length -l 2000 -r 500 -s > $outdir/$prefix.promoter.bed +echo "|-- $prefix.promoter.bed is done..." + +#####1 genebody +echo "|-- $prefix.genebody.bed begin..." +less -S $ref | perl -alne 'print join "\t", @F[2,4,5,1,12,3];' | sort -k1,1 -k2,2n > $outdir/$prefix.genebody.bed +echo "--| $prefix.genebody.bed is done..." + +#####2 intergenic +echo "|-- $prefix.intergenic.bed begin..." +sort -k1,1 -k2,2n -u $prefix.genebody.bed | perl -alne ' $g="$F[3];$F[4];$F[5]"; $end=$F[2]; if($F[0] eq $tem_chr){ print $F[0],"\t", $tem_end+1,"\t", $F[1]-1,"\t", $tem_gene,"\t", $g; } $tem_gene=$g; $tem_end=$end; $tem_chr=$F[0];' | perl -alne 'print if $F[2]-$F[1]>1000' | sort -k1,1 -k2,2n > $outdir/$prefix.intergenic.bed +echo "--| $prefix.intergenic.bed is done..." + +####3 up2k +echo "|-- $prefix.up2k.bed begin..." +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ print join "\t",$F[0],$F[1]-2000, $F[1]-1, @F[3,4,5] }else{print join "\t",$F[0], $F[2]+1, $F[2]+2000,@F[3,4,5]}' > $outdir/$prefix.up2k.bed +echo "--| $prefix.up2k.bed is done..." + +####3 up1k +echo "|-- $prefix.up1k.bed begin..." +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ print join "\t",$F[0],$F[1]-1000, $F[1]-1, @F[3,4,5] }else{print join "\t",$F[0], $F[2]+1, $F[2]+1000,@F[3,4,5]}' > $outdir/$prefix.up1k.bed +echo "--| $prefix.up1k.bed is done..." + +###4 down2k +echo "|-- $prefix.down2k.bed begin..." +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ print join "\t",$F[0],$F[2]+1, $F[2]+2000, @F[3,4,5] }else{print join "\t",$F[0], $F[1]-2000, $F[1]-1,@F[3,4,5]}' > $outdir/$prefix.down2k.bed +echo "--| $prefix.down2k.bed is done..." + +###4 down1k +echo "|-- $prefix.down1k.bed begin..." +cat $prefix.genebody.bed | perl -alne 'if($F[5] eq "+"){ print join "\t",$F[0],$F[2]+1, $F[2]+1000, @F[3,4,5] }else{print join "\t",$F[0], $F[1]-1000, $F[1]-1,@F[3,4,5]}' > $outdir/$prefix.down1k.bed +echo "--| $prefix.down1k.bed is done..." + +####5 exon +echo "|-- $prefix.exon.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); + @b=split(/,/,$F[10]); + if($F[3] eq "+"){ + foreach $i (0..$#a){ + if($F[1]=~/;/){@f=split(/;/,$F[1]);$F[1]=$f[0]}; + print join "\t", $F[2],$a[$i],$b[$i],$F[1],$F[12],$F[3]; + } + } + else{ + foreach $i (0..$#a){ + if($F[1]=~/;/){@f=split(/;/,$F[1]);$F[1]=$f[0]}; + print join "\t", $F[2],$a[$i],$b[$i],$F[1],$F[12],$F[3]; + } + } ' | sort -k1,1 -k2,2n > $outdir/$prefix.exon.bed +echo "--| $prefix.exon.bed is done..." + +####5.1 first exon +echo "|-- $prefix.exon1.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); + @b=split(/,/,$F[10]); + if($F[3] eq "+"){ + foreach $i (0..$#a){ + if($i==0){print join "\t", $F[2],$a[$i],$b[$i],$F[1],$F[12],$F[3],exon1;} + } + } + else{ + foreach $i (0..$#a){ + if($i==$#a){ + print join "\t", $F[2],$a[$i],$b[$i],$F[1],$F[12],$F[3],exon1; + } + } + } ' | sort -k1,1 -k2,2n > $outdir/$prefix.exon1.bed + +#####6 intron +echo "|-- $prefix.intron.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); + @b=split(/,/,$F[10]); + next if @a==1; + if($F[3] eq "+"){ + foreach $i (0..($#b-1)){ + next if ($b[$i]+1) > ($a[$i+1]-1); + if($i==0){ + print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3]; + #print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3],intron1; + } + else{ + print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3]; + } + } + } + else{ + foreach $i (0..($#b-1)){ + next if ($b[$i]+1) > ($a[$i+1]-1); + if($i==($#b-1)){ + #print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3],intron1; + print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3]; + } + else{ + print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3]; + } + } + }' | sort -k1,1 -k2,2n > $outdir/$prefix.intron.bed +echo "--| $prefix.intron.bed is done..." + +#####6.1 first intron +echo "|-- $prefix.intron1.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); +@b=split(/,/,$F[10]); +next if @a==1; +if($F[3] eq "+"){ + foreach $i (0..($#b-1)){ + next if ($b[$i]+1) > ($a[$i+1]-1); + if($i==0){ + #print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3],intron1; + } + } +}else{ + foreach $i (0..($#b-1)){ + next if ($b[$i]+1) > ($a[$i+1]-1); + if($i==($#b-1)){ + print join "\t", $F[2],$b[$i]+1,$a[$i+1]-1,$F[1],$F[12],$F[3],intron1; + } + } + }' | sort -k1,1 -k2,2n > $outdir/$prefix.intron1.bed + +####7 cds +echo "|-- $prefix.cds.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); + @b=split(/,/,$F[10]); + $st = $F[6]; + $end = $F[7]; + foreach $i (0..$#a){ + $s = $a[$i]; + $e = $b[$i]; + next if $e < $st || $s > $end; + @arr = sort { $a<=>$b }($st, $end, $s, $e); + print join "\t", $F[2],$arr[1],$arr[2],$F[1],$F[12],$F[3]; + } ' | sort -k1,1 -k2,2n > $outdir/$prefix.cds.bed +echo "--| $prefix.cds.bed is done..." + +####8 5utr +echo "|-- $prefix.5utr.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); + @b=split(/,/,$F[10]); + $st = $F[6]; + $end = $F[7]; + if($F[3] eq "+"){ + foreach $i (0..$#a){ + $s = $a[$i]; + $e = $b[$i]; + if( $st>=$s && $st <=$e){ + print join "\t",$F[2], $s, $st, $F[1], $F[12], $F[3]; + } + } + } + else{ + foreach $i (0..$#a){ + $s = $a[$i]; + $e = $b[$i]; + if( $end>=$s && $end <=$e){ + print join "\t", $F[2], $end, $e, $F[1], $F[12], $F[3]; + } + } + }' | perl -alne 'print if $F[2]-$F[1]>1' | sort -k1,1 -k2,2n > $outdir/$prefix.5utr.bed +echo "--| $prefix.5utr.bed is done..." + +####9 3utr +echo "|-- $prefix.3utr.bed begin..." +less -S $ref | perl -F"\t" -alne '@a=split(/,/,$F[9]); + @b=split(/,/,$F[10]); + $st = $F[6]; + $end = $F[7]; + if($F[3] eq "+"){ + foreach $i (0..$#a){ + $s = $a[$i]; + $e = $b[$i]; + if( $end>=$s && $end <=$e){ + print join "\t",$F[2], $end, $e, $F[1], $F[12], $F[3]; + } + } + } + else{ + foreach $i (0..$#a){ + $s = $a[$i]; + $e = $b[$i]; + if( $st>=$s && $st <=$e){ + print join "\t", $F[2], $s, $st, $F[1], $F[12], $F[3]; + } + } + }' | perl -alne 'print if $F[2]-$F[1]>1' | sort -k1,1 -k2,2n > $outdir/$prefix.3utr.bed +echo "--| $prefix.3utr.bed is done..." +#### diff --git a/script/parse_UCSC_repeats.sh b/script/parse_UCSC_repeats.sh new file mode 100644 index 0000000..2236def --- /dev/null +++ b/script/parse_UCSC_repeats.sh @@ -0,0 +1,12 @@ +file=$1 #RepeatMaser.txt.gz +outdir=$2 + +for type in DNA LINE LTR Retroposon RNA Satellite SINE Low_complexity Simple_repeat + do + echo -e "Extracting repeat elements from genome: $type ..." + if [ $type == "RNA" ];then + zcat $file |perl -alne 'if($F[11]=~/RNA$/){print join "\t",@F[5..7],@F[10..12],$F[9]}' > $outdir/Repeats.$type.bed + else + zcat $file |perl -alne 'if($F[11] eq '$type'){print join "\t",@F[5..7],@F[10..12],$F[9]}' > $outdir/Repeats.$type.bed + fi + done diff --git a/script/plot_DMR.annotate.R b/script/plot_DMR.annotate.R new file mode 100644 index 0000000..0b08406 --- /dev/null +++ b/script/plot_DMR.annotate.R @@ -0,0 +1,89 @@ +### This is script for pieplot of DMR annotation results #### +### Author: zhangdu +### date: 2018-08-14 +### version:v1.0 + +arg <- commandArgs(T) +if(length(arg) < 2){ + cat("Argument: **.annotate.xls DMR_annotate.pdf\n") + quit('no') +} +library(dplyr) +library(ggplot2) +tab<-read.table(arg[1],header=T) +tab1<-tab[,9:15] +total<-nrow(tab1) + +x0 <- apply(tab1,1,function(i) sum(i=="--")==length(i)) +tab2<-tab1[x0,] +unanno<-nrow(tab2) + +## stat the annotation results: +aa<-data.frame(promoter=nrow(filter(tab1,!promoter %in% "--")), + up2k=nrow(filter(tab1,!up2k %in% "--")), + down2k=nrow(filter(tab1,!down2k %in% "--")), + genebody=nrow(filter(tab1,!genebody %in% "--")), + exon=nrow(filter(tab1,!exon %in% "--")), + intron=nrow(filter(tab1,!intron %in% "--")), + cgi=nrow(filter(tab1,!cgi %in% "--")), + other_region=unanno + ) +rownames(aa)<-"Number" +aa1<-t(aa) +aa1<-as.data.frame(aa1) +aa1$Element<-colnames(aa) +aa1 = aa1[order(aa1$Number, decreasing = TRUE),] +aa1$Number<-as.numeric(aa1$Number) +aa1$Percent<-paste(round(aa1$Number / total * 100, 2), "%", sep = "") +aa1 <- select(aa1, Element, everything()) +write.table(aa1,file=paste(arg[2],"stat.xls",sep="."),row.names=F,col.names=T,sep="\t",quote=F) + + +# plot +myLabel = as.vector(aa1$Element) +myLabel = paste(myLabel, "(",aa1$Number," DMRs,",round(aa1$Number / sum(aa1$Number) * 100, 2), "%)", sep = "") + +M<-max(aa1$Number) + +#barplot +pdf(paste(arg[2],"barplot.pdf",sep="_")) + ggplot(aa1, aes(x=Element,y=Number,fill = Element)) + + geom_bar(stat="identity",width=0.9,color="black")+ + #coord_polar(theta = "y",start=0)+ + scale_fill_discrete(breaks = aa1$Element, labels = myLabel)+ + #scale_fill_brewer(palette="Greens")+ + guides(fill = guide_legend(reverse = TRUE))+ + geom_text(aes(label=Number), vjust=1.5, color="white",position = position_dodge(0.9), size=3.5)+ + theme(axis.text.x = element_text(angle = 45,hjust = 1)) + + #expand_limits(m)+ + theme( + # panel.grid = element_blank(), + panel.background = element_blank(), + # axis.text.y = element_blank(), + # axis.ticks= element_blank(), + # axis.title = element_blank() + )+ + labs(y="Annotated DMR Number",title="DMR annotation results") + +dev.off() + +# rose plot +pdf(paste(arg[2],"roseplot.pdf",sep="_")) + ggplot(aa1, aes(x=Element,y=Number,fill = Element)) + + geom_bar(stat="identity",width=0.95)+ + coord_polar(theta = "x",start=0)+ + scale_fill_discrete(breaks = aa1$Element, labels = myLabel)+ + guides(fill = guide_legend(reverse = TRUE))+ + geom_text(aes(label=Number), vjust=-1.5, color="black",position = position_dodge(0.9), size=3.5)+ + #expand_limits(-M/5)+ + ylim(c(-M/5,M))+ + theme( + panel.grid = element_blank(), + panel.background = element_blank(), + axis.text.y = element_blank(), + axis.ticks= element_blank(), + axis.title = element_blank() + )+ + labs(title="DMR annotation results") +dev.off() + diff --git a/script/plot_element_methy.R b/script/plot_element_methy.R new file mode 100644 index 0000000..ffd486b --- /dev/null +++ b/script/plot_element_methy.R @@ -0,0 +1,42 @@ +# A script for mutisamples vioplot of element methy +# author: zhangdu +# date: 2018-8-14 + +args<-commandArgs(T) +if(length(args) < 3){ + cat("Argument: all.sample.genome.cgi.methy.txt all.sample.genome.cgi.methy_vioplot CGI\n") + quit('no') +} +infile<-args[1] +outpdf<-args[2] +element<-args[3] + +library(ggplot2) +tab<-read.table(infile,header=F) +colnames(tab)<-c("Group","Sample_ID","Methylation_level") +n<-length(levels(factor(tab$Sample_ID))) +tab$Methylation_level<-as.numeric(as.vector(tab$Methylation_level)) +#all sample plot +pdf(file=paste("all.sample",outpdf,sep="."),height=5,width=0.7*n) +ggplot(tab, aes(x=Sample_ID, y=Methylation_level)) + +geom_violin(fill="deepskyblue",size=0.5) + +geom_boxplot(width=0.6/n,fill="black",outlier.size=0,size=0.5) + +theme_minimal() + +stat_summary(fun.y=median, geom="point", size=15/n,shape=21,fill="white") + +theme(axis.text.x = element_text(angle = -45,hjust = 0.5, vjust=0.5)) + +theme(panel.border = element_rect(fill = NA)) + +labs(title = paste("Sample methylation level distribution of",element,sep=" ")) +dev.off() +#group plot +m<-length(levels(factor(tab$Group))) +pdf(file=paste("all.group",outpdf,sep="."),height=5,width=0.2*m) +ggplot(tab, aes(x=Group, y=Methylation_level)) + +geom_violin(fill="green4",size=0.5) + +geom_boxplot(width=0.05/m,fill="black",outlier.size=0,size=0.5) + +theme_minimal() + +stat_summary(fun.y=median, geom="point", size=10/m,shape=21,fill="white") + +theme(axis.text.x = element_text(angle = -45,hjust = 0.5, vjust=0.5)) + +theme(panel.border = element_rect(fill = NA)) + +labs(title = paste("Group methylation level distribution of",element,sep=" ")) +dev.off() + diff --git a/script/plot_element_methy.v1.0.pl b/script/plot_element_methy.v1.0.pl new file mode 100644 index 0000000..98d4a70 --- /dev/null +++ b/script/plot_element_methy.v1.0.pl @@ -0,0 +1,82 @@ +#!