Regarding FASTdRNA

[PRIYANKA-22091995]

Hello Team,

I have a basic query regarding the tool, to run the command for the alternative splicing, do i always need a two samples with duplicates as well. Because i was planning to run the alternative splicing command as i only have one sample without duplicates.It would be nice to have your insights on the same.

Thanks & Regards
Priyanka Roy

[Tomcxf]

Hi Priyanka,
Sorry to reply so late. That's OK if you don't have duplicates. The pipeline will also work. The only difference may be that it will not have p-value.
Best wishes!

[Tomcxf]

I have updated the code, called dRNAas_single.py.
Below is my discussion with Suppa's developer, how can help you! comprna/SUPPA#147
If there's any problems and error, please tell me :)

[PRIYANKA-22091995]

Thanks for your response, this will be really helpful for my analysis.
For running the alternative splicing, i have to first run following command first, so as to proceed with alternative splicing command.
snakemake -s {dRNAmain.py}
-s the snakemake file you want to run
--cores / -c : the number of cores to use (necessary)
--set-threads myrule=XXX set threads XXX for running

But, the only question i have is where should i provide the input file in the above command. Looking forward to your reply.

Thanks & Regards
Priyanka Roy

[Tomcxf]

OK,
you can add your input file information at the file called configForModifAS.yaml.
Since you don't have duplications, you can just fill the information of ref , S1R1, S2R1.
I also change details of dRNAas_single.py, hope it can help you!

[PRIYANKA-22091995]

Thank you so much for the information. I want to clarify one more information i.e. I only have one sample to run, so for my analysis, it is only S1R1. Will that work with the pipeline? Thanks & Regards Priyanka Roy

…

On Fri, Dec 15, 2023 at 2:53 PM Tomcxf ***@***.***> wrote: OK, you can add your input file information at the file called configForModifAS.yaml <https://github.com/Tomcxf/FASTdRNA/blob/main/configForModifAS.yaml>. Since you don't have duplications, you can just fill the information of ref , S1R1, S2R1. I also change details of dRNAas_single.py, hope it can help you! — Reply to this email directly, view it on GitHub <#2 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/A2FSZXRIANSEV332V7WTLGTYJQJKZAVCNFSM6AAAAAA7HCSI5SVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTQNJXGU2DQNJTGU> . You are receiving this because you authored the thread.Message ID: ***@***.***>

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[Tomcxf]

yes, it will run.
you can change dRNAas_single.py.
add '#' to whole 'rule PSI_Con2' and line16.

[ArnavBharti]

I am trying to do alternative splicing with one sample and no duplicates.

1. I got an error while running the program:

Building DAG of jobs...
ChildIOException:
File/directory is a child to another output:
('/home/.../FASTdRNA/AlternativeSplicing/localAS', generateEvents)
('/home/.../FASTdRNA/AlternativeSplicing/localAS/Condition1', PSI_Con1)

2. Changes to dRNAas_single.py

• Logs
  • Commented line 16 according to above conversation,
  • Commented lines 61-68 according to above conversation,
  • Changed extension to .smk for VS Code Snakemake linter to work,
  • Replaced "：" with ":" in lines 31, 58 (The character U+ff1a "：" could be confused with the ASCII character U+003a ":", which is more common in source code.)
  • Replaced config.yaml with configForModifAS.yaml (line 10)
  • line 80 column 1 replaced "3" with "#"
  • Uncommented line 9
  • Uncommented line 4
  • Replaced "RGF" with config["ref"] in line 9
  • Replaced "S1R1" with config["S1R1"] in line 4
  • Moved line 10 to line 4

##################################
# A pipeline for ONT_dRNA_seq written by Chen X.F
##################################
configfile: "configForModifAS.yaml"
S1R1=config["S1R1"]
#S1R2="S1R2"
#S2R1="S2R1"
#S2R2="S2R2"

ref=config["ref"]

#################################
rule all :
    input:  
        "AlternativeSplicing/localAS/Condition1",
        # "AlternativeSplicing/localAS/Condition2"

rule generateEvents:
    input:
        {ref}
    output:
        "AlternativeSplicing/localAS"
    shell:
        "mkdir AlternativeSplicing | mkdir AlternativeSplicing/localAS | suppa.py generateEvents -i {input} -o {output}/local -f ioe -e {SE,SS,MX,RI,FL}"

rule generateEvents2:
    input:
        "AlternativeSplicing/localAS"
    output:
        "AlternativeSplicing/localAS/allevents.ioe"
    shell:
        "awk 'FNR==1 && NR!=1 { while (/^<header>/) getline; } 1 {print}' {input}/*.ioe > output[0]"

#rule joinFilesCond1:
#    input:
        #{S1R1}
        #{S1R2}
#    output:
#        "AlternativeSplicing/localAS/Condition1"
#    shell：
#        "suppa.py joinFiles -f tpm -i {input[0]} {input[1]} -o {output}"

