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revamp.sh
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#!/bin/bash
#NOTES: Require users to supply raw sequence data files in format "samplename_R[12].fastq.gz".
## These names should be identical to names in the sample metadata file.
## Names will be cleaned of illegal characters (only alphanumeric and underline allowed).
## This cleaning step will also be applied to the sample names in the metadata file (so they are the same).
#
#NO spaces or quotes or other weird characters in file paths
#revamp.sh must be in PATH
#Pro tip: steps that use memory only use 70% of what max you give
#Will need nt and taxonomydmp - run prepscript beforehand
#Can provide own blastn output on whatever database. Must be formatted -outfmt '6 qseqid pident length staxids'
unset parameterfilepath
unset samplemetafilepath
unset readfolderpath
unset outdirectory
unset optionalUserBLASTResult
unset figureparamfilepath
unset silvangsInputExportFile
unset silvangsInputClusterFile
unset silvaRefMap
workingdirectory=`pwd`
unset revampdir
unset tempprogdir
tempprogdir=`which revamp.sh`
revampdir=`echo $tempprogdir | sed -E 's/\/revamp\.sh$//'`
myInvocation="$(printf %q "$BASH_SOURCE")$((($#)) && printf ' %q' "$@")"
##########################################################################################
##
## Get command line options
##
##########################################################################################
pflag=0
sflag=0
rflag=0
oflag=0
fflag=0
cflag=0
blastflag=FALSE
silvaASVflag=FALSE
bypassflag=FALSE
keepIntermediateFiles=TRUE
while getopts ":p:s:r:o:b:f:yke" opt; do
case ${opt} in
p ) pflag=1
parameterfilepath=$OPTARG #revamp_config.txt (see README)
;;
s ) sflag=1
samplemetafilepath=$OPTARG #Sample metadata file (see README)
;;
r ) rflag=1
readfolderpath=$OPTARG #Folder with fastq.gz raw read files
;;
o ) oflag=1
outdirectory=$OPTARG #Location for output files
outdirectory=`echo $outdirectory | sed -E 's/\/$//'`
;;
f ) fflag=1
figureparamfilepath=$OPTARG #Location of figure config file (see README)
;;
b ) blastflag=TRUE
optionalUserBLASTResult=$OPTARG #Location of user blastn input (optional)
;;
e ) silvaASVflag=TRUE
;;
y ) bypassflag=TRUE
;;
k ) keepIntermediateFiles=FALSE
;;
\? ) echo "Invalid option: -$OPTARG"
echo "Usage: revamp.sh" #Invalid option provided
echo " -p Config File"
echo " -f Figure config file"
echo " -s Sample metadata file"
echo " -r Read folder"
echo " -o Output directory"
echo " -b User-supplied BLASTn btab result file (optional)"
echo " -e Toggle use of SILVAngs taxonomy assignments by ASV (optional)"
echo " -y Bypass all terminal prompts (optional)"
echo " -k Remove all intermediate files (optional)"
echo " Not recommended for first run"
echo " (best to rerun with -k after successful completion)"
echo " See README for details"
exit
;;
: ) echo "Option is missing an argument: -$OPTARG"
echo "Usage: revamp.sh" #Arg for a called option not provided
echo " -p Config File"
echo " -f Figure config file"
echo " -s Sample metadata file"
echo " -r Read folder"
echo " -o Output directory"
echo " -b User-supplied BLASTn btab result file (optional)"
echo " -e Toggle use of SILVAngs taxonomy assignments by ASV (optional)"
echo " -y Bypass all terminal prompts (optional)"
echo " -k Remove all intermediate files (optional)"
echo " Not recommended for first run"
echo " (best to rerun with -k after successful completion)"
echo " See README for details"
exit
;;
esac
done
shift $((OPTIND -1))
if [ $OPTIND -eq 1 ]
then echo "Usage: revamp.sh" #No options passed
echo " -p Config File"
echo " -f Figure config file"
echo " -s Sample metadata file"
echo " -r Read folder"
echo " -o Output directory"
echo " -b User-supplied BLASTn btab result file (optional)"
echo " -e Toggle use of SILVAngs taxonomy assignments by ASV (optional)"
echo " -y Bypass all terminal prompts (optional)"
echo " -k Remove all intermediate files (optional)"
echo " Not recommended for first run"
echo " (best to rerun with -k after successful completion)"
echo " See README for details"
exit
fi
if [[ $pflag -eq 0 || $sflag -eq 0 || $rflag -eq 0 || $oflag -eq 0 || $fflag -eq 0 ]]
then echo "All options except -b, -y, and -k are required."