/usr/bin/perl +use strict; +use FindBin qw/$Bin/; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: plot_element_methy + + It is designed for plot gene element methylation level vioplot of BS-seq. + version:1.0 + usage: perl $0 + -i map file; + -p element methy file path; + -e element name list, split with "," like: cgi,cgi_shore,promoter; + -o results file path; + -Rscript Rscript program; + -h help; + author: zhangdu + date: 2018-05-29 +USAGE + +my ($ref,$file,$outpath,$ele,$elepath,$Rscript); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$elepath, + "e=s" => \$ele, + "o=s" => \$outpath, + "Rscript=s" => \$Rscript, + "h=s" => \$help +); + +die $usage if $help; +die $usage unless $file; +die $usage unless $outpath; +die $usage unless $ele; +die $usage unless $elepath; +die $usage unless $Rscript; + +my @elelist=split(/,/,$ele); +foreach my $element(@elelist){ + print "#################################\n|-----Analysising $element methylation distribution for all samples...\n"; + system ("date"); + my $out=$elepath."/all.sample.genome.".$element.".methy.txt"; + open FILE, $file or die "can not open $file:$!"; + open OUT, ">$out" or die "can not open $out:$!"; + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + #my $in=$elepath."/".$sampleinfo[1]."/genome.".$element.".methy.txt"; + my $in=$elepath."/".$sampleinfo[1]."/$sampleinfo[1].genome.".$element.".methy.txt"; + if(-e $in){ + $in=$elepath."/".$sampleinfo[1]."/$sampleinfo[1].genome.".$element.".methy.txt"; + }else{ + $in=$elepath."/".$sampleinfo[1]."/$sampleinfo[1].Repeats.".$element.".methy.txt"; + } + open IN, $in or die "can not open $in:$!"; + my $n=0; + while(){ + chomp $_; + my @info=split(/\t/,$_); + next if($info[-1] eq "NA"); + print OUT "$sampleinfo[0]\t$sampleinfo[1]\t$info[-1]\n"; + $n++; + } + print "|-----Total read $n $element from sample: $sampleinfo[1]...\n"; + close IN; + } + close OUT; + close FILE; +#plot +print "|-----violin ploting...\n"; +my $pdf1=$outpath."/all.sample.genome.".$element.".methy_vioplot.pdf"; +my $pdf2=$outpath."/all.group.genome.".$element.".methy_vioplot.pdf"; +system ("$Rscript $Bin/plot_sample_element_methy.v1.0.R $out $pdf1 $element"); +system ("$Rscript $Bin/plot_group_element_methy.v1.0.R $out $pdf2 $element"); +print "-----|$element analysis finished...\n"; +system ("date"); +print "#################################\n\n"; +} +########################################################################################################################### diff --git a/script/plot_group_element_methy.v1.0.R b/script/plot_group_element_methy.v1.0.R new file mode 100644 index 0000000..1e16719 --- /dev/null +++ b/script/plot_group_element_methy.v1.0.R @@ -0,0 +1,30 @@ +# A script for mutisamples vioplot of element methy +# author: zhangdu +# date: 2018-8-14 + +args<-commandArgs(T) +if(length(args) < 3){ + cat("Argument: all.sample.genome.cgi.methy.txt all.sample.genome.cgi.methy_vioplot CGI\n") + quit('no') +} +infile<-args[1] +outpdf<-args[2] +element<-args[3] + +library(ggplot2) +tab<-read.table(infile,header=F) +colnames(tab)<-c("Group","Sample_ID","Methylation_level") +tab$Methylation_level<-as.numeric(as.vector(tab$Methylation_level)) +#group plot +m<-length(levels(factor(tab$Group))) +pdf(file=outpdf) +ggplot(tab, aes(x=Group, y=Methylation_level)) + +geom_violin(fill="green4",size=0.5) + +geom_boxplot(width=0.01,fill="black",outlier.size=0,size=0.5) + +theme_minimal() + +stat_summary(fun.y=median, geom="point", size=1,shape=21,fill="white") + +theme(axis.text.x = element_text(angle = -45,hjust = 0.5, vjust=0.5)) + +theme(panel.border = element_rect(fill = NA)) + +labs(title = paste("Group methylation level distribution of",element,sep=" ")) +dev.off() + diff --git a/script/plot_sample_element_methy.v1.0.R b/script/plot_sample_element_methy.v1.0.R new file mode 100644 index 0000000..f64b573 --- /dev/null +++ b/script/plot_sample_element_methy.v1.0.R @@ -0,0 +1,30 @@ +# A script for mutisamples vioplot of element methy +# author: zhangdu +# date: 2018-8-14 + +args<-commandArgs(T) +if(length(args) < 3){ + cat("Argument: all.sample.genome.cgi.methy.txt all.sample.genome.cgi.methy_vioplot CGI\n") + quit('no') +} +infile<-args[1] +outpdf<-args[2] +element<-args[3] + +library(ggplot2) +tab<-read.table(infile,header=F) +colnames(tab)<-c("Group","Sample_ID","Methylation_level") +n<-length(levels(factor(tab$Sample_ID))) +tab$Methylation_level<-as.numeric(as.vector(tab$Methylation_level)) +#all sample plot +pdf(file=outpdf,height=5,width=0.7*n) +ggplot(tab, aes(x=Sample_ID, y=Methylation_level)) + +geom_violin(fill="deepskyblue",size=0.2) + +geom_boxplot(width=0.2/n,fill="black",outlier.size=1e-50,size=0.2) + +theme_minimal() + +stat_summary(fun.y=median, geom="point", size=12/n,shape=21,fill="white") + +theme(axis.text.x = element_text(angle = -45,hjust = 0.5, vjust=0.5)) + +theme(panel.border = element_rect(fill = NA)) + +labs(title = paste("Sample methylation level distribution of",element,sep=" ")) +dev.off() + diff --git a/script/pointTopoint.pl b/script/pointTopoint.pl new file mode 100644 index 0000000..dd80da1 --- /dev/null +++ b/script/pointTopoint.pl @@ -0,0 +1,26 @@ +#!/usr/bin/perl -w +use strict; +die "Usage: perl file1 file2 > out_file\n" unless @ARGV == 2; + +my %geneList; +open IN, $ARGV[0]; +#chr1 refSeq mRNA 6407435 6443590 . 6443171 6444542 +while () { + chomp; + my @info = split /\t/; + my $key = "$info[0]\t$info[1]\t$info[2]"; + #$geneList{$key}="$info[1]\t$info[2]\t$info[3]"; + $geneList{$key}=join "\t", @info[3..$#info]; +} +close IN; +#chr1 56754 59454 + ID=NM_001005484; name=OR4F5; ICPs + +open IN, $ARGV[1]; +while () { + chomp; + my @info = split /\t/; + my $key = "$info[0]\t$info[1]\t$info[2]"; + if(exists $geneList{$key}) + {print join "\t", $_,$geneList{$key},"\n";} +} +close IN; diff --git a/script/qc_stat.v1.0.sh b/script/qc_stat.v1.0.sh new file mode 100644 index 0000000..1b9a3fa --- /dev/null +++ b/script/qc_stat.v1.0.sh @@ -0,0 +1,31 @@ +# name: qc_stat +# usage: sh qc_stat.v1.0.sh +# dir 04_qc_stat abs dir; +# prefix common prefix of sample id. +# author: zhangdu +# e-mail: zhangducsu\@163.com +# date: +# version:v1.0 + +statdir=$1 # 04_qc_stat abs dir +prefix=$2 # common prefix of sample id + +cd $statdir +echo -e "Sample_ID\tRawReadsNum\tRawBaseNum\tRawQ20Rate\tRawQ30Rate\tAdapterCleanBaseRate\tCleanReadsNum\tCleanBaseNum\tCleanQ20Rate\tCleanQ30Rate\tCleanReadsRate\tCleanBaseRate\tCleanData(Gb)" >> QC_result.xls +for sam in `ls -d $prefix*` +do +Sample_ID=$sam +RawReadsNum=$(less -S $sam/$sam.raw.xls | awk '$0!~/^R/{print $1}') +RawBaseNum=$(less -S $sam/$sam.raw.xls | awk '$0!~/^R/{print $2}') +RawQ20Rate=$(less -S $sam/$sam.raw.xls | awk '$0!~/^R/{printf "%.2f%\n",$4*100}') +RawQ30Rate=$(less -S $sam/$sam.raw.xls | awk '$0!~/^R/{printf "%.2f%\n",$6*100}') +AdapterCleanBaseRate=$(less -S $sam/$sam.noAdapter.xls | awk '$0!~/^R/{printf "%.2f%\n",($2/'$RawBaseNum')*100}') +CleanReadsNum=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{print $1}') +CleanBaseNum=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{print $2}') +CleanQ20Rate=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{printf "%.2f%\n",$4*100}') +CleanQ30Rate=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{printf "%.2f%\n",$6*100}') +CleanReadsRate=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{printf "%.2f%\n",($1/'$RawReadsNum')*100}') +CleanBaseRate=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{printf "%.2f%\n",($2/'$RawBaseNum')*100}') +CleanData=$(less -S $sam/$sam.clean.xls | awk '$0!~/^R/{printf "%.3f\n",$2/10^9}') +echo -e "$Sample_ID\t$RawReadsNum\t$RawBaseNum\t$RawQ20Rate\t$RawQ30Rate\t$AdapterCleanBaseRate\t$CleanReadsNum\t$CleanBaseNum\t$CleanQ20Rate\t$CleanQ30Rate\t$CleanReadsRate\t$CleanBaseRate\t$CleanData" >> QC_result.xls +done diff --git a/script/rrbs_control_batch.v1.0.pl b/script/rrbs_control_batch.v1.0.pl new file mode 100644 index 0000000..94098f5 --- /dev/null +++ b/script/rrbs_control_batch.v1.0.pl @@ -0,0 +1,115 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: rrbs_control_batch + + It is designed for running rrbs Control DNA CT convertion rate in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p fastq files folder path; + -o results file path; + -m s for single end; p for pair end; + -r control.fa; + -bsmap bsmap program dir(bsmap-2.73/ abs dir); + -t Specifies the number of files which can be processed simultaneously. + Each thread will be allocated 250MB of memory so you shouldn't run more + threads than your available memory will cope with, and not more than 6 threads on a 32 bit machine + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my ($file,$outfile,$fqpath,$threads,$mode,$ref,$bsmap); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "m=s" => \$mode, + "r=s" => \$ref, + "bsmap=s" => \$bsmap, + "t=i" => \$threads, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $fqpath; +die $usage unless $ref; +die $usage unless $bsmap; +$threads=8 unless $threads; +$mode="p" unless $mode; +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($file)); + $outfile=abs_path($file); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $path1=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.clean.1.fq.gz'; + my $path2=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.clean.2.fq.gz'; + my $path3=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.clean.single.fq.gz'; + my $sam1=$opath.