echo "Usage: revamp.sh" #Missing required options
echo " -p Config File"
echo " -f Figure config file"
echo " -s Sample metadata file"
echo " -r Read folder"
echo " -o Output directory"
echo " -b User-supplied BLASTn btab result file (optional)"
echo " -e Toggle use of SILVAngs taxonomy assignments by ASV (optional)"
echo " -y Bypass all terminal prompts (optional)"
echo " -k Remove all intermediate files (optional)"
echo " Not recommended for first run"
echo " (best to rerun with -k after successful completion)"
echo " See README for details"
exit
fi
##########################################################################################
##########################################################################################
##########################################################################################
##
## Get program config file options
##
##########################################################################################
unset primerF
unset primerR
unset blastLengthCutoff
unset systemmemoryMB
unset locationNTdatabase
unset taxonomyCutoffs
unset dada_minlength
unset dada_phix
unset dada_trunQ
unset dada_maxEE1
unset dada_maxEE2
unset dada_trimRight
unset dada_trimLeft
unset blastMode
failedMerge_useDirection=NA
removeASVsFILE=NULL
source $parameterfilepath
temp_holder=$taxonomyCutoffs
taxonomyCutoffs=`echo $temp_holder | sed -E 's/[^0-9]/,/g'`
#TEMP Holding:
#echo "<<<$taxonomyCutoffs>>>"
#sed -E 's/^\D//' | sed -E 's/\D$//'
##########################################################################################
##########################################################################################
##########################################################################################
##
## Cleanup and prep
##
##########################################################################################
cutadaptFinished=FALSE
dada2_Finished=FALSE
blastFinished=FALSE
blastformattingFinished=FALSE
taxonomyscriptFinished=FALSE
figuresFinished=FALSE
if [ -d "${outdirectory}" ]; then
configcompare1=`cat ${parameterfilepath}`
configcompare2=`cat ${outdirectory}/config_file.txt`
samplecompare1=`cat ${samplemetafilepath}`
samplecompare2=`cat ${outdirectory}/sample_metadata.txt`
if [[ "${configcompare1}" = "${configcompare2}" ]]; then
echo "Config files identical"
else
echo "Config files differ between runs, choose a different out directory"
exit
fi
if [[ "${samplecompare1}" = "${samplecompare2}" ]]; then
echo "Sample metadata files identical"
else
echo "Sample metadata files differ between runs, choose a different out directory"
exit
fi
source ${outdirectory}/progress.txt
mkdir -p ${outdirectory}/run_logs
currenttime=`date | sed -E 's/[^A-Za-z0-9_]/_/g'`
mv ${outdirectory}/run.log ${outdirectory}/run_logs/runlog_${currenttime}.txt
touch ${outdirectory}/run.log
echo >> ${outdirectory}/Rscript_arguments.log
echo "New Run" >> ${outdirectory}/Rscript_arguments.log
else
mkdir ${outdirectory}
touch ${outdirectory}/progress.txt
touch ${outdirectory}/run.log
echo "First Run" >> ${outdirectory}/Rscript_arguments.log
cp ${parameterfilepath} ${outdirectory}/config_file.txt
cp ${samplemetafilepath} ${outdirectory}/sample_metadata.txt
fi
exec &> >(tee -a ${outdirectory}/run.log)
echo
echo "Start of run:"
date
echo
echo "Invoked script options:"
echo "$myInvocation"
echo
#Clean raw read files to remove illegal characters
cd ${readfolderpath}
for f in *fastq.gz
do cleanprior=`echo $f | sed -E 's/^MP_//'`
base=$(basename $cleanprior .fastq.gz)
newname=`echo ${base} | sed -E 's/[^A-Za-z0-9_]/_/g'`
mv $f MP_${newname}.fastq.gz
done
cd ${workingdirectory}
#Create sample metadata file with identical manipulation of sample names for downstream R work
perl ${revampdir}/assets/sampleMetadata_fileCleanup.pl -i ${samplemetafilepath} > ${outdirectory}/sample_metadata_forR.txt
#Create ordered sample name file
cat ${outdirectory}/sample_metadata_forR.txt | cut -f1 | grep -v "Sample" > ${outdirectory}/sample_order.txt
##########################################################################################
##########################################################################################
##########################################################################################
##
## Run CUTADAPT (v.3.7)
##
##########################################################################################
if [[ "${cutadaptFinished}" = "TRUE" ]]; then
echo "Cutadapt from prior run"
else
echo "Running Cutadapt: `date`"
if [ -d "${outdirectory}/cutadapt" ]; then
rm -r ${outdirectory}/cutadapt
mkdir ${outdirectory}/cutadapt
else
mkdir ${outdirectory}/cutadapt
fi
#Reverse complement primers (I is converted to N; T/U ok)
revcomp_primerF=`echo $primerF | tr ACGTUWSMKRYBDHVNIacgtuwsmkrybdhvni TGCAAWSKMYRVHDBNNtgcaawskmyrvhdbnn | rev`
revcomp_primerR=`echo $primerR | tr ACGTUWSMKRYBDHVNIacgtuwsmkrybdhvni TGCAAWSKMYRVHDBNNtgcaawskmyrvhdbnn | rev`
temp_primerF=`echo $primerF | tr Ii Nn`
temp_primerR=`echo $primerR | tr Ii Nn`
primerF=$temp_primerF
primerR=$temp_primerR
#Run cutadapt
if [[ "${failedMerge_useDirection}" = "NA" ]]; then
for sample in $(cat ${outdirectory}/sample_order.txt)
do
echo "======================================" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo "On sample: $sample" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo "======================================" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
cutadapt -a "${primerF};required...${revcomp_primerR};optional" \
-A "${primerR};required...${revcomp_primerF};optional" \
--discard-untrimmed \
-j 0 \
-m 1 \
-o ${outdirectory}/cutadapt/${sample}_R1_trimmed.fq.gz -p ${outdirectory}/cutadapt/${sample}_R2_trimmed.fq.gz \
${readfolderpath}/${sample}_R1.fastq.gz ${readfolderpath}/${sample}_R2.fastq.gz \
>> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt 2>&1
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
done
echo "Finished Cutadapt: `date`"
fi
if [[ "${failedMerge_useDirection}" = "FORWARD" ]]; then
for sample in $(cat ${outdirectory}/sample_order.txt)
do
echo "======================================" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo "On sample: $sample" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo "======================================" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
cutadapt -a "${primerF};required...${revcomp_primerR};optional" \
--discard-untrimmed \
-j 0 \
-m 1 \
-o ${outdirectory}/cutadapt/${sample}_R1_trimmed.fq.gz \
${readfolderpath}/${sample}_R1.fastq.gz \
>> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt 2>&1
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
done
echo "Finished Cutadapt: `date`"
fi
if [[ "${failedMerge_useDirection}" = "REVERSE" ]]; then
for sample in $(cat ${outdirectory}/sample_order.txt)
do
echo "======================================" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo "On sample: $sample" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo "======================================" >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
cutadapt -a "${primerR};required...${revcomp_primerF};optional" \
--discard-untrimmed \
-j 0 \
-m 1 \
-o ${outdirectory}/cutadapt/${sample}_R2_trimmed.fq.gz \
${readfolderpath}/${sample}_R2.fastq.gz \
>> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt 2>&1
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
echo >> ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt
done
echo "Finished Cutadapt: `date`"
fi
#print cutadapt stats
echo "Sample Passing Reads Passing bp"
paste ${outdirectory}/sample_order.txt <(grep "passing" ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt | cut -f3 -d "(" | tr -d ")") <(grep "filtered" ${outdirectory}/cutadapt/cutadapt_primer_trimming_stats.txt | cut -f3 -d "(" | tr -d ")")
if [[ "$bypassflag" = "FALSE" ]]; then
echo
echo "Please check Cutadapt success. Proceed? [y/n]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, exiting"
exit
fi
fi
echo "cutadaptFinished=TRUE" >> ${outdirectory}/progress.txt
fi
#TODO: Deal with samples where ALL reads filtered?