$sampleinfo[1].'.control.pe.sam'; + my $sam2=$opath.$sampleinfo[1].'.control.se.sam'; + my $bam1=$opath.$sampleinfo[1].'.control.pe.bam'; + my $bam2=$opath.$sampleinfo[1].'.control.se.bam'; + my $bam3=$opath.$sampleinfo[1].'.control.merge.bam'; + my $stat=$opath.$sampleinfo[1].'.control.bsmap.result'; + my $cout=$opath.$sampleinfo[1].'.control.cout'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + if($mode eq "p"){ + print SIGNAL "$bsmap/bsmap -a $path1 -b $path2 -d $ref -o $sam1 -v 0.1 -g 1 -p $threads && echo bsmap pe is done.. + cd $bsmap + sh $bsmap/sam2bam.sh $sam1 && echo sam 2 bam pe is done.. + rm $sam1 + $bsmap/bsmap -a $path3 -d $ref -o $sam2 -v 0.1 -g 1 -p $threads && echo bsmap se is done.. + sh $bsmap/sam2bam.sh $sam2 && echo sam 2 bam se is done.. + rm $sam2 + $bsmap/samtools/samtools merge -f $bam3 $bam1 $bam2 && echo merge is done.. + $bsmap/samtools/samtools index $bam3 && echo index merge is done.. + $bsmap/samtools/samtools flagstat $bam3 > $stat && echo stat is done.. + /public/software/bin/python $bsmap/methratio.py --ref=$ref --sam-path=$bsmap/samtools --out=$cout $bam3 && echo cout done...\necho Start gzip $cout ... + gzip -f $cout && echo gzip $cout done\n"; + }elsif($mode eq "s"){ + print SIGNAL "$bsmap/bsmap -a $path3 -d $ref -o $sam2 -v 0.1 -g 1 -p $threads && echo bsmap se is done.. + cd $bsmap + sh $bsmap/sam2bam.sh $sam2 && echo sam 2 bam se is done.. + mv $bam2 $bam3 && echo bam rename is done.. + $bsmap/samtools/samtools index $bam3 && echo index merge is done.. + $bsmap/samtools/samtools flagstat $bam3 > $stat && echo stat is done.. + /public/software/bin/python $bsmap/methratio.py --ref=$ref --sam-path=$bsmap/samtools --out=$cout $bam3 && echo cout done...\necho Start gzip $cout ... + gzip -f $cout && echo gzip $cout done\n"; + } + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=$threads $signalpbs\n"; + print SGEall "qsub -cwd -l vf=8G -q all.q -pe smp $threads $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/rrbs_cout_batch.v1.0.pl b/script/rrbs_cout_batch.v1.0.pl new file mode 100644 index 0000000..6207138 --- /dev/null +++ b/script/rrbs_cout_batch.v1.0.pl @@ -0,0 +1,76 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: rrbs_cout_batch + + It is designed for running rrbs bsmap cout in batch model. + version:1.0 + usage: perl $0 + -i map file; + -b bsmap bam files folder path; + -o results file path; + -r reference genome fa; + -py2 python2 program; + -bsmap mapping program dir(bsmap-2.73/ abs dir); + -h help; + author: zhangdu + date: 2017-03-20 +USAGE + +my ($file,$outfile,$bam,$group,$ref,$python2,$bsmap); +my $help; +GetOptions("i=s" => \$file, + "b=s" => \$bam, + "o=s" => \$outfile, + "r=s" => \$ref, + "py2=s" => \$python2, + "bsmap=s" => \$bsmap, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $bam; +die $usage unless $ref; +die $usage unless $bsmap; +die $usage unless $python2; + +$bam=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +print "Reading map file...\n"; +#my $totalpath; +system ("date"); +open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if(/^#Group/); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $path1=$bam."/".$sampleinfo[1]."/".$sampleinfo[1].'.merge.bam'; + my $cout=$opath.$sampleinfo[1].'.cout'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "$python2 $bsmap/methratio.py --ref=$ref --sam-path=$bsmap/samtools --out=$cout $path1 && echo cout done...\necho Start gzip $cout ... + gzip -f $cout && echo gzip $cout done\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=1 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=20G -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/rrbs_final.stat.v1.1.sh b/script/rrbs_final.stat.v1.1.sh new file mode 100644 index 0000000..47b8c2c --- /dev/null +++ b/script/rrbs_final.stat.v1.1.sh @@ -0,0 +1,153 @@ +#!/bin/sh +if [ $# -lt 3 ] +then + echo "Name: rrbs_final.stat.v1.3.sh" + echo "Usage: $0 " + echo "This script is for RRBS stat on login2" + echo "Author: zhangdu" + exit +fi + +pjdir=$1 +prefix=$2 +control=$3 +outdir=$pjdir/02_methycalling/04_stat +mkdir -p $pjdir/02_methycalling/04_stat/jobs +result1=$outdir/01.QC-mapping_stat.xls +result2=$outdir/02.Global_methy.stat.xls +result3=$outdir/03.CG_global_methy.stat.xls + +if [ "$control" = "YES" ] +then + + echo "NOTE: CT covertion rate will be stat..." + echo -e "Table 1. QC and mapping summery" > $result1 + echo -e "Sample_ID\tRawReadsNum\tRawBaseNum\tRawQ20Rate\tRawQ30Rate\tAdapterCleanBaseRate\tCleanReadsNum\tCleanBaseNum\tCleanQ20Rate\tCleanQ30Rate\tCleanReadsRate\tCleanBaseRate\tCleanData(Gb)\tMappedReads\tMappedRate\tCT ConvertRatio" >> $result1 + echo -e "Table 2. Global average depth and methylation level in C site " > $result2 + echo -e "Sample_ID\tAverageDepC\tAverageDepCG\tAverageDepCHG\tAverageDepCHH\tMethyLevelC\tMethyLevelCG\tMethyLevelCHG\tMethyLevelCHH" >> $result2 + echo -e "Table 3. Methylation summery on C and CpG sites " > $result3 + echo -e "Sample_ID\tC>1x\tC>1xdep\tC>1xcoverage%\tC>1xmethy\tCG>1x\tCG>1xdep\tCG>1xcoverage%\tCG>1xmethy\tC>=5x\tC>=5xdep\tC>=5xcoverage%\tC>=5xmethy\tCG>=5x\tCG>=5xdep\tCG>=5xcoverage%\tCG>=5xmethy" >> $result3 + cd $pjdir/01_cleandata/02_clean + for sam in `ls -d $prefix*` + do + echo -n " + echo -e \"||-------$sam stastics started\\n\" + +######################################################################################################################################## + #QC stat + cd $pjdir/01_cleandata/04_qc_stat + Sample_ID=$sam + RawReadsNum=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{print \$1}') + RawBaseNum=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{print \$2}') + RawQ20Rate=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$4*100}') + RawQ30Rate=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$6*100}') + AdapterCleanBaseRate=\$(less -S $sam/$sam.noAdapter.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",(\$2/'\$RawBaseNum')*100}') + CleanReadsNum=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{print \$1}') + CleanBaseNum=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{print \$2}') + CleanQ20Rate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$4*100}') + CleanQ30Rate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$6*100}') + CleanReadsRate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",(\$1/'\$RawReadsNum')*100}') + CleanBaseRate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",(\$2/'\$RawBaseNum')*100}') + CleanData=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.3f\\n\",\$2/10^9}') + +######################################################################################################################################## + #bsmap stat + cd $pjdir/02_methycalling/01_bsmap/$sam + file1=$sam.mapping.stat + info1=\$(less -S \$file1 | awk 'BEGIN{FS=\"[ \\t(:]\";OFS=\"\\t\"}NR==3{print \$1,\$6}') + + cd $pjdir/02_methycalling/03_control/$sam + file2=$sam.control.cout.gz + info2=\$(zcat \$file2 | awk '\$6~/[.]/{ratio+=1-\$6;count++}END{printf \"%.2f%\\n\",(ratio/count)*100}') + + echo -e \"\$Sample_ID\\t\$RawReadsNum\\t\$RawBaseNum\\t\$RawQ20Rate\\t\$RawQ30Rate\\t\$AdapterCleanBaseRate\\t\$CleanReadsNum\\t\$CleanBaseNum\\t\$CleanQ20Rate\\t\$CleanQ30Rate\\t\$CleanReadsRate\\t\$CleanBaseRate\\t\$CleanData\\t\$info1\\t\$info2\" >> $result1 + +######################################################################################################################################### + #average methy stat + cd $pjdir/02_methycalling/02_cout/$sam + file3=$sam.cout.gz + info3=\$(zcat \$file3 | awk 'BEGIN{OFS=\"\\t\"}\$4~/^C/{depthC+=(\$7+\$8);ratioC+=\$6;countC++;}\$4~/^CG$/{depthCG+=(\$7+\$8);ratioCG+=\$6;countCG++;}\$4~/^CHG$/{depthCHG+=(\$7+\$8);ratioCHG+=\$6;countCHG++;}\$4~/^CHH$/{depthCHH+=(\$7+\$8);ratioCHH+=\$6;countCHH++;}END{print depthC/countC,depthCG/countCG,depthCHG/countCHG,depthCHH/countCHH,ratioC/countC,ratioCG/countCG,ratioCHG/countCHG,ratioCHH/countCHH}') + + echo -e \"\$Sample_ID\\t\$info3\" >> $result2 + +########################################################################################################################################### +#coverage and depth stat + info4=\$(zcat \$file3 | awk 'BEGIN{OFS=\"\\t\"}\$4~/^C/{totalC++;if(\$7+\$8>1){depthC+=\$7+\$8;ratioC+=\$6;countC++;}}\$4~/^CG$/{totalCG++;if(\$7+\$8>1){depthCG+=\$7+\$8;ratioCG+=\$6;countCG++;}}\$4~/^C/{totalCF++;if(\$7+\$8>4){depthCF+=\$7+\$8;ratioCF+=\$6;countCF++;}}\$4~/^CG$/{totalCGF++;if(\$7+\$8>4){depthCGF+=\$7+\$8;ratioCGF+=\$6;countCGF++;}}END{print countC,depthC/countC,(countC/totalC)*100,ratioC/countC,countCG,depthCG/countCG,(countCG/totalCG)*100,ratioCG/countCG,countCF,depthCF/countCF,(countCF/totalCF)*100,ratioCF/countCF,countCGF,depthCGF/countCGF,(countCGF/totalCGF)*100,ratioCGF/countCGF}') + echo -e \"\$Sample_ID\\t\$info4\" >> $result3 + +########################################################################################################################################### + + echo -e \"||-------$sam stastics finished\\n\" + " > $outdir/jobs/$sam.pbs + echo -e "qsub -cwd -l vf=10G -q all.q -pe smp 1 $outdir/jobs/$sam.pbs" >> $outdir/jobs/all.sge + echo -e "qsub -N $sam -l nodes=1:ppn=1 $outdir/jobs/$sam.pbs" >> $outdir/jobs/all.pbs + + done + +elif [ "$control" = "NO" ] +then + echo "NOTE: CT covertion rate was set to not stat!!!!!" + echo -e "Table 1. QC and mapping summery" > $result1 + echo -e "Sample_ID\tRawReadsNum\tRawBaseNum\tRawQ20Rate\tRawQ30Rate\tAdapterCleanBaseRate\tCleanReadsNum\tCleanBaseNum\tCleanQ20Rate\tCleanQ30Rate\tCleanReadsRate\tCleanBaseRate\tCleanData(Gb)\tMappedReads\tMappedRate" >> $result1 + echo -e "Table 2. Global average depth and methylation level in C site " > $result2 + echo -e "Sample_ID\tAverageDepC\tAverageDepCG\tAverageDepCHG\tAverageDepCHH\tMethyLevelC\tMethyLevelCG\tMethyLevelCHG\tMethyLevelCHH" >> $result2 + echo -e "Table 3. Methylation summery on C and CpG sites " > $result3 + echo -e "Sample_ID\tC>1x\tC>1xdep\tC>1xcoverage%\tC>1xmethy\tCG>1x\tCG>1xdep\tCG>1xcoverage%\tCG>1xmethy\tC>=5x\tC>=5xdep\tC>=5xcoverage%\tC>=5xmethy\tCG>=5x\tCG>=5xdep\tCG>=5xcoverage%\tCG>=5xmethy" >> $result3 + cd $pjdir/01_cleandata/02_clean + for sam in `ls -d $prefix*` + do + echo -n " + echo -e \"||-------$sam stastics started\\n\" + +######################################################################################################################################## + #QC stat + cd $pjdir/01_cleandata/04_qc_stat + Sample_ID=$sam + RawReadsNum=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{print \$1}') + RawBaseNum=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{print \$2}') + RawQ20Rate=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$4*100}') + RawQ30Rate=\$(less -S $sam/$sam.raw.