##########################################################################################
##
## Run DADA2
##
##########################################################################################
if [[ "${dada2_Finished}" = "TRUE" ]]; then
echo "DADA2 from prior run"
else
echo
echo "Running DADA2: `date`"
if [ -d "${outdirectory}/dada2" ]; then
rm -r ${outdirectory}/dada2
mkdir ${outdirectory}/dada2
else
mkdir ${outdirectory}/dada2
fi
echo "Trim and filter in DADA2..."
if [[ "${failedMerge_useDirection}" = "NA" ]]; then
dada2continue=FALSE
while [ $dada2continue = FALSE ]
do
Rscript --vanilla ${revampdir}/assets/dada2_step1.R ${workingdirectory}/${outdirectory}/dada2 $dada_minlength $dada_phix $dada_trunQ $dada_maxEE1 $dada_maxEE2 $dada_trimRight $dada_trimLeft \
1>> ${workingdirectory}/${outdirectory}/dada2/dada2_rscripts_out.log 2>&1
echo "dada2_step1.R ${workingdirectory}/${outdirectory}/dada2 $dada_minlength $dada_phix $dada_trunQ $dada_maxEE1 $dada_maxEE2 $dada_trimRight $dada_trimLeft" >> ${outdirectory}/Rscript_arguments.log
echo
echo "DADA2 Filtering results:"
echo "Sample % Reads Passing"
awk 'NR>1 { print $1, 100 * ( $3 / $2 ) }' ${workingdirectory}/${outdirectory}/dada2/filtered_out_stats.txt
echo
echo "Parameters to modify:"
echo "minLen,rm.phix,truncQ,maxEE-primer1,maxEE-primer2,trimRight,trimLeft"
echo "Current settings:"
echo "${dada_minlength},${dada_phix},${dada_trunQ},${dada_maxEE1},${dada_maxEE2},${dada_trimRight},${dada_trimLeft}"
if [[ "$bypassflag" = "FALSE" ]]; then
echo "Please check DADA2 filtering success. Proceed? [y/n/m]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
dada2continue=TRUE
elif [[ "$mainmenuinput" = "m" || "$mainmenuinput" = "M" ]]; then
echo "You have chosen to redo DADA2 filtering with modified settings."
echo "Input new settings separated by commas in the same order as above."
read secondmainmenuinput
IFS=','
read -ra ADDR <<< "$secondmainmenuinput"
dada_minlength=${ADDR[0]}
dada_phix=${ADDR[1]}
dada_trunQ=${ADDR[2]}
dada_maxEE1=${ADDR[3]}
dada_maxEE2=${ADDR[4]}
dada_trimRight=${ADDR[5]}
dada_trimLeft=${ADDR[6]}
echo "New settings:"
echo "${dada_minlength},${dada_phix},${dada_trunQ},${dada_maxEE1},${dada_maxEE2},${dada_trimRight},${dada_trimLeft}"
echo "Rerunning DADA2 filtering"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, try again"
fi
else
dada2continue=TRUE
fi
done
echo
echo "Learning error, Dereplication, Merge, and ASVs in DADA2..."
echo "Please be patient, may take a while. Messages printed to Rscript log."
echo
Rscript --vanilla ${revampdir}/assets/dada2_step2.R ${workingdirectory}/${outdirectory}/dada2 $systemmemoryMB \
1>> ${workingdirectory}/${outdirectory}/dada2/dada2_rscripts_out.log 2>&1
echo "dada2_step2.R ${workingdirectory}/${outdirectory}/dada2 $systemmemoryMB" >> ${outdirectory}/Rscript_arguments.log
cat ${outdirectory}/dada2/ASVs_counts.tsv | sed -E 's/^ /x /' > ${outdirectory}/dada2/ASVs_counts_mod.tsv
mv ${outdirectory}/dada2/ASVs_counts_mod.tsv ${outdirectory}/dada2/ASVs_counts.tsv
#TODO: minOverlap currently set to 20bp, which seems reasonable. Option for change?
echo
echo "FINAL DADA2 STATS"
echo "Note: Please check for a failed merge of forward/reverse sequences"
echo "Sample %Reads Retained"
awk 'NR>1 { print $1, $8 }' ${workingdirectory}/${outdirectory}/dada2/ReadTrimSummary.txt
if [[ "$bypassflag" = "FALSE" ]]; then
echo
echo "Do you wish to Proceed? [y/n]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, exiting"
exit
fi
fi
fi
if [[ "${failedMerge_useDirection}" = "FORWARD" ]]; then
dada2continue=FALSE
while [ $dada2continue = FALSE ]
do
Rscript --vanilla ${revampdir}/assets/dada2_step1_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $dada_minlength $dada_phix $dada_trunQ $dada_maxEE1 $dada_maxEE2 $dada_trimRight $dada_trimLeft forward \
1>> ${workingdirectory}/${outdirectory}/dada2/dada2_rscripts_out.log 2>&1
echo "dada2_step1_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $dada_minlength $dada_phix $dada_trunQ $dada_maxEE1 $dada_maxEE2 $dada_trimRight $dada_trimLeft forward" >> ${outdirectory}/Rscript_arguments.log
echo
echo "DADA2 Filtering results:"
echo "Sample % Reads Passing"
awk 'NR>1 { print $1, 100 * ( $3 / $2 ) }' ${workingdirectory}/${outdirectory}/dada2/filtered_out_stats.txt
echo
echo "Parameters to modify:"
echo "minLen,rm.phix,truncQ,maxEE-primer1,maxEE-primer2,trimRight,trimLeft"
echo "Current settings:"
echo "${dada_minlength},${dada_phix},${dada_trunQ},${dada_maxEE1},${dada_maxEE2},${dada_trimRight},${dada_trimLeft}"
if [[ "$bypassflag" = "FALSE" ]]; then
echo "Please check DADA2 filtering success. Proceed? [y/n/m]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
dada2continue=TRUE
elif [[ "$mainmenuinput" = "m" || "$mainmenuinput" = "M" ]]; then
echo "You have chosen to redo DADA2 filtering with modified settings."