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$6*100}') + AdapterCleanBaseRate=\$(less -S $sam/$sam.noAdapter.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",(\$2/'\$RawBaseNum')*100}') + CleanReadsNum=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{print \$1}') + CleanBaseNum=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{print \$2}') + CleanQ20Rate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$4*100}') + CleanQ30Rate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",\$6*100}') + CleanReadsRate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",(\$1/'\$RawReadsNum')*100}') + CleanBaseRate=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.2f%\\n\",(\$2/'\$RawBaseNum')*100}') + CleanData=\$(less -S $sam/$sam.clean.xls | awk '\$0!~/^R/{printf \"%.3f\\n\",\$2/10^9}') + +######################################################################################################################################## + #bsmap stat + cd $pjdir/02_methycalling/01_bsmap/$sam + file1=$sam.mapping.stat + info1=\$(less -S \$file1 | awk 'BEGIN{FS=\"[ \\t(:]\";OFS=\"\\t\"}NR==3{print \$1,\$6}') + + echo -e \"\$Sample_ID\\t\$RawReadsNum\\t\$RawBaseNum\\t\$RawQ20Rate\\t\$RawQ30Rate\\t\$AdapterCleanBaseRate\\t\$CleanReadsNum\\t\$CleanBaseNum\\t\$CleanQ20Rate\\t\$CleanQ30Rate\\t\$CleanReadsRate\\t\$CleanBaseRate\\t\$CleanData\\t\$info1\" >> $result1 +######################################################################################################################################### + #average methy stat + cd $pjdir/02_methycalling/02_cout/$sam + file3=$sam.cout.gz + info3=\$(zcat \$file3 | awk 'BEGIN{OFS=\"\\t\"}\$4~/^C/{depthC+=(\$7+\$8);ratioC+=\$6;countC++;}\$4~/^CG$/{depthCG+=(\$7+\$8);ratioCG+=\$6;countCG++;}\$4~/^CHG$/{depthCHG+=(\$7+\$8);ratioCHG+=\$6;countCHG++;}\$4~/^CHH$/{depthCHH+=(\$7+\$8);ratioCHH+=\$6;countCHH++;}END{print depthC/countC,depthCG/countCG,depthCHG/countCHG,depthCHH/countCHH,ratioC/countC,ratioCG/countCG,ratioCHG/countCHG,ratioCHH/countCHH}') + echo -e \"\$Sample_ID\\t\$info3\" >> $result2 + +########################################################################################################################################### + #coverage and depth stat + info4=\$(zcat \$file3 | awk 'BEGIN{OFS=\"\\t\"}\$4~/^C/{totalC++;if(\$7+\$8>1){depthC+=\$7+\$8;ratioC+=\$6;countC++;}}\$4~/^CG$/{totalCG++;if(\$7+\$8>1){depthCG+=\$7+\$8;ratioCG+=\$6;countCG++;}}\$4~/^C/{totalCF++;if(\$7+\$8>4){depthCF+=\$7+\$8;ratioCF+=\$6;countCF++;}}\$4~/^CG$/{totalCGF++;if(\$7+\$8>4){depthCGF+=\$7+\$8;ratioCGF+=\$6;countCGF++;}}END{print countC,depthC/countC,(countC/totalC)*100,ratioC/countC,countCG,depthCG/countCG,(countCG/totalCG)*100,ratioCG/countCG,countCF,depthCF/countCF,(countCF/totalCF)*100,ratioCF/countCF,countCGF,depthCGF/countCGF,(countCGF/totalCGF)*100,ratioCGF/countCGF}') + echo -e \"\$Sample_ID\\t\$info4\" >> $result3 + +########################################################################################################################################### + + echo -e \"||-------$sam stastics finished\\n\" + " > $outdir/jobs/$sam.pbs + + echo -e "qsub -cwd -l vf=2G -pe smp 1 $outdir/jobs/$sam.pbs" >> $outdir/jobs/all.sge + echo -e "qsub -N $sam -l nodes=1:ppn=1 $outdir/jobs/$sam.pbs" >> $outdir/jobs/all.pbs + + done + +else + echo "Unkown argument..." + exit +fi + +echo -e "||-------All stastics task qsub build done..\n" diff --git a/script/rrbs_fq_clean.v1.1.pl b/script/rrbs_fq_clean.v1.1.pl new file mode 100644 index 0000000..fb474bf --- /dev/null +++ b/script/rrbs_fq_clean.v1.1.pl @@ -0,0 +1,116 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: $0 + + It is designed for running RRBS data clean in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p clean data folder path, gz file path; + -o results file path; + -m 0 for RRBS(default); 1 for DRRBS; + -py3 python3 program; + -cut cutadapt program; + -h help; + author: zhangdu + date: 2018-05-02 +USAGE + +my $p1="$Bin/00_FQ_trim.2.pl"; +my $p2="$Bin/cut_rrbs.v1.0.pl"; +my $p3="$Bin/02_FQ_clean.2.pl"; +my $p4="$Bin/01_FQ_pas.pl"; + +my ($map,$outfile,$fqpath,$python3,$cutadapt,$mode); +my $help; +GetOptions("i=s" => \$map, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "m=s" => \$mode, + "py3=s" => \$python3, + "cut=s" => \$cutadapt, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $map; +die $usage unless $fqpath; +die $usage unless $python3; +die $usage unless $cutadapt; +$mode ||=0; + +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($map)); + $outfile=abs_path($map); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; +my $jobpath="$outfile/jobs"; +if (-e $jobpath){ + print STDOUT "$jobpath exists!"; +}else{ + system("mkdir -p $jobpath"); +} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +system ("date"); +open FILE, $map + or die "can not open $map:$!"; #打开文件 +while(){ + chomp $_; + if (/^#Group/){next}; + my @sampleinfo=split(/\t/,$_); + print "Deal with $sampleinfo[1] : $sampleinfo[2]\t$sampleinfo[3]\n"; + if($sampleinfo[3] eq ""){ # for single end sequencing + my $path1=$fqpath."/".$sampleinfo[2]; + my $opath=$outfile."/".$sampleinfo[1]; + system("mkdir -p $opath"); + my $temp1=$opath."/".$sampleinfo[1].".fq.gz"; + my $temp4=$opath."/".$sampleinfo[1].".cut1.fq.gz"; + my $temp5=$opath."/".$sampleinfo[1].".cut2.fq.gz"; + my $ofile=$opath."/".$sampleinfo[1].".clean"; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "perl $p1 $path1 1 $temp1 && echo fq1 head revise done.. + $python3 $cutadapt -a GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA -f fastq -m 35 -n 2 -o $temp4 $temp1 && echo cutting adapter is done.. + perl $p2 -i $temp4 -m $mode -o $temp5 && echo cut rrbs is done.. + perl $p3 $temp5 $ofile && echo Clean is done..\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=2 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=10G -q all.q $signalpbs\n"; + } + else{ # for pair end sequencing + my $path1=$fqpath."/".$sampleinfo[2]; + my $path2=$fqpath."/".$sampleinfo[3]; + my $opath=$outfile."/".$sampleinfo[1]; + system("mkdir -p $opath"); + my $temp1=$opath."/".$sampleinfo[1].".1.fq.gz"; + my $temp2=$opath."/".$sampleinfo[1].".2.fq.gz"; + my $temp3=$opath."/".$sampleinfo[1].".fq.gz"; + my $temp4=$opath."/".$sampleinfo[1].".cut1.fq.gz"; + my $temp5=$opath."/".$sampleinfo[1].".cut2.fq.gz"; + my $ofile=$opath."/".$sampleinfo[1].".clean"; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "perl $p1 $path1 1 $temp1 && echo fq1 head revise done.. + perl $p1 $path2 2 $temp2 && echo fq2 head revise done.. + perl $p4 $temp1 $temp2 $temp3 && echo fq1 and fq2 merge done.. + $python3 $cutadapt -a GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA -f fastq -m 35 -n 2 -o $temp4 $temp3 && echo cutting adapter is done.. + perl $p2 -m $mode -i $temp4 -o $temp5 && echo cut rrbs is done.. + perl $p3 $temp5 $ofile && echo Clean is done..\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=2 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=10G -q all.q $signalpbs\n"; + } +} +close FILE; +close SGEall; +close PBSall; +#END +########################################################################################################################### diff --git a/script/rrbs_fq_clean.v1.2.pl b/script/rrbs_fq_clean.v1.2.pl new file mode 100644 index 0000000..9b4e5e2 --- /dev/null +++ b/script/rrbs_fq_clean.v1.2.pl @@ -0,0 +1,116 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use FindBin qw($Bin); +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + + name: $0 + + It is designed for running RRBS data clean in batch model. + version:1.0 + usage: perl $0 + -i map file; + -p clean data folder path, gz file path; + -o results file path; + -m 0 for RRBS(default); 1 for DRRBS; + -py3 python3 program; + -cut cutadapt program; + -h help; + author: zhangdu + date: 2018-05-02 +USAGE + +my $p1="$Bin/00_FQ_trim.2.pl"; +my $p2="$Bin/cut_rrbs.v1.0.pl"; +my $p3="$Bin/02_FQ_clean.2.pl"; +my $p4="$Bin/01_FQ_pas.pl"; + +my ($map,$outfile,$fqpath,$python3,$cutadapt,$mode); +my $help; +GetOptions("i=s" => \$map, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "m=s" => \$mode, + "py3=s" => \$python3, + "cut=s" => \$cutadapt, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $map; +die $usage unless $fqpath; +die $usage unless $python3; +die $usage unless $cutadapt; +$mode ||=0; + +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($map)); + $outfile=abs_path($map); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; +my $jobpath="$outfile/jobs"; +if (-e $jobpath){ + print STDOUT "$jobpath exists!"; +}else{ + system("mkdir -p $jobpath"); +} + +open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; +open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; +system ("date"); +open FILE, $map + or die "can not open $map:$!"; #打开文件 +while(){ + chomp $_; + if (/^#Group/){next}; + my @sampleinfo=split(/\t/,$_); + print "Deal with $sampleinfo[1] : $sampleinfo[2]\t$sampleinfo[3]\n"; + if($sampleinfo[3] eq ""){ # for single end sequencing + my $path1=$fqpath."/".$sampleinfo[2]; + my $opath=$outfile."/".$sampleinfo[1]; + system("mkdir -p $opath"); + my $temp1=$opath."/".$sampleinfo[1].".fq.gz"; + my $temp4=$opath."/".$sampleinfo[1].".cut1.fq.gz"; + my $temp5=$opath."/".$sampleinfo[1].".cut2.fq.gz"; + my $ofile=$opath."/".$sampleinfo[1].".clean"; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "perl $p1 $path1 1 $temp1 && echo fq1 head revise done.. + $python3 $cutadapt -a AGATCGGAAGAGC -a AGATCGGAAGAGC -f fastq -m 35 -n 2 -o $temp4 $temp1 && echo cutting adapter is done.. + perl $p2 -i $temp4 -m $mode -o $temp5 && echo cut rrbs is done.. + perl $p3 $temp5 $ofile && echo Clean is done..\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=2 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=10G -q all.q $signalpbs\n"; + } + else{ # for pair end sequencing + my $path1=$fqpath."/".$sampleinfo[2]; + my $path2=$fqpath."/".$sampleinfo[3]; + my $opath=$outfile."/".$sampleinfo[1]; + system("mkdir -p $opath"); + my $temp1=$opath."/".$sampleinfo[1].".1.fq.gz"; + my $temp2=$opath."/".$sampleinfo[1].".2.fq.gz"; + my $temp3=$opath."/".$sampleinfo[1].".fq.gz"; + my $temp4=$opath."/".$sampleinfo[1].".cut1.fq.gz"; + my $temp5=$opath."/".$sampleinfo[1].".cut2.fq.gz"; + my $ofile=$opath."/".$sampleinfo[1].".clean"; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + print SIGNAL "perl $p1 $path1 1 $temp1 && echo fq1 head revise done.. + perl $p1 $path2 2 $temp2 && echo fq2 head revise done.. + perl $p4 $temp1 $temp2 $temp3 && echo fq1 and fq2 merge done.. + $python3 $cutadapt -a AGATCGGAAGAGC -a AGATCGGAAGAGC -f fastq -m 35 -n 2 -o $temp4 $temp3 && echo cutting adapter is done.. + perl $p2 -m $mode -i $temp4 -o $temp5 && echo cut rrbs is done.. + perl $p3 $temp5 $ofile && echo Clean is done..\n"; + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=2 $signalpbs\n"; + print SGEall "qsub -cwd -l vf=10G -q all.q $signalpbs\n"; + } +} +close FILE; +close SGEall; +close PBSall; +#END +########################################################################################################################### diff --git a/script/rrbs_mapping_batch.v1.0.pl b/script/rrbs_mapping_batch.v1.0.pl new file mode 100644 index 0000000..3daa3a9 --- /dev/null +++ b/script/rrbs_mapping_batch.v1.0.pl @@ -0,0 +1,132 @@ +#!/usr/bin/perl +use strict; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + +name: rrbs_mapping_batch + + It is designed for running rrbs mapping in batch model. +version:1.0 +usage: perl $0 + -i map file; + -p fastq files folder path; + -o results file path; + -m s for single end; p for pair end; + -r reference genome fa;\ + -t threads to use. default is 8; + -soft mapping program. 0: bsmap(default); 1: bsmark; + For bsmap: + -v if this value is between 0 and 1, it's interpreted as the mismatch rate w.r.t to the read length. + otherwise it's interpreted as the maximum number of mismatches allowed on a read, <=15. + example: -v 5 (max #mismatches = 5), -v 0.1 (max #mismatches = read_length * 10%) + default=0.08. + -g gap size, BSMAP only allow 1 continuous gap (insert or deletion) with up to 3 nucleotides + default=1 + -n [0,1] set mapping strand information. default: 0 + -n 0: only map to 2 forward strands, i.e. BSW(++) and BSC(-+), + for PE sequencing, map read#1 to ++ and -+, read#2 to +- and --. + -n 1: map SE or PE reads to all 4 strands, i.e. ++, +-, -+, -- + generally normal BS-seq is 0; single cell BS-seq is 1. + -softdir mapping program dir(bsmap-2.73/ abs dir); + -h help; +author: zhangdu +date: 2017-03-20 +USAGE + +my ($file,$outfile,$fqpath,$threads,$mode,$ref,$soft,$mismatch,$gap,$softdir,$n); +my $help; +GetOptions("i=s" => \$file, + "p=s" => \$fqpath, + "o=s" => \$outfile, + "m=s" => \$mode, + "r=s" => \$ref, + "t=i" => \$threads, + "soft=s" => \$soft, + "v=s" => \$mismatch, + "g=s" => \$gap, + "n=s" => \$n, + "softdir=s" => \$softdir, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $file; +die $usage unless $fqpath; +die $usage unless $ref; +$threads=4 unless $threads; +die $usage unless $softdir; +$soft ||=0; +if($soft == 0){ + $mismatch ||=0.08; + $gap ||=1; + $n ||=0; +} +$mode="p" unless $mode; +if ($fqpath eq ''){ + my @outtmp=split(/\//,abs_path($file)); + $outfile=abs_path($file); + $outfile=~s/$outtmp[-1]//; + $outfile=~s/\/\//\//; +} +$fqpath=~s/\/\//\//; + +my $jobpath="$outfile/jobs"; +#if (-e $jobpath){ +# print STDOUT "$jobpath exists!"; +#}else{ + system("mkdir -p $jobpath"); +#} + + open PBSall, ">$jobpath/all.pbs" or die "$! : all.pbs\n"; + open SGEall, ">$jobpath/all.sge" or die "$! : all.sge\n"; + print "Reading map file...\n"; +#my $totalpath; + system ("date"); + open FILE, $file + or die "can not open $file:$!"; #打开文件 + while(){ + chomp $_; + next if($.==1); + my @sampleinfo=split(/\t/,$_); + print $sampleinfo[1]."\n"; + my $opath=$outfile."/".$sampleinfo[1]."/"; + system("mkdir -p $opath"); + my $path1=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.clean.1.fq.gz'; + my $path2=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.clean.2.fq.gz'; + my $path3=$fqpath.$sampleinfo[1]."/".$sampleinfo[1].'.clean.single.fq.gz'; + my $sam1=$opath.$sampleinfo[1].'.pe.sam'; + my $sam2=$opath.$sampleinfo[1].'.se.sam'; + my $bam1=$opath.$sampleinfo[1].'.pe.bam'; + my $bam2=$opath.$sampleinfo[1].'.se.bam'; + my $bam3=$opath.$sampleinfo[1].'.merge.bam'; + my $stat=$opath.$sampleinfo[1].'.mapping.stat'; + my $signalpbs=$jobpath."/".$sampleinfo[1].".pbs"; + open SIGNAL, ">$signalpbs" or die "$! : $signalpbs\n"; + if($soft ==0){ + if($mode eq "p"){ + print SIGNAL "$softdir/bsmap -a $path1 -b $path2 -d $ref -o $sam1 -v $mismatch -g $gap -n $n -p $threads -R -u && echo bsmap pe is done.. + cd $softdir + sh $softdir/sam2bam.sh $sam1 && echo sam 2 bam pe is done.. + mv $bam1 $bam3 && echo bam rename is done.. + $softdir/samtools/samtools index $bam3 && echo index merge is done.. + $softdir/samtools/samtools flagstat $bam3 > $stat && echo stat is done..\n"; + }elsif($mode eq "s"){ + print SIGNAL "$softdir/bsmap -a $path3 -d $ref -o $sam2 -v $mismatch -g $gap -n $n -p $threads -R -u && echo bsmap se is done.. + cd $softdir + sh $softdir/sam2bam.sh $sam2 && echo sam 2 bam se is done.. + mv $bam2 $bam3 && echo bam rename is done.. + $softdir/samtools/samtools index $bam3 && echo index merge is done.. + $softdir/samtools/samtools flagstat $bam3 > $stat && echo stat is done..\n"; + } + }elsif($soft ==1){ + #bismark mapping + } + + close SIGNAL; + print PBSall "qsub -N $sampleinfo[1] -l nodes=1:ppn=$threads $signalpbs\n"; + print SGEall "qsub -cwd -l vf=8G -pe smp $threads -q all.q $signalpbs\n"; + } +close FILE; +close SGEall; +close PBSall; +########################################################################################################################### diff --git a/script/stat-fa_GC_detect.pl b/script/stat-fa_GC_detect.pl new file mode 100644 index 0000000..a3991a7 --- /dev/null +++ b/script/stat-fa_GC_detect.pl @@ -0,0 +1,60 @@ +#!usr/bin/perl +use warnings; +use strict; +use Getopt::Long; +#use FindBin qw($Bin $Script); +#use File::Basename qw(basename dirname); +#use Cwd; +#use PerlIO::gzip; +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 *.fa + Input : fasta files + ouput : + Seq_ID Len C_# G_# [C+G]_# CpG_# (G+C)% OE +author: luhanlin\@genomics.org.cn +date: +USAGE +die $usage unless $ARGV[0]; +open (IN,"<","$ARGV[0]") || die "can't open $ARGV[0]"; +my ($name, $sequence); +print "Seq_ID\tLen\tC_#\tG_#\t[C+G]_#\tCpG_#\t(G+C)%\tOE\n"; +while(){ + chomp; + if(/^>(\S+)/){ + if($. > 1){ + CG_detect($name,$sequence); + } + $name = $1; + $sequence = ""; + } + else{ + $sequence .= $_; + } +} +CG_detect($name, $sequence); +close IN; +#### +sub CG_detect{ + my ($id, $str) = @_; + my ($c,$g,$cg,$oe,$gc,$tgc,$len) = (0,0,0,0,0,0,0); + $c = ($str=~s/c/c/gi); + $g = ($str=~s/g/g/gi); + $cg = ($str=~s/cg/cg/gi); + $gc = $c + $g; + $len = length($str); + $tgc = $len==0 ? 0 : $gc/$len; + $cg = $cg ? $cg : 0; + $c = $c ? $c : 0; + $g = $g ? $g : 0; + $gc = $gc ? $gc : 0; + if($c && $g){ + $oe = $len==0 ? 0 : $cg*$len/($c*$g); + } + else{ + $oe=0; + } + $tgc = sprintf("%.4f", $tgc); + $oe = sprintf("%.4f", $oe); + print "$id\t$len\t$c\t$g\t$gc\t$cg\t$tgc\t$oe\n"; +} diff --git a/script/venn.main.v1.0.pl b/script/venn.main.v1.0.pl new file mode 100644 index 0000000..b7f924d --- /dev/null +++ b/script/venn.main.v1.0.pl @@ -0,0 +1,52 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use FindBin qw($Bin $Script); +use File::Basename qw(basename dirname); +use Cwd; +use PerlIO::gzip; +#~~~~~~~~ USAGE ~~~~~~~~# +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 + -I <> list1 + or list1,list2 + or list1,list2,list3 + or list1,list2,list3,list4 + or list1,list2,list3,list4,list5 + list[1..5] is a input file containing one column (e.g., integers, chars), + with each component corresponding to a separate circle in the Venn diagram + -N [] name1,name2,..name5 + -Rscript [] Rscript program + -O [] out prefix + -h [] help +author: zhangdu +date: +USAGE +#~~~~~~~~ Globle Parameters ~~~~~~~~# +my ($filelist, $namelist, $out, $help,$Rscript); +GetOptions( + "help|?|h" => \$help, + "I=s" => \$filelist, + "N:s" => \$namelist, + "Rscript:s" => \$Rscript, + "O:s" => \$out, +); +die $usage if $help; +die $usage unless ($filelist); +die $usage unless ($namelist); +die $usage unless $Rscript; +$out ||= "venn"; + +#~~~~~~~~~ Main Process ~~~~~~~~# +my @collist; +my @flist = split(/,/, $filelist); +for (my $i=0; $i<=$#flist; $i++){ + push @collist, 0; +} +my $clist=join(",",@collist); + +system("perl $Bin\/Venn.pl -I $filelist -C $clist \> $out.venn.xls"); +system("perl $Bin\/Draw_venn.pl -I $filelist -N $namelist -Rscript $Rscript -O $out.venn.pdf"); + diff --git a/script/z01.accu_cover.R b/script/z01.accu_cover.R new file mode 100644 index 0000000..a9a49e4 --- /dev/null +++ b/script/z01.accu_cover.R @@ -0,0 +1,28 @@ +################ +# 绘制平å‡ç”²åŸºåŒ–水平与测åºæ·±åº¦ä¹‹é—´çš„关系曲线图 +# 用法: +# z01.accu_cover.R infile outfile maxpotdepth +# 输入文件格å¼ï¼š +######################################### +##Depth CG CHG CHH +#1 1 1 1 +#2 0.95060021 0.95155783 0.95493366 +#3 0.91336506 0.91503184 0.92008996 +#4 0.88390236 0.88590425 0.89146675 +########################################## + + +args <- commandArgs(T) +file <- args[1] +outpdf <- args[2] +maxpotdepth <-as.numeric(args[3]) + +rt <- read.table(file=file) +pdf(file=outpdf, height=4, width=6) +par(mar=c(5,5,5,4), las=1) +plot(rt[,1], rt[,2] * 100, type="l", lwd=2, col="royalblue", xlim=c(1,maxpotdepth), + xlab=" >= Sequencing Depth ", ylab="Coverage (%)", main="Accumulated Coverage") +points(rt[,1], rt[,3] * 100, type="l", lwd=2, col="firebrick") +points(rt[,1], rt[,4] * 