echo "Input new settings separated by commas in the same order as above."
read secondmainmenuinput
IFS=','
read -ra ADDR <<< "$secondmainmenuinput"
dada_minlength=${ADDR[0]}
dada_phix=${ADDR[1]}
dada_trunQ=${ADDR[2]}
dada_maxEE1=${ADDR[3]}
dada_maxEE2=${ADDR[4]}
dada_trimRight=${ADDR[5]}
dada_trimLeft=${ADDR[6]}
echo "New settings:"
echo "${dada_minlength},${dada_phix},${dada_trunQ},${dada_maxEE1},${dada_maxEE2},${dada_trimRight},${dada_trimLeft}"
echo "Rerunning DADA2 filtering"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, try again"
fi
else
dada2continue=TRUE
fi
done
echo
echo "Learning error, Dereplication, and ASVs in DADA2..."
echo "Please be patient, may take a while. Messages printed to Rscript log."
echo
Rscript --vanilla ${revampdir}/assets/dada2_step2_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $systemmemoryMB forward \
1>> ${workingdirectory}/${outdirectory}/dada2/dada2_rscripts_out.log 2>&1
echo "dada2_step2_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $systemmemoryMB forward" >> ${outdirectory}/Rscript_arguments.log
cat ${outdirectory}/dada2/ASVs_counts.tsv | sed -E 's/^ /x /' > ${outdirectory}/dada2/ASVs_counts_mod.tsv
mv ${outdirectory}/dada2/ASVs_counts_mod.tsv ${outdirectory}/dada2/ASVs_counts.tsv
touch ${outdirectory}/dada2/00_MERGEFAILED_ForwardReadsOnly_usedFor_ASVs_EOM
echo
echo "FINAL DADA2 STATS"
echo "Note: Please check for a failed merge of forward/reverse sequences"
echo "Sample %Reads Retained"
awk 'NR>1 { print $1, $6 }' ${workingdirectory}/${outdirectory}/dada2/ReadTrimSummary.txt
if [[ "$bypassflag" = "FALSE" ]]; then
echo
echo "Do you wish to Proceed? [y/n]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, exiting"
exit
fi
fi
fi
if [[ "${failedMerge_useDirection}" = "REVERSE" ]]; then
dada2continue=FALSE
while [ $dada2continue = FALSE ]
do
Rscript --vanilla ${revampdir}/assets/dada2_step1_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $dada_minlength $dada_phix $dada_trunQ $dada_maxEE1 $dada_maxEE2 $dada_trimRight $dada_trimLeft reverse \
1>> ${workingdirectory}/${outdirectory}/dada2/dada2_rscripts_out.log 2>&1
echo "dada2_step1_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $dada_minlength $dada_phix $dada_trunQ $dada_maxEE1 $dada_maxEE2 $dada_trimRight $dada_trimLeft reverse" >> ${outdirectory}/Rscript_arguments.log
echo
echo "DADA2 Filtering results:"
echo "Sample % Reads Passing"
awk 'NR>1 { print $1, 100 * ( $3 / $2 ) }' ${workingdirectory}/${outdirectory}/dada2/filtered_out_stats.txt
echo
echo "Parameters to modify:"
echo "minLen,rm.phix,truncQ,maxEE-primer1,maxEE-primer2,trimRight,trimLeft"
echo "Current settings:"
echo "${dada_minlength},${dada_phix},${dada_trunQ},${dada_maxEE1},${dada_maxEE2},${dada_trimRight},${dada_trimLeft}"
if [[ "$bypassflag" = "FALSE" ]]; then
echo "Please check DADA2 filtering success. Proceed? [y/n/m]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
dada2continue=TRUE
elif [[ "$mainmenuinput" = "m" || "$mainmenuinput" = "M" ]]; then
echo "You have chosen to redo DADA2 filtering with modified settings."
echo "Input new settings separated by commas in the same order as above."
read secondmainmenuinput
IFS=','
read -ra ADDR <<< "$secondmainmenuinput"
dada_minlength=${ADDR[0]}
dada_phix=${ADDR[1]}
dada_trunQ=${ADDR[2]}
dada_maxEE1=${ADDR[3]}
dada_maxEE2=${ADDR[4]}
dada_trimRight=${ADDR[5]}
dada_trimLeft=${ADDR[6]}
echo "New settings:"
echo "${dada_minlength},${dada_phix},${dada_trunQ},${dada_maxEE1},${dada_maxEE2},${dada_trimRight},${dada_trimLeft}"
echo "Rerunning DADA2 filtering"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, try again"
fi
else
dada2continue=TRUE
fi
done
echo
echo "Learning error, Dereplication, and ASVs in DADA2..."
echo "Please be patient, may take a while. Messages printed to Rscript log."
echo
Rscript --vanilla ${revampdir}/assets/dada2_step2_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $systemmemoryMB reverse \
1>> ${workingdirectory}/${outdirectory}/dada2/dada2_rscripts_out.log 2>&1
echo "dada2_step2_mergeFail.R ${workingdirectory}/${outdirectory}/dada2 $systemmemoryMB reverse" >> ${outdirectory}/Rscript_arguments.log
cat ${outdirectory}/dada2/ASVs_counts.tsv | sed -E 's/^ /x /' > ${outdirectory}/dada2/ASVs_counts_mod.tsv
mv ${outdirectory}/dada2/ASVs_counts_mod.tsv ${outdirectory}/dada2/ASVs_counts.tsv
touch ${outdirectory}/dada2/00_MERGEFAILED_ReverseReadsOnly_usedFor_ASVs_EOM
echo
echo "FINAL DADA2 STATS"
echo "Note: Please check for a failed merge of forward/reverse sequences"
echo "Sample %Reads Retained"
awk 'NR>1 { print $1, $6 }' ${workingdirectory}/${outdirectory}/dada2/ReadTrimSummary.txt
if [[ "$bypassflag" = "FALSE" ]]; then
echo
echo "Do you wish to Proceed? [y/n]"
read mainmenuinput
if [[ "$mainmenuinput" = "y" || "$mainmenuinput" = "Y" ]]; then
echo "Continuing!"
elif [[ "$mainmenuinput" = "n" || "$mainmenuinput" = "N" ]]; then
echo "You have chosen to exit"
exit
else
echo "Invalid selection, exiting"
exit
fi
fi
fi
echo "dada2_Finished=TRUE" >> ${outdirectory}/progress.txt
fi
##########################################################################################
##
## Blastn ASVs
##
##########################################################################################
if [[ "${silvaASVflag}" = "TRUE" ]]; then
echo "Skipping BLASTn to use SILVAngs taxonomy downstream"
else
if [[ "${blastFinished}" = "TRUE" ]]; then
echo "BLASTn from prior run"
else
if [ -d "${outdirectory}/blast_results" ]; then
rm -r ${outdirectory}/blast_results
mkdir ${outdirectory}/blast_results
else
mkdir ${outdirectory}/blast_results
fi
if [[ "$blastflag" = "FALSE" ]]; then
echo
echo "Running BLASTn: `date`"
passblastscrutiny=FALSE
maxtargetseqs=4000
runthroughcount=3 #temp TODO
while [ $passblastscrutiny = FALSE ]
do
let "runthroughcount=runthroughcount+1"
if [[ "${blastMode}" = "allIN" ]]; then
blastn -db ${locationNTdatabase}/nt -query ${outdirectory}/dada2/ASVs.fa -outfmt '6 qseqid pident length staxids sacc' -subject_besthit -max_target_seqs $maxtargetseqs -num_threads 6 -out ${outdirectory}/blast_results/ASV_blastn_nt.btab
elif [[ "${blastMode}" = "mostEnvOUT" ]]; then
blastn -db ${locationNTdatabase}/nt -query ${outdirectory}/dada2/ASVs.fa -outfmt '6 qseqid pident length staxids sacc' -subject_besthit -max_target_seqs $maxtargetseqs -num_threads 6 -out ${outdirectory}/blast_results/ASV_blastn_nt.btab -negative_taxidlist ${locationNTdatabase}/taxdump/taxid_exclusion_list_leavesinUnclassified.txt
elif [[ "${blastMode}" = "allEnvOUT" ]]; then
blastn -db ${locationNTdatabase}/nt -query ${outdirectory}/dada2/ASVs.fa -outfmt '6 qseqid pident length staxids sacc' -subject_besthit -max_target_seqs $maxtargetseqs -num_threads 6 -out ${outdirectory}/blast_results/ASV_blastn_nt.btab -negative_taxidlist ${locationNTdatabase}/taxdump/taxid_exclusion_list_removesUnclassified.txt
else
echo "Incorrect blastMode specified"
exit
fi
perl ${revampdir}/assets/blast_assessment.pl -i ${outdirectory}/blast_results/ASV_blastn_nt.btab -c `grep -c ">" ${outdirectory}/dada2/ASVs.fa` > ${outdirectory}/blast_results/checkmaxtargetseqs.txt
highestnumber=`cat ${outdirectory}/blast_results/checkmaxtargetseqs.txt | sort -k2 -nr | head -1 | cut -f2`
if [[ "$highestnumber" -lt "$maxtargetseqs" ]]; then
passblastscrutiny=TRUE
elif [[ "$highestnumber" -ge "$maxtargetseqs" && "$runthroughcount" -le 3 ]]; then
echo "Rerun BLAST Number ${runthroughcount} - Max Target Not High Enough (${maxtargetseqs})"
let "multiplierseqs=runthroughcount+2"
let "maxtargetseqs=highestnumber*multiplierseqs"
echo "New max target seqs = ${maxtargetseqs}"
else
echo "Max target seqs inclusive of all top hits cannot be reached"
echo "Used max target seqs parameter = ${maxtargetseqs}"
passblastscrutiny=TRUE
fi
done
elif [[ "$blastflag" = "TRUE" ]]; then
echo "Using user supplied BLASTn btab result"
cp $optionalUserBLASTResult ${outdirectory}/blast_results/ASV_blastn_nt.btab
fi
echo
echo "blastFinished=TRUE" >> ${outdirectory}/progress.txt
fi
fi
##########################################################################################
##
## Reformat BLAST output
##
##########################################################################################
if [[ "${silvaASVflag}" = "TRUE" ]]; then
echo "Skipping BLAST formatting to use SILVAngs taxonomy downstream"
else
if [[ "${blastformattingFinished}" = "TRUE" ]]; then
echo "BLAST formatting from prior run"
else
if [ -f "${outdirectory}/blast_results/ASV_blastn_nt_formatted.txt" ]; then
rm ${outdirectory}/blast_results/ASV_blastn_nt_formatted.txt
fi
echo
echo "Reformatting BLAST output: `date`"
#let "insertSize=ampliconSize - ${#primerF} - ${#primerR}"
Rscript --vanilla ${revampdir}/assets/reformat_blast.R ${workingdirectory}/${outdirectory}/blast_results $blastLengthCutoff \
1>> ${workingdirectory}/${outdirectory}/blast_results/blastreformatting_rscript_out.log 2>&1
echo "reformat_blast.R ${workingdirectory}/${outdirectory}/blast_results $blastLengthCutoff" >> ${outdirectory}/Rscript_arguments.log
echo
echo "blastformattingFinished=TRUE" >> ${outdirectory}/progress.txt
fi
fi
##########################################################################################
##
## Running perl script for taxonomy decisions, standard table outs, and KRONA plots
##
##########################################################################################
if [[ "${silvaASVflag}" = "TRUE" ]]; then
if [[ "${taxonomyscriptFinished}" = "TRUE" ]]; then
echo "ASV-2-Taxonomy Script results from prior run"