100, type="l", lwd=2, col="gold") +legend("topright", c("CG", "CHG", "CHH"), col=c("royalblue", "firebrick", "gold"), lwd=2) +dev.off() diff --git a/script/z01.dep_coverage.pl b/script/z01.dep_coverage.pl new file mode 100644 index 0000000..f9fad2e --- /dev/null +++ b/script/z01.dep_coverage.pl @@ -0,0 +1,131 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use FindBin qw($Bin $Script); #将本程åºçš„目录ä¿å­˜åœ¨$Bin +use File::Basename qw(basename dirname); +use Cwd; +use PerlIO::gzip; + +my $usage=<<"USAGE"; + + name: dep_coverage.v1.0.pl + It is designed for: + 1. plot Depth-coverage relationship of BS-seq; + 2. plot >=5x methylation level distribution of BS-seq; + 3. generate sampleid.coverage_depth.xls (all C sites used) + 4. generate sampleif.average_methy.xls (>=1xC sites used) + version:1.0 + usage: perl $0 + -i input cout file, cout.gz is ok; + -o outdir; + -n samplename; + -d max plot depth,default is 10x; + -r which Rscript use to plot; + -h help; + author: zhangdu + date: 2018-5-27 +USAGE + +my ($infile,$outdir, $name,$maxplotdepth, $R,$help); +GetOptions("i=s" => \$infile, + "o=s" => \$outdir, + "n=s" => \$name, + "d=s" => \$maxplotdepth, + "r=s" => \$R, + "h=s" => \$help + ); +die $usage if $help; +die $usage unless $infile; +die $usage unless $outdir; +die $usage unless $name; +$maxplotdepth ||=10; +$R ||="~/src/R-3.4.2/builddir/bin/Rscript"; +my %h = (); +my %c = (); +my %m = (); +my %r = (); +my %d = (); +my %dd = (); +my %d5 = (); +my %d10 = (); + +open IN, "<:gzip(autopop)", "$infile" || die "Cannot open $infile:$!\n"; + +while(){ + chomp; +################cout format############################################ +#chr1 3379656 + CG GACGG 1.000 2 0 2 0 0 0.342 1.000 +#chr1 3379657 - CG ACGGT NA 0 0 0 2 2 NA NA +#chr1 3379658 - CHG CGGTA NA 0 0 0 2 2 NA NA +#chr1 3379661 - CHH TAGTC NA 0 0 0 2 2 NA NA +############################################################# + my @a = split; + next if /^#/; + my $d = $a[6] + $a[7]; + $h{$a[3]}{$d} += 1; # $h{Ctype}{depth}=number + $c{$a[3]} += 1;# $c{Ctype}=number + if($d>0){ + $m{$a[3]}{"meth"} += $a[5]; # >=1xçš„æ‰ç”¨æ¥è®¡ç®—甲基化率 $m{Ctype}=totalmethy + $m{$a[3]}{"numb"} += 1;# $c{Ctype}{"numb"}=number + $d{$a[3]} += 1; + $dd{$a[3]} += $d; + } + if($d>4){ #only considering >=5 x + my $win = int( $a[5] / 0.1 ); # 用$win记录该甲基化水平处于哪个窗å£ï¼š$win=0代表处于[0,0.1);$win=1代表处于[0.1,0.2) + $r{$a[3]}{$win} += 1; # $r{Ctype}{win}=number + $d5{$a[3]} += 1; #d5{Ctype}=number + } + $d10{$a[3]} += 1 if $d > 9; #only considering >=10 x + +} +close IN; + +open OA, ">$outdir/$name.accu_cover.xls" or die $!; +print OA "#Depth\tCG\tCHG\tCHH\n"; +foreach my $d (1..100){ + my @out = (); + push @out, $d; + foreach my $t ("CG", "CHG", "CHH"){ + my $n = 0; + foreach my $p (keys %{$h{$t}}){ + $n += $h{$t}{$p} if $p >= $d + } + push @out, sprintf("%.8f", $n/$c{$t}); # @out=(depth,CGcoverage,CHGcoverage,CHHcoverage) + } + print OA join("\t", @out), "\n"; +} +close OA; +system("$R $Bin/z01.accu_cover.R $outdir/$name.accu_cover.xls $outdir/$name.accu_cover.pdf $maxplotdepth"); + + +open OB, ">$outdir/$name.averge_methyl.xls" or die $!; +print OB "Sample\tC\tCG\tCHG\tCHH\n"; +my $mean_meth = ($m{"CG"}{"meth"} + $m{"CHG"}{"meth"} + $m{"CHH"}{"meth"}) / ($m{"CG"}{"numb"} + $m{"CHG"}{"numb"} + $m{"CHH"}{"numb"}); +print OB join("\t", $name, $mean_meth, $m{"CG"}{"meth"}/$m{"CG"}{"numb"}, $m{"CHG"}{"meth"}/$m{"CHG"}{"numb"}, $m{"CHH"}{"meth"}/$m{"CHH"}{"numb"} ), "\n"; +close OB; + + + +open OC, ">$outdir/$name.distribution_methyl.xls" or die $!; +print OC "\tCG\tCHG\tCHH\n"; +foreach my $w (0..9){ + my @out = (); + my $range = $w == 9 ? "[" . $w * 0.1 . "," . ($w+1) * 0.1 . "]" : "[" . $w * 0.1 . "," . ($w+1) * 0.1 . ")"; + push @out, $range; + foreach my $t ("CG", "CHG", "CHH"){ + my $r = $w == 9 ? ( $r{$t}{$w} + $r{$t}{ $w + 1 } ) / $d5{$t} : $r{$t}{$w} / $d5{$t}; + push @out, $r; + } + print OC join("\t", @out), "\n"; +} +close OC; +system("$R $Bin/z01.methyl_distr.R $outdir/$name.distribution_methyl.xls $outdir/$name.distribution_methyl.pdf"); + + +open OD, ">$outdir/$name.coverage_depth.xls" or die $!; +print OD "Sample\ttotal_CG\tmean_depthCG\teffect_depthCG(>=1X)\tcovrageCG\tcoverageCG(d>=5X)\tcoverageCG(d>=10X)\ttotal_CHG\tmean_depthCHG\teffect_depthCHG(>=1X)\tcovrageCHG\tcoverageCHG(d>=5X)\tcoverageCHG(d>=10X)\ttotal_CHH\tmean_depthCHH\teffect_depthCHH(>=1X)\tcovrageCHH\tcoverageCHH(d>=5X)\tcoverageCHH(d>=10X)\n"; +print OD join("\t",$name, $c{"CG"}, $dd{"CG"}/$c{"CG"}, $dd{"CG"}/$d{"CG"}, $d{"CG"}/$c{"CG"}, $d5{"CG"}/$c{"CG"}, $d10{"CG"}/$c{"CG"}, $c{"CHG"}, $dd{"CHG"}/$c{"CHG"}, $dd{"CHG"}/$d{"CHG"}, $d{"CHG"}/$c{"CHG"}, $d5{"CHG"}/$c{"CHG"}, $d10{"CHG"}/$c{"CHG"}, $c{"CHH"}, $dd{"CHH"}/$c{"CHH"}, $dd{"CHH"}/$d{"CHH"}, $d{"CHH"}/$c{"CHH"}, $d5{"CHH"}/$c{"CHH"}, $d10{"CHH"}/$c{"CHH"}, "\n"); +close OD; + + diff --git a/script/z01.methyl_distr.R b/script/z01.methyl_distr.R new file mode 100644 index 0000000..01f39f4 --- /dev/null +++ b/script/z01.methyl_distr.R @@ -0,0 +1,32 @@ +################ +# 绘制Cä½ç‚¹ç”²åŸºåŒ–水平的分布 +# 用法: +# z01.z01.methyl_distr.R infile outfile +# 输入文件格å¼ï¼š +######################################### +# CG CHG CHH +#[0,0.1) 0.120946785 0.138971474 0.650840649 +#[0.1,0.2) 0.077062877 0.089175356 0.116816655 +#[0.2,0.3) 0.056911997 0.076997818 0.059114109 +#[0.3,0.4) 0.045222842 0.072850296 0.037000598 +#[0.4,0.5) 0.042275573 0.075972856 0.027260841 +#[0.5,0.6) 0.050512477 0.087672859 0.022682883 +#[0.6,0.7) 0.064611636 0.089312718 0.017492999 +#[0.7,0.8) 0.101704028 0.101019993 0.013923476 +#[0.8,0.9) 0.151598535 0.107528524 0.010420904 +#[0.9,1] 0.289153251 0.160498107 0.044446884 +########################################## + + +args <- commandArgs(T) +file <- args[1] +outpdf <- args[2] + +##### +rt <- read.table(file=file, header=T) +rt <- t(as.matrix(rt)) +pdf(file=outpdf, height=4, width=6) +par(las=2) +barplot(rt, beside=T, col=c("royalblue", "firebrick","gold"), ylab="Methylation level", + legend=T) +dev.off() diff --git a/script/z02.get_seq.pl b/script/z02.get_seq.pl new file mode 100644 index 0000000..e3d50cc --- /dev/null +++ b/script/z02.get_seq.pl @@ -0,0 +1,115 @@ +#!/usr/bin/perl +use strict; +use warnings; +use Getopt::Long; +use PerlIO::gzip; +my $usage = < + -f [input_2_format: + chrID-start-end [0-1-2] + regionID-chrID-start-end [3-0-1-2] + regionID-chrID-start-end-strand [3-0-1-2-5] ] + -O [output: region.fa [out.fa] ] + -T [sequence in output is continuous ] + -N [number of bases per line in output.[50] ] + +example: perl $0 -D refrence.fa -R region.bed -f 0-1-2-3 -O region.fa +USAGE + +my ($ref, $reg, $out, $format, $One, $baseN); + +GetOptions ( + "D:s" => \$ref, + "R=s" => \$reg, + "O:s" => \$out, + "f:s" => \$format, + "T" => \$One, + "N:i" => \$baseN +); + +die $usage unless ($reg); + +################################################################################ +###################### ######################## +################################################################################# +$ref ||= "/public/agis/gaofei_group/luhanlin/02_data/hg19/fa/hg19.fa"; +$out ||= "out.fa"; +$format ||= "0-1-2"; +$baseN ||= 50; + +my ($regionID_c, $chr_c, $start_c, $end_c, $strand_c); +my @f = split("-", $format); +if(@f==3){ + ($chr_c, $start_c, $end_c) = @f; +} +elsif(@f==4){ + ($regionID_c, $chr_c, $start_c, $end_c) = @f; +} +elsif(@f==5){ + ($regionID_c, $chr_c, $start_c, $end_c, $strand_c) = @f; +} +else{ + print STDERR "Incorrect parameter: -f\n"; + die $usage; +} + +print STDERR "|--> Reading the Ref : $ref\n"; +if($ref=~m/\.gz$/){ + open IN,"<:gzip", "$ref" or die "cannot open the reference file:\t$ref\n"; +} +else{ + open IN, "<$ref" or die "cannot open the reference file:\t$ref\n"; +} +my %ref; +my $chr; + +while () { + chomp; + if (/>(\S+)/) { + $chr = $1; + } + else{ + $ref{$chr} .= $_; + } +} +close IN; + +open IN, "$reg" or die "cannot open the region file:\t$reg\n"; + +open OUT, ">$out" or die $!; + +print STDERR "|--> Extract the sequence ...\n"; + +while (){ + chomp; + next if /^\s*$/; + my @arr = split(/\s+/, $_); + my ($chr, $start, $end) = @arr[$chr_c, $start_c, $end_c]; + my $regionID = $regionID_c ? $arr[$regionID_c] : "R$."; + my $strand = $strand_c ? $arr[$strand_c] : "+"; + my $seq = substr($ref{$chr}, $start-1, $end - $start + 1); + print OUT ">$regionID:$chr:$start-$end:$strand\n"; + if($strand eq "-"){ + $seq =~tr/ACGTacgt/TGCAtgca/; + $seq = join("", reverse( split( //, $seq) ) )."\n"; + } + unless(defined $One){ + my $i=0; + while ( $i < length($seq) - $baseN ) { + print OUT substr($seq, $i, $baseN),"\n"; + $i += $baseN; + } + print OUT substr($seq, $i, ), "\n"; + } + else{ + print OUT "$seq\n"; + } +} +close IN; +close OUT; +print STDERR "|--> All done ...\n"; + diff --git a/script/z02.mC_seqlogo.sh b/script/z02.mC_seqlogo.sh new file mode 100644 index 0000000..4f02feb --- /dev/null +++ b/script/z02.mC_seqlogo.sh @@ -0,0 +1,22 @@ +sample=$1 +cout=$2 +outdir=$3 +ref=$4 +Rscript=$5 + +shell=$0 +Bin=${shell%/*} + + +for t in CG CHH CHG +do + ### get mc bedformat : 筛选 >=10x && methy >0.75 çš„ä½ç‚¹ + zcat $cout | awk -v c=$t 'BEGIN{OFS="\t"}{ if($4==c && $7+$8 > 9 && $6>0.75){n+=1; if($3=="+"){ print $1, $2-4, $2+4, $3, n; } } } ' > $outdir/$sample.seqlogo.m$t.bed + ### get sequences pwm + perl $Bin/z02.get_seq.pl -D $ref -R $outdir/$sample.seqlogo.m$t.bed -f 3-0-1-2-4 -O $outdir/$sample.seqlogo.m$t.fa + perl -lne 'next if /^>/; next if length($_)<9; @a=split(//, $_); foreach $i (0..8){ $h{$i}{$a[$i]}+=1;} END{ foreach $i (0..8){ $a = $h{$i}{"A"} ? $h{$i}{"A"} : 0; $c = $h{$i}{"C"} ? $h{$i}{"C"} : 0; $g = $h{$i}{"G"} ? $h{$i}{"G"} : 0; $t = $h{$i}{"T"} ? $h{$i}{"T"} : 0; print join "\t", $a, $c, $g, $t; } } ' $outdir/$sample.seqlogo.m$t.fa > $outdir/$sample.seqlogo.m$t.pwm + ### plot + $Rscript $Bin/z02.seqlogo.R $outdir/$sample.seqlogo.m$t.pwm T $outdir/$sample.m$t.seqlogo.pdf + rm -vf $outdir/$sample.seqlogo.m$t.bed $outdir/$sample.seqlogo.m$t.fa +done + diff --git a/script/z02.seqlogo.R b/script/z02.seqlogo.R new file mode 100644 index 0000000..e677b66 --- /dev/null +++ b/script/z02.seqlogo.R @@ -0,0 +1,30 @@ +args<-commandArgs(TRUE) +if(length(args) != 3){ + stop("seqlogo.R pwm [T|F] pdf + pwm : The W * 4 position frequence matrix: + 12 12 12 12 + 48 0 0 0 + 0 40 2 6 + 0 0 0 48 + 12 12 12 12 + ic.scale : T, the height of each column is proportional to its information content. + F, all columns have the same height. + pdf : the output of pdf.") +} +library(seqLogo) +a<-read.table(file=args[1]) +a<-t(a) +for( i in 1:ncol(a) ){ a[,i] = a[,i]/sum(a[,i]) } +pdf(file=args[3], width=15) +seqLogo(a,ic.scale=args[2]) +dev.off() + +######### Introduction for information content ######### +#The information content is measured in bits and, in the case of DNA sequences, ranges from 0 +#to 2 bits. A position in the motif at which all nucleotides occur with equal probability has an +#information content of 0 bits, while a position at which only a single nucleotide can occur has +#an information content of 2 bits. The information content at a given position can therefore be +#thought of as giving a measure of the tolerance for substitutions in that position: Positions +#that are highly conserved and thus have a low tolerance for substitutions correspond to high +#information content, while positions with a high tolerance for substitutions correspond to +#low information content. diff --git a/script/z03.global_pattern.R b/script/z03.global_pattern.R new file mode 100644 index 0000000..e4fe7c9 --- /dev/null +++ b/script/z03.global_pattern.R @@ -0,0 +1,46 @@ +args <- commandArgs(T) +file <- args[1] +chr <- args[2] +outpdf <- args[3] + +##### +rt <- read.table(file=file) +pdf(file=outpdf, height=6, width=17) + par(mar=c(4,6,5,6), las=1) + plot(0, type="n", + xlab="", xlim=c(0, rt[nrow(rt),1]), + ylim=c(-1, 1), ylab="CpG methylation level", + bty="n", yaxt="n", + main=chr) + abline(h=0) + axis(2, at=c(-1, -0.8, -0.6, -0.4, -0.2, 0, 0.2, 0.4, 0.6, 0.8, 1), + lab=c(1, 0.8, 0.6, 0.4, 0.2, 0, 0.2, 0.4, 0.6, 0.8, 1), + col="royalblue") + axis(4, at=c(-1, -0.8, -0.6, -0.4, -0.2, 0, 0.2, 0.4, 0.6, 0.8, 1), + lab=c(100, 80, 60, 40, 20, 0, 20, 40, 60, 80, 100), + col="green3") + mtext(side=4, "GC content (%)", las=3, line=3) + + points(rt[,1], rt[,2], type="h", col=rgb(0.2,0.8,0.2,0.1), lwd=0.1, lend=2) + points(rt[,1], -rt[,2], type="h", col=rgb(0.2,0.8,0.2,0.1), lwd=0.1, lend=2) + + ## CG + s<-smooth.spline(rt[,1], rt[,3],df=100) + points(rt[,1], s$y, type="l", col="royalblue", lwd=2, xpd=T) + s<-smooth.spline(rt[,1], -rt[,6],df=100) + points(rt[,1], s$y, type="l", col="royalblue", lwd=2, xpd=T) + + ## CHG + s<-smooth.spline(rt[,1], rt[,4],df=100) + points(rt[,1], s$y, type="l", col="firebrick", lwd=2, xpd=T) + s<-smooth.spline(rt[,1], -rt[,7],df=100) + points(rt[,1], s$y, type="l", col="firebrick", lwd=2, xpd=T) + + ## CHH + s<-smooth.spline(rt[,1], rt[,5]*50,df=100) + points(rt[,1], s$y, type="l", col="gold", lwd=2, xpd=T) + s<-smooth.spline(rt[,1], -rt[,8]*50,df=100) + points(rt[,1], s$y, type="l", col="gold", lwd=2, xpd=T) + + legend("topright", c("CG", "CHG", "CHH"), col=c("royalblue", "firebrick", "gold"), lwd=2,bty="n") +dev.off() diff --git a/script/z03.global_pattern.pl b/script/z03.global_pattern.pl new file mode 100644 index 0000000..75feb60 --- /dev/null +++ b/script/z03.global_pattern.pl @@ -0,0 +1,88 @@ +#!/usr/bin/perl +use strict; +use warnings; +use PerlIO::gzip; +use Getopt::Long; +use FindBin qw($Bin $Script); + +my $usage=<<"USAGE"; +name: $0 +usage: perl $0 + -I file input *.cout + -N name chr name + -B number the statistics window (bp), default 10000; + -O [str] dir output dir of file + -R [str] prom path of Rscript +author: luhanlin\@genomics.org.cn +date: +USAGE + +my ($help, $cout, $bin,$name, $outdir, $Rscript); + +GetOptions( + "H|help|?" => \$help, + "I=s" => \$cout, + "N:s" => \$name, + "B=i" => \$bin, + "O:s" => \$outdir, + "R:s" => \$Rscript, +); + +$name ||= "test"; +$outdir ||= "."; +$Rscript ||= "/public/home/zhangdu/src/R-3.4.2/builddir/bin/Rscript"; +$bin ||=10000; + +if($help){ + print $usage; + exit 0; +} + +unless($cout){ + print $usage; + exit 1; +} + +if($cout=~/\.gz$/){ + open IN, "<:gzip", $cout or die $!; +} +else{ + open IN, $cout or die $!; +} + +my %n; +my %m; +my %cg_content; + +while(){ + next if /^#/; + chomp; + my @a = split(/\t/, $_); + next unless $a[0] eq $name; + my $d += $a[6] + $a[7]; + my $win = int( $a[1] / $bin );# 常用æ¥ç¡®å®šå¯¹æ•°æ®è¿›è¡Œçª—å£åŒ–分的方法 + $n{$win}{$a[3]}{$a[2]} += $d; #窗å£å†…测到的CG总次数 + $m{$win}{$a[3]}{$a[2]} += $a[6];#窗å£å†…测到mCG的总次数 + $cg_content{$win} += 1; #窗å£å†…çš„CG总数 +} +close IN; + +open OA, ">$outdir/$name.global_pattern.xls" or die $!; +print OA "##bins\tcg_content\tcg+\tchg+\tchh+\tcg-\tchg-\tchh-\n"; +foreach my $win (sort{$a<=>$b} keys %n){ + + my $cgcont = $cg_content{$win} ? sprintf("%.6f", $cg_content{$win} / $bin) : 0; #CGå«é‡ï¼šCG总数/窗å£é•¿åº¦ + + my $cg_p = $n{$win}{"CG"}{"+"} ? sprintf("%.6f", $m{$win}{"CG"}{"+"} / $n{$win}{"CG"}{"+"}) : 0; #窗å£å†…正链CG甲基化水平 + my $chg_p = $n{$win}{"CHG"}{"+"} ? sprintf("%.6f", $m{$win}{"CHG"}{"+"} / $n{$win}{"CHG"}{"+"}) : 0; + my $chh_p = $n{$win}{"CHH"}{"+"} ? sprintf("%.6f", $m{$win}{"CHH"}{"+"} / $n{$win}{"CHH"}{"+"}) : 0; + + my $cg_n = $n{$win}{"CG"}{"-"} ? sprintf("%.6f", $m{$win}{"CG"}{"-"} / $n{$win}{"CG"}{"-"}) : 0;#窗å£å†…负链CG甲基化水平 + my $chg_n = $n{$win}{"CHG"}{"-"} ? sprintf("%.6f", $m{$win}{"CHG"}{"-"} / $n{$win}{"CHG"}{"-"}) : 0; + my $chh_n = $n{$win}{"CHH"}{"-"} ? sprintf("%.6f", $m{$win}{"CHH"}{"-"} / $n{$win}{"CHH"}{"-"}) : 0; + print OA join("\t", $win * $bin, $cgcont, $cg_p, $chg_p, $chh_p, $cg_n, $chg_n, $chh_n), "\n"; +} +close OA; + +system("$Rscript $Bin/z03.global_pattern.R $outdir/$name.global_pattern.xls $name $outdir/$name.global_pattern.pdf"); +## diff --git a/script/z04.elements_methyl.R b/script/z04.elements_methyl.R new file mode 100644 index 0000000..c1e7be3 --- /dev/null +++ b/script/z04.elements_methyl.R @@ -0,0 +1,83 @@ +args <- commandArgs(T) +indir <- args[1] +elementlist <- args[2] +outprefix <- args[3] +sample <- args[4] + +elementlist1<- unlist(strsplit(elementlist,split="-")) +#elementlist2<-paste("genome",elementlist1,"methy.txt",sep=".") +#elementdir <- paste(indir,elementlist2,sep="/") + +a <- matrix(c(1,1),nrow=1) +for(i in elementlist1){ + elementlist2<-paste(sample,"genome",i,"methy.txt",sep=".") + elementdir <- paste(indir,elementlist2,sep="/") + print(paste("Reading methylation information:",i,sep=" ")) + x1<-read.table(elementdir,sep="\t") + x1<-as.matrix(x1) + xa<-x1[,ncol(x1)] + xb<-matrix(xa,ncol=1) + a<- rbind(a,cbind(i,xb)) +} + +#col <- rainbow(length(elementlist1)) +colo <- c("red3","green4","yellow3","purple3","blue3","orange3","pink3","deepskyblue","grey") +color <- colo[1:length(elementlist1)] +a <-a[-1,] +colnames(a)<-c("Element","Methylation_level") +a <- na.omit(a) +a <- as.data.frame(a) + +#write.table(a,file="plot.txt") +#a<-read.table("plot.txt") + +library(ggplot2) +pdf(file=paste(outprefix,"boxplot.pdf",sep=".")) +a$Methylation_level<-as.numeric(as.vector(a$Methylation_level)) +ggplot(a, aes(x=Element, y=Methylation_level,fill=Element)) + +geom_boxplot(outlier.size=0.0015,outlier.alpha=0.3,outlier.color="red") + +theme_minimal() + +stat_summary(fun.y=median, geom="point", size=2,shape=22,fill="white") + + #annotate("text", label = paste("DMR = ",n), x = 2, y = 1, size = 5) + +theme(panel.border = element_rect(fill = NA)) + +theme(axis.text.x = element_text(angle = 45,hjust = 1)) + +labs(title = paste("Sample",sample,"element methylation level distribution",sep=" ")) + +dev.off() + +pdf(file=paste(outprefix,"density_plot.pdf",sep=".")) +p <- ggplot(a, aes(x=Methylation_level)) +p <- p + geom_density(aes(fill=Element,color=Element),alpha=0.6) +p <- p + facet_grid(Element~.) +p +dev.off() + +pdf(file=paste(outprefix,"vioplot.pdf",sep=".")) +e <- ggplot(data = a, aes(x=Element, y=Methylation_level)) +e <- e + geom_violin(alpha=0.4,aes(fill=Element,color=Element)) +e + geom_boxplot(width=0.01,outlier.size=0.0005,outlier.alpha=0.3,outlier.color="red") +dev.off() + +library(ggridges) +pdf(file=paste(outprefix,"ridges.pdf",sep=".")) +ggplot(a, aes(x=Methylation_level, y=Element, fill=..x..))+ + geom_density_ridges_gradient(scale=3, rel_min_height=0.01, gradient_lwd = 1.)+ + scale_x_continuous(breaks=seq(0,1,0.1))+ + scale_y_discrete(expand = c(0.01,0))+ + labs(title=paste("Sample",sample,"element methylation level distribution",sep=" "))+ + theme_ridges(font_size = 13, grid = FALSE)+ + theme(axis.title.y = element_blank()) +dev.off() + +pdf(file=paste(outprefix,"ridges2.pdf",sep=".")) +ggplot(a, aes(x=Methylation_level, y=Element, fill=Element))+ + geom_density_ridges(scale=4,alpha=0.4)+ + scale_x_continuous(breaks=seq(0,1,0.1))+ + scale_y_discrete(expand = c(0.01,0))+ + scale_fill_cyclical(values = colo)+ + labs(title=paste("Sample",sample,"element methylation level distribution",sp=" "))+ + theme_ridges(font_size = 13, grid = FALSE) + + xlim(0,1)+ + theme(axis.title.y = element_blank()) +dev.off() + diff --git a/script/z04.elements_methyl.pl b/script/z04.elements_methyl.pl new file mode 100644 index 0000000..e7bc1c1 --- /dev/null +++ b/script/z04.elements_methyl.pl @@ -0,0 +1,90 @@ +#!/usr/bin/perl +use strict; +use warnings; +use PerlIO::gzip; +use Getopt::Long; +use Cwd 'abs_path'; +my $usage=<<"USAGE"; + name: rrbs_GlobalMethy_batch + It is designed for running Global Methylation profile of BS-seq in batch model. + (note: global used all sites; element used >=5x sites) + + version:1.0 + usage: perl $0 + -c cout file; + -b element.bed file; "global" for genome wide stat; + -t C type: CG,CHG or CHH; + -o out file; + -h help; + author: zhangdu + date: 2018-05-29 +USAGE + + + +my ($cout, $bed, $out, $cytosine,$help); +my %h; +GetOptions("b=s" => \$bed, + "c=s" => \$cout, + "o=s" => \$out, + "t=s" => \$cytosine, + "h=s" => \$help +); +die $usage if $help; +die $usage unless $bed; +die $usage unless $cout; +die $usage unless $out; +die $usage unless $cytosine; + +if( $bed eq "global"){ + open IN,"<:gzip", $cout or die $!; + while(){ + chomp; + my @a = split; + #next if $a[0] ne $chr; + next if($a[3] ne $cytosine); + my $d = $a[6] + $a[7]; + $h{"d"} += $d; + $h{"m"} += $a[6] + } + close IN; + my $methyl = $h{"d"} == 0 ? "NA" : sprintf("%.6f", $h{"m"} / $h{"d"}); + open OUT, ">$out" or die $!; + print OUT join("\t", "Global", $h{"m"}, $h{"d"}, $methyl), "\n"; #global的甲基化水平是用全基因组 总mC/C算出æ¥çš„ + # global C总深度 mC总深度 mC总深度/C总深度 + close OUT; +}else{ + + open IN,"<:gzip", $cout or die " !! can not open file: $cout \n"; + while(){ + chomp; + my @a = split; + #next if $a[0] ne $chr; + next if $a[3] ne $cytosine; + my $d = $a[6] + $a[7]; + next if $d < 5; + $h{$a[0]}{$a[1]} = $a[5]; # >=5x Cä½ç‚¹çš„ç”²åŸºåŒ–æ°´å¹³è®°å½•ä¸‹æ¥ + } + close IN; + + open OUT, ">$out" or die $!; + open BB, $bed or die " !! can not open file: $bed \n"; + while(){ + chomp; + my @a = split; + #next if $a[0] ne $chr; + my $n = 0; + my $m = 0; + foreach my $i ($a[1]..