else
if [ -d "${outdirectory}/ASV2Taxonomy" ]; then
rm -r ${outdirectory}/ASV2Taxonomy
mkdir ${outdirectory}/ASV2Taxonomy
else
mkdir ${outdirectory}/ASV2Taxonomy
fi
echo "Enter the location of the SILVAngs ssu or lsu results directory (i.e. ~/Downloads/results/ssu)"
read silvaNGSdirectory
silvangsInputExportFile=${silvaNGSdirectory}/exports/*---otus.csv
silvangsInputClusterFile=${silvaNGSdirectory}/stats/sequence_cluster_map/data/*.fa.clstr
echo "Enter the location of the reference taxonomy map for current SILVA database: i.e. tax_slv_ssu_138.1.txt"
read silvaRefMap
echo
echo "Running ASV-2-Taxonomy Script: `date`"
cd ${outdirectory}/ASV2Taxonomy
perl ${revampdir}/assets/asv_taxonomy_processing_figureOuts.pl -a ../dada2/ASVs_counts.tsv \
-n ${outdirectory} -o ../sample_order.txt -y $silvangsInputExportFile -z $silvangsInputClusterFile -r $silvaRefMap -m $revampdir -e
cat ${outdirectory}_asvTaxonomyTable.txt | grep -v "Unknown" > ${outdirectory}_asvTaxonomyTable_NOUNKNOWNS.txt
cd ${workingdirectory}
cat ${outdirectory}/dada2/ASVs_counts.tsv | grep -v -f ${outdirectory}/ASV2Taxonomy/${outdirectory}_unknown_asvids.txt > ${outdirectory}/ASV2Taxonomy/ASVs_counts_NOUNKNOWNS.tsv
perl ${revampdir}/assets/merge_on_taxonomy.pl -a ${outdirectory}/dada2/ASVs_counts.tsv -t ${outdirectory}/ASV2Taxonomy/${outdirectory}_asvTaxonomyTable.txt -o ${outdirectory}/ASV2Taxonomy > ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv
cat ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv | grep -v -f ${outdirectory}/ASV2Taxonomy/${outdirectory}_unknown_asvids.txt > ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy_NOUNKNOWNS.tsv
mkdir ${outdirectory}/ASV2Taxonomy/KRONA_plots
mkdir ${outdirectory}/ASV2Taxonomy/KRONA_plots/KRONA_inputs
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_allin_KRONA.txt ${outdirectory}/ASV2Taxonomy/${outdirectory}_allin_TaxonomyASVSampleCount_byline.txt
mv ${outdirectory}/ASV2Taxonomy/MP_*_KRONA.txt ${outdirectory}/ASV2Taxonomy/KRONA_plots/KRONA_inputs/
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_wholeKRONA.txt ${outdirectory}/ASV2Taxonomy/KRONA_plots/KRONA_inputs/${outdirectory}_samplesSummedKRONA.txt
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_master_krona.html ${outdirectory}/ASV2Taxonomy/KRONA_plots/
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_wholeKRONA.html ${outdirectory}/ASV2Taxonomy/KRONA_plots/${outdirectory}_samplesSummedKRONA.html
perl ${revampdir}/assets/stats.pl -a ${outdirectory}/dada2/ASVs_counts.tsv -t ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv -i ${outdirectory}/ASV2Taxonomy/${outdirectory}_asvTaxonomyTable.txt > ${outdirectory}/ASV2Taxonomy/basic_ASV_taxonomy_stats.txt
echo "taxonomyscriptFinished=TRUE" >> ${outdirectory}/progress.txt
fi
else
if [[ "${taxonomyscriptFinished}" = "TRUE" ]]; then
echo "ASV-2-Taxonomy Script results from prior run"
else
if [ -d "${outdirectory}/ASV2Taxonomy" ]; then
rm -r ${outdirectory}/ASV2Taxonomy
mkdir ${outdirectory}/ASV2Taxonomy
if [[ "$removeASVsFILE" != "NULL" ]]; then
mv ${outdirectory}/dada2/ASVs_unfiltered.fa ${outdirectory}/dada2/ASVs.fa
mv ${outdirectory}/dada2/ASVs_counts_unfiltered.tsv ${outdirectory}/dada2/ASVs_counts.tsv
#WILL THROW AN ERROR: If you run with removeASVsFILE given then remove that parameter without moving the *unfiltered* back to their original names.
fi
else
mkdir ${outdirectory}/ASV2Taxonomy
fi
echo
echo "Running ASV-2-Taxonomy Script: `date`"
#prep taxon ids for taxonkit
cat ${outdirectory}/blast_results/ASV_blastn_nt_formatted.txt | cut -f4 | sed -E "s/ //g" | tr ',' '\n' | tr ';' '\n' | sort | uniq | grep -v "taxid" > ${outdirectory}/ASV2Taxonomy/taxids.txt
taxonkit lineage ${outdirectory}/ASV2Taxonomy/taxids.txt | awk '$2!=""' > ${outdirectory}/ASV2Taxonomy/taxonkit_out.txt
taxonkit reformat ${outdirectory}/ASV2Taxonomy/taxonkit_out.txt | cut -f1,3 > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt
if [[ "$bypassflag" = "FALSE" ]]; then
echo
echo "Reformatted taxon strings created. Options:"
echo "Continue without changes [c]"
echo "Manually edit file and replace in same location with identical file structure [m]"
echo " (Make choice when file is modified and you are ready to proceed)"
echo "Automatically fill gaps in reformatted taxonkit hierarchy [a]"
read mainmenuinput
if [[ "$mainmenuinput" = "c" || "$mainmenuinput" = "C" ]]; then
cat ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt | sed -E 's/[^A-Za-z0-9;[:blank:]]/_/g' > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
mv ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out_ORIGINAL.txt
mv ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt
echo "Continuing!"
elif [[ "$mainmenuinput" = "m" || "$mainmenuinput" = "M" ]]; then
echo "Continuing!"
cat ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt | tr '\r' '\n' | tr -s '\n' > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
cat ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp | sed -E 's/[^A-Za-z0-9;[:blank:]]/_/g' > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt
rm ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
elif [[ "$mainmenuinput" = "a" || "$mainmenuinput" = "A" ]]; then
echo "Reformatting..."
echo "Original reformatted taxonkit out stored at ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out_ORIGINAL.txt"
perl ${revampdir}/assets/fillIn_taxonkit.pl -i ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
mv ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out_ORIGINAL.txt
cat ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp | sed -E 's/[^A-Za-z0-9;[:blank:]]/_/g' > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt
rm ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
echo "Continuing!"
else
echo "Invalid selection, exiting"
exit
fi
elif [[ "$bypassflag" = "TRUE" ]]; then
echo "Automatically reformatting taxonkit hierarchy..."
echo "Original reformatted taxonkit out stored at ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out_ORIGINAL.txt"
perl ${revampdir}/assets/fillIn_taxonkit.pl -i ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
mv ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out_ORIGINAL.txt
cat ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp | sed -E 's/[^A-Za-z0-9;[:blank:]]/_/g' > ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt
rm ${outdirectory}/ASV2Taxonomy/reformatted_taxonkit_out.txt_temp
echo "Continuing!"
fi
if [[ "$removeASVsFILE" = "NULL" ]]; then
cd ${outdirectory}/ASV2Taxonomy
perl ${revampdir}/assets/asv_taxonomy_processing_figureOuts.pl -a ../dada2/ASVs_counts.tsv -s ../blast_results/ASV_blastn_nt_formatted.txt \
-t reformatted_taxonkit_out.txt -f $taxonomyCutoffs -n ${outdirectory} -c ${locationNTdatabase}/taxdump/common_names.dmp -o ../sample_order.txt
cat ${outdirectory}_asvTaxonomyTable.txt | grep -v "Unknown" > ${outdirectory}_asvTaxonomyTable_NOUNKNOWNS.txt
cd ${workingdirectory}
cat ${outdirectory}/ASV2Taxonomy/${outdirectory}_unknown_asvids.txt | cut -f1 | sed -E 's/$/ /' > ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns
cat ${outdirectory}/dada2/ASVs_counts.tsv | grep -v -f ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns > ${outdirectory}/ASV2Taxonomy/ASVs_counts_NOUNKNOWNS.tsv
perl ${revampdir}/assets/merge_on_taxonomy.pl -a ${outdirectory}/dada2/ASVs_counts.tsv -t ${outdirectory}/ASV2Taxonomy/${outdirectory}_asvTaxonomyTable.txt -o ${outdirectory}/ASV2Taxonomy > ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv
cat ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv | grep -v -f ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns > ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy_NOUNKNOWNS.tsv
rm ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns
mkdir ${outdirectory}/ASV2Taxonomy/KRONA_plots
mkdir ${outdirectory}/ASV2Taxonomy/KRONA_plots/KRONA_inputs
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_allin_KRONA.txt ${outdirectory}/ASV2Taxonomy/${outdirectory}_allin_TaxonomyASVSampleCount_byline.txt
mv ${outdirectory}/ASV2Taxonomy/MP_*_KRONA.txt ${outdirectory}/ASV2Taxonomy/KRONA_plots/KRONA_inputs/
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_wholeKRONA.txt ${outdirectory}/ASV2Taxonomy/KRONA_plots/KRONA_inputs/${outdirectory}_samplesSummedKRONA.txt
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_master_krona.html ${outdirectory}/ASV2Taxonomy/KRONA_plots/
mv ${outdirectory}/ASV2Taxonomy/${outdirectory}_wholeKRONA.html ${outdirectory}/ASV2Taxonomy/KRONA_plots/${outdirectory}_samplesSummedKRONA.html
perl ${revampdir}/assets/stats.pl -a ${outdirectory}/dada2/ASVs_counts.tsv -t ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv -i ${outdirectory}/ASV2Taxonomy/${outdirectory}_asvTaxonomyTable.txt > ${outdirectory}/ASV2Taxonomy/basic_ASV_taxonomy_stats.txt
else
cd ${outdirectory}/ASV2Taxonomy
perl ${revampdir}/assets/asv_taxonomy_processing_figureOuts.pl -a ../dada2/ASVs_counts.tsv -s ../blast_results/ASV_blastn_nt_formatted.txt -d $removeASVsFILE \
-t reformatted_taxonkit_out.txt -f $taxonomyCutoffs -n ${outdirectory} -c ${locationNTdatabase}/taxdump/common_names.dmp -o ../sample_order.txt
#Move non-filtered files to folder, replace with files from "IGNORING_ASVs" directory
mkdir unfiltered_files
mkdir -p KRONA_plots/KRONA_inputs
mv ${outdirectory}_allin_KRONA.txt ${outdirectory}_allin_TaxonomyASVSampleCount_byline.txt
mv MP_*_KRONA.txt KRONA_plots/KRONA_inputs/
mv ${outdirectory}_wholeKRONA.txt KRONA_plots/KRONA_inputs/${outdirectory}_samplesSummedKRONA.txt
mv ${outdirectory}_master_krona.html KRONA_plots/
mv ${outdirectory}_wholeKRONA.html KRONA_plots/${outdirectory}_samplesSummedKRONA.html
mv KRONA_plots unfiltered_files/