$a[2]){ + if(exists $h{$a[0]}{$i}){ + $n += 1; + $m += $h{$a[0]}{$i}; + } + } + my $meth = $n== 0 ? "NA" : sprintf("%.6f", $m/$n); + print OUT join("\t", $_, $meth),"\n"; # 输出bed中æ¯æ¡åºåˆ—çš„å¹³å‡ç”²åŸºåŒ–æ°´å¹³(>=5xæ‰ç®—) + } + close BB; + close OUT; +} + diff --git a/script/z04.elements_methyl.repeats.R b/script/z04.elements_methyl.repeats.R new file mode 100644 index 0000000..8e8fada --- /dev/null +++ b/script/z04.elements_methyl.repeats.R @@ -0,0 +1,90 @@ +args <- commandArgs(T) +indir <- args[1] +elementlist <- args[2] +outprefix <- args[3] +sample <- args[4] + +elementlist1<- unlist(strsplit(elementlist,split="-")) +#elementlist2<-paste("genome",elementlist1,"methy.txt",sep=".") +#elementdir <- paste(indir,elementlist2,sep="/") + +a <- matrix(c(1,1),nrow=1) +for(i in elementlist1){ + elementlist2<-paste(sample,"Repeats",i,"methy.txt",sep=".") + elementdir <- paste(indir,elementlist2,sep="/") + print(paste("Reading methylation information:",i,sep=" ")) + x1<-read.table(elementdir) + x1<-as.matrix(x1) + xa<-x1[,ncol(x1)] + xb<-matrix(xa,ncol=1) + a<- rbind(a,cbind(i,xb)) +} + +#col <- rainbow(length(elementlist1)) +colo <- c("red3","green4","yellow3","purple3","blue3","orange3","pink3","deepskyblue","grey") +color <- colo[1:length(elementlist1)] +a <-a[-1,] +colnames(a)<-c("Element","Methylation_level") +a <- na.omit(a) +a <- as.data.frame(a) + +#write.table(a,file="plot.txt") +#a<-read.table("plot.txt") + +library(ggplot2) +pdf(file=paste(outprefix,"boxplot.pdf",sep=".")) +a$Methylation_level<-as.numeric(as.vector(a$Methylation_level)) +ggplot(a, aes(x=Element, y=Methylation_level,fill=Element)) + +geom_boxplot(outlier.size=0.0015,outlier.alpha=0.3,outlier.color="red") + +theme_minimal() + +stat_summary(fun.y=median, geom="point", size=2,shape=22,fill="white") + + #annotate("text", label = paste("DMR = ",n), x = 2, y = 1, size = 5) + +theme(panel.border = element_rect(fill = NA)) + +theme(axis.text.x = element_text(angle = 45,hjust = 1)) + +labs(title = paste("Sample",sample,"element methylation level distribution",sep=" ")) + +dev.off() + +pdf(file=paste(outprefix,"density_plot.pdf",sep=".")) +p <- ggplot(a, aes(x=Methylation_level)) +p <- p + geom_density(aes(fill=Element,color=Element),alpha=0.6) +p <- p + facet_grid(Element~.) +p +dev.off() + +pdf(file=paste(outprefix,"density_plot.pdf",sep=".")) +p <- ggplot(a, aes(x=Methylation_level)) +p <- p + geom_density(aes(fill=Element,color=Element),alpha=0.6) +p <- p + facet_grid(Element~.) +p +dev.off() + +pdf(file=paste(outprefix,"vioplot.pdf",sep=".")) +e <- ggplot(data = a, aes(x=Element, y=Methylation_level)) +e <- e + geom_violin(alpha=0.4,aes(fill=Element,color=Element)) +e + geom_boxplot(width=0.01,outlier.size=0.0005,outlier.alpha=0.3,outlier.color="red") +dev.off() + +library(ggridges) +pdf(file=paste(outprefix,"ridges.pdf",sep=".")) +ggplot(a, aes(x=Methylation_level, y=Element, fill=..x..))+ + geom_density_ridges_gradient(scale=3, rel_min_height=0.01, gradient_lwd = 1.)+ + scale_x_continuous(breaks=seq(0,1,0.1))+ + scale_y_discrete(expand = c(0.01,0))+ + labs(title=paste("Sample",sample,"element methylation level distribution",sep=" "))+ + theme_ridges(font_size = 13, grid = FALSE)+ + theme(axis.title.y = element_blank()) +dev.off() + +pdf(file=paste(outprefix,"ridges2.pdf",sep=".")) +ggplot(a, aes(x=Methylation_level, y=Element, fill=Element))+ + geom_density_ridges(scale=4,alpha=0.4)+ + scale_x_continuous(breaks=seq(0,1,0.1))+ + scale_y_discrete(expand = c(0.01,0))+ + scale_fill_cyclical(values = colo)+ + labs(title=paste("Sample",sample,"element methylation level distribution",sp=" "))+ + theme_ridges(font_size = 13, grid = FALSE) + + xlim(0,1)+ + theme(axis.title.y = element_blank()) +dev.off() + diff --git a/script/z04.elements_methyl.sh b/script/z04.elements_methyl.sh new file mode 100644 index 0000000..29e5d71 --- /dev/null +++ b/script/z04.elements_methyl.sh @@ -0,0 +1,67 @@ +#### average methlation level for gene elements of samples + +# Usage: +# z04.elements_methyl.sh <"cgi" or ""> +# +# details: +# generally analysis "promoter down1k down2k exon genebody intergenic intron up2k up1k"; +# add "cgi" in args 6 if you want to analysis "cgi cgi_shelf cgi_shore". + +shell=$0 +Bin=${shell%/*} + + ele_dir=$1 + cout=$2 + outdir=$3 + type=$4 + sample=$5 + Rscript=$6 + CGI=$7 + + + mkdir -p $outdir + +### global +#echo -e "|-----Start analysis $sample of global $type methylation level..." +#echo `date` +#perl $Bin/z04.elements_methyl.pl -c $cout -b global -t $type -o $outdir/$sample.$type.global_methy.txt +#echo "-----|done..." + +### cgi +if [ $CGI ];then + for i in cgi cgi_shelf cgi_shore + #for i in cgi cgi_shore + do + echo -e "##################################################\n|-----Start analysis $sample $i methylation level..." + echo `date` + echo -e "[COMAND]: perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t CG -o $outdir/$sample.genome.$i.methy.txt" + perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t CG -o $outdir/$sample.genome.$i.methy.txt + echo "-----|done..." + done + #plot + echo -e "|-----Violin plotting...\n" + echo -e "[COMAND]: $Rscript $Bin/z04.elements_methyl.R $outdir cgi-cgi_shelf-cgi_shore $sample.genome.CGI $sample" + $Rscript $Bin/z04.elements_methyl.R $outdir cgi-cgi_shelf-cgi_shore $outdir/$sample.genome.CG $sample +fi + + +##### genic +#for i in downstream2k downstream5k exon genebody intergenic intron upstream2k upstream5k +for i in promoter down1k down2k exon genebody intergenic intron up2k up1k 5utr 3utr cds exon1 intron1 +do + echo -e "##################################################\n|-----Start analysis $sample $i methylation level..." + echo `date` + perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t $type -o $outdir/$sample.genome.$i.methy.txt + echo -e "[COMAND]: perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t $type -o $outdir/$sample.genome.$i.methy.txt" + echo "-----|done..." +done +echo -e "|-----Violin plotting...\n" +echo -e "[COMAND]: $Rscript $Bin/z04.elements_methyl.R $outdir promoter-down1k-down2k-exon-genebody-intergenic-intron-up2k-up1k-intron1-exon1 $sample.genome.genetic $sample" +$Rscript $Bin/z04.elements_methyl.R $outdir promoter-down1k-down2k-exon-genebody-intergenic-intron-up2k-up1k-5utr-3utr-cds-intron1-exon1 $outdir/$sample.genome.genetic $sample + + +## repeat +for j in DNA LINE Low_complexity LTR RNA Satellite Simple_repeat SINE +#do +# perl $p1 chr$i.combinedCpG.filter.txt $d/repeat/$j.mbed chr$i > z02_stat/chr$i.repeat.$j.txt +#done diff --git a/script/z04.elements_methyl.v1.1.sh b/script/z04.elements_methyl.v1.1.sh new file mode 100644 index 0000000..acd16ef --- /dev/null +++ b/script/z04.elements_methyl.v1.1.sh @@ -0,0 +1,75 @@ +#### average methlation level for gene elements of samples + +# Usage: +# z04.elements_methyl.sh <"cgi" or ""> +# +# details: +# generally analysis "promoter down1k down2k exon genebody intergenic intron up2k up1k"; +# add "cgi" in args 6 if you want to analysis "cgi cgi_shelf cgi_shore". + +shell=$0 +Bin=${shell%/*} + + ele_dir=$1 + cout=$2 + outdir=$3 + type=$4 + sample=$5 + Rscript=$6 + CGI=$7 + + + mkdir -p $outdir + +### global +#echo -e "|-----Start analysis $sample of global $type methylation level..." +#echo `date` +#perl $Bin/z04.elements_methyl.pl -c $cout -b global -t $type -o $outdir/$sample.$type.global_methy.txt +#echo "-----|done..." + +### cgi +if [ $CGI ];then + for i in cgi cgi_shelf cgi_shore + #for i in cgi cgi_shore + do + echo -e "##################################################\n|-----Start analysis $sample $i methylation level..." + echo `date` + echo -e "[COMAND]: perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t CG -o $outdir/$sample.genome.$i.methy.txt" + perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t CG -o $outdir/$sample.genome.$i.methy.txt + echo "-----|done..." + done + #plot + echo -e "|-----Violin plotting...\n" + echo -e "[COMAND]: $Rscript $Bin/z04.elements_methyl.R $outdir cgi-cgi_shelf-cgi_shore $sample.genome.CGI $sample" + $Rscript $Bin/z04.elements_methyl.R $outdir cgi-cgi_shelf-cgi_shore $outdir/$sample.genome.CG $sample +fi + + +##### genic +#for i in downstream2k downstream5k exon genebody intergenic intron upstream2k upstream5k +for i in promoter down1k down2k exon genebody intergenic intron up2k up1k 5utr 3utr cds exon1 intron1 +do + echo -e "##################################################\n|-----Start analysis $sample $i methylation level..." + echo `date` + perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t $type -o $outdir/$sample.genome.$i.methy.txt + echo -e "[COMAND]: perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/genome.$i.bed -t $type -o $outdir/$sample.genome.$i.methy.txt" + echo "-----|done..." +done +echo -e "|-----Violin plotting...\n" +echo -e "[COMAND]: $Rscript $Bin/z04.elements_methyl.R $outdir promoter-down1k-down2k-exon-genebody-intergenic-intron-up2k-up1k-intron1-exon1 $sample.genome.genetic $sample" +$Rscript $Bin/z04.elements_methyl.R $outdir promoter-down1k-down2k-exon-genebody-intergenic-intron-up2k-up1k-5utr-3utr-cds-intron1-exon1 $outdir/$sample.genome.genetic $sample + + +## repeat +for j in DNA LINE LTR Retroposon RNA Satellite SINE Low_complexity Simple_repeat +do + echo -e "##################################################\n|-----Start analysis $sample $j methylation level..." + echo `date` + perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/Repeats.$j.bed -t $type -o $outdir/$sample.Repeats.$j.methy.txt + echo -e "[COMAND]: perl $Bin/z04.elements_methyl.pl -c $cout -b $ele_dir/Repeats.$j.bed -t $type -o $outdir/$sample.Repeats.$j.methy.txt" + echo "-----|done..." +done +echo -e "|-----Violin plotting...\n" +echo -e "[COMAND]: $Rscript $Bin/z04.elements_methyl.repeats.R $outdir DNA-LINE-LTR-Retroposon-RNA-Satellite-SINE-Low_complexity-Simple_repeat $sample.genome.repeats $sample" +$Rscript $Bin/z04.elements_methyl.repeats.R $outdir 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