mv ${outdirectory}_allin_TaxonomyASVSampleCount_byline.txt unfiltered_files/
mv ${outdirectory}_asvTaxonomyTable.txt unfiltered_files/
mv ${outdirectory}_barchart_forR.txt unfiltered_files/
mv ${outdirectory}_barchart.txt unfiltered_files/
mv ${outdirectory}_heatmap_multiASV.txt unfiltered_files/
mv ${outdirectory}_NO_UNKNOWNS_barchart.txt unfiltered_files/
mkdir -p IGNORING_ASVs/KRONA_plots/KRONA_inputs
mv IGNORING_ASVs/${outdirectory}_IGNORE_allin_KRONA.txt ${outdirectory}_allin_TaxonomyASVSampleCount_byline.txt
mv IGNORING_ASVs/MP_*_KRONA.txt IGNORING_ASVs/KRONA_plots/KRONA_inputs/
mv IGNORING_ASVs/${outdirectory}_IGNORE_master_krona.html IGNORING_ASVs/KRONA_plots/
mv IGNORING_ASVs/KRONA_plots ./
mv IGNORING_ASVs/${outdirectory}_ASVs_to_IGNORE.txt ./
mv IGNORING_ASVs/${outdirectory}_IGNORE_asvTaxonomyTable.txt ./${outdirectory}_asvTaxonomyTable.txt
mv IGNORING_ASVs/${outdirectory}_IGNORE_barchart_forR.txt ./${outdirectory}_barchart_forR.txt
mv IGNORING_ASVs/${outdirectory}_IGNORE_barchart.txt ./${outdirectory}_barchart.txt
mv IGNORING_ASVs/${outdirectory}_IGNORE_heatmap_multiASV.txt ./${outdirectory}_heatmap_multiASV.txt
mv IGNORING_ASVs/${outdirectory}_IGNORE_NO_UNKNOWNS_barchart.txt ./${outdirectory}_NO_UNKNOWNS_barchart.txt
rmdir IGNORING_ASVs
cat ${outdirectory}_asvTaxonomyTable.txt | grep -v "Unknown" > ${outdirectory}_asvTaxonomyTable_NOUNKNOWNS.txt
cd ${workingdirectory}
mv ${outdirectory}/dada2/ASVs.fa ${outdirectory}/dada2/ASVs_unfiltered.fa
mv ${outdirectory}/dada2/ASVs_counts.tsv ${outdirectory}/dada2/ASVs_counts_unfiltered.tsv
cat ${outdirectory}/ASV2Taxonomy/${outdirectory}_ASVs_to_IGNORE.txt | sed -E 's/$/\$/' > ${outdirectory}/temp
grep -A 1 -f ${outdirectory}/temp ${outdirectory}/dada2/ASVs_unfiltered.fa | sed -E 's/$/\$/' > ${outdirectory}/temp2
grep -v "^--\$$" ${outdirectory}/temp2 > ${outdirectory}/temp
grep -v -f ${outdirectory}/temp ${outdirectory}/dada2/ASVs_unfiltered.fa > ${outdirectory}/dada2/ASVs.fa
rm ${outdirectory}/temp
rm ${outdirectory}/temp2
#Note for potential bug: Will remove the sequence (but not the header) for sequences identical to an ASV in the IGNORE list.
cat ${outdirectory}/ASV2Taxonomy/${outdirectory}_ASVs_to_IGNORE.txt | sed -E 's/$/ /' > ${outdirectory}/temp
cat ${outdirectory}/dada2/ASVs_counts_unfiltered.tsv | grep -v -f ${outdirectory}/temp > ${outdirectory}/dada2/ASVs_counts.tsv
rm ${outdirectory}/temp
cat ${outdirectory}/ASV2Taxonomy/${outdirectory}_unknown_asvids.txt | cut -f1 | sed -E 's/$/ /' > ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns
cat ${outdirectory}/dada2/ASVs_counts.tsv | grep -v -f ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns > ${outdirectory}/ASV2Taxonomy/ASVs_counts_NOUNKNOWNS.tsv
perl ${revampdir}/assets/merge_on_taxonomy.pl -a ${outdirectory}/dada2/ASVs_counts.tsv -t ${outdirectory}/ASV2Taxonomy/${outdirectory}_asvTaxonomyTable.txt -o ${outdirectory}/ASV2Taxonomy > ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv
cat ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv | grep -v -f ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns > ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy_NOUNKNOWNS.tsv
rm ${outdirectory}/ASV2Taxonomy/temp_grep_unknowns
perl ${revampdir}/assets/stats.pl -a ${outdirectory}/dada2/ASVs_counts.tsv -t ${outdirectory}/ASV2Taxonomy/ASVs_counts_mergedOnTaxonomy.tsv -i ${outdirectory}/ASV2Taxonomy/${outdirectory}_asvTaxonomyTable.txt > ${outdirectory}/ASV2Taxonomy/basic_ASV_taxonomy_stats.txt
fi
echo "taxonomyscriptFinished=TRUE" >> ${outdirectory}/progress.txt
fi
fi
##########################################################################################
##
## File cleanup
##
##########################################################################################
if [[ "$keepIntermediateFiles" = "FALSE" ]]; then
rm -f ${outdirectory}/cutadapt/*_trimmed.fq.gz
rm -f ${outdirectory}/dada2/*_filtered.fq.gz
if [[ "${silvaASVflag}" != "TRUE" ]]; then
gzip -q -9 ${outdirectory}/blast_results/ASV_blastn_nt.btab
fi
fi
##########################################################################################
##
## FIGURES & ANALYSIS
##
##########################################################################################
#Defaults, user can override:
filterPercent=5
filterLowQualSamples="FALSE"
filterPercentLowQualSamples=30
pieScale=6 #TODO assign appropriate default
removeNA="FALSE"
providedTaxaOfInterest="FALSE"
taxaOfInterestFile="NULL"
taxaOfInterestLevel="NULL"
#Interpreted from sample metadata file:
controlPos="FALSE"
controlNeg="FALSE"
replicates="FALSE"
sites="FALSE"
chemData="FALSE"
controlspresent="FALSE"
groupsDefinedFlag="FALSE"
locationChemHeaders="NULL"
source $figureparamfilepath
cp $figureparamfilepath ${outdirectory}/figure_config_file.txt
#TODO Check previous and current config files and skip this if finished. Likely will want this to be more granular.
if [[ "${figuresFinished}" = "TRUE" ]]; then
echo "Figure creation from prior run"
else