#rule joinFilesCond1:
#    input:
#        {S2R1}
#        {S2R2}
#    output:
#        "AlternativeSplicing/localAS/Condition2"
#    shell：
#        "suppa.py joinFiles -f tpm -i {input[0]} {input[1]} -o {output}"

rule PSI_Con1:
    input:
        "AlternativeSplicing/localAS/allevents.ioe",
        {S1R1}
    output:
        "AlternativeSplicing/localAS/Condition1"
    shell:
        "suppa.py psiPerEvent --ioe-file {input[0]} --expression-file {input[1]}.tpm -o {output}"

# rule PSI_Con2:
#     input:
#         "AlternativeSplicing/localAS/allevents.ioe",
#         {S2R1}
#     output:
#         "AlternativeSplicing/localAS/Condition2"
#     shell：
#         "suppa.py psiPerEvent --ioe-file {input[0]} --expression-file {input[1]}.tpm -o {output}"

#rule diffsplice:
#    input:
#        "AlternativeSplicing/localAS/allevents.ioe",
#        "AlternativeSplicing/localAS/Condition1",
#        "AlternativeSplicing/localAS/Condition2",
#        "AlternativeSplicing/localAS/Condition1",
#        "AlternativeSplicing/localAS/Condition2"
#    output:
#        "AlternativeSplicing/localAS/Con1vs2"
#    shell： 
#        "suppa.py diffSplice --method empirical --input {input[0]} --psi {input[1]}.psi {input[2]}.psi --tpm {input[3]}.tpm {input[4]}.tpm --area 1000 --lower-bound 0.05 -gc -o {output}"

#rule cluster:
#    input:
#        "AlternativeSplicing/localAS/Con1vs2"
#    output:
#        "AlternativeSplicing/localAS/cluster"
#    shell:
#        "suppa.py clusterEvents --dpsi {input}.dpsi --psivec {input}.psivec --sig-threshold 0.1 --eps 0.1 --min-pts 20 --groups 1-2,3-4 -o <output-file>" 

#rule trans_gE:
#    input:
#        {ref}
#    output:
#        "AlternativeSplicing/transcript"
#    shell:
#        "mkdir AlternativeSplicing/transcript | suppa.py generateEvents -i {input} -o {output}/local -f ioe -e {SE,SS,MX,RI,FL}"

4. configForModifAS.yaml

ref: /home/.../PlasmoDB-66_PvivaxP01_AnnotatedTranscripts.fasta
S1R1: /home/.../FASTQ

5. Commands ran:

$ mamba activate FdR
$ snakemake -s dRNAas_single.smk -c 2

[Tomcxf]

I am trying to do alternative splicing with one sample and no duplicates.

1. I got an error while running the program:

Building DAG of jobs...
ChildIOException:
File/directory is a child to another output:
('/home/.../FASTdRNA/AlternativeSplicing/localAS', generateEvents)
('/home/.../FASTdRNA/AlternativeSplicing/localAS/Condition1', PSI_Con1)

2. Changes to dRNAas_single.py

• Logs

  • Commented line 16 according to above conversation,
  • Commented lines 61-68 according to above conversation,
  • Changed extension to .smk for VS Code Snakemake linter to work,
  • Replaced "：" with ":" in lines 31, 58 (The character U+ff1a "：" could be confused with the ASCII character U+003a ":", which is more common in source code.)
  • Replaced config.yaml with configForModifAS.yaml (line 10)
  • line 80 column 1 replaced "3" with "#"
  • Uncommented line 9
  • Uncommented line 4
  • Replaced "RGF" with config["ref"] in line 9
  • Replaced "S1R1" with config["S1R1"] in line 4
  • Moved line 10 to line 4

##################################
# A pipeline for ONT_dRNA_seq written by Chen X.F
##################################
configfile: "configForModifAS.yaml"
S1R1=config["S1R1"]
#S1R2="S1R2"
#S2R1="S2R1"
#S2R2="S2R2"

ref=config["ref"]

#################################
rule all :
    input:  
        "AlternativeSplicing/localAS/Condition1",
        # "AlternativeSplicing/localAS/Condition2"

rule generateEvents:
    input:
        {ref}
    output:
        "AlternativeSplicing/localAS"
    shell:
        "mkdir AlternativeSplicing | mkdir AlternativeSplicing/localAS | suppa.py generateEvents -i {input} -o {output}/local -f ioe -e {SE,SS,MX,RI,FL}"

rule generateEvents2:
    input:
        "AlternativeSplicing/localAS"
    output:
        "AlternativeSplicing/localAS/allevents.ioe"
    shell:
        "awk 'FNR==1 && NR!=1 { while (/^<header>/) getline; } 1 {print}' {input}/*.ioe > output[0]"

#rule joinFilesCond1:
#    input:
        #{S1R1}
        #{S1R2}
#    output:
#        "AlternativeSplicing/localAS/Condition1"
#    shell：
#        "suppa.py joinFiles -f tpm -i {input[0]} {input[1]} -o {output}"

#rule joinFilesCond1:
#    input:
#        {S2R1}
#        {S2R2}
#    output:
#        "AlternativeSplicing/localAS/Condition2"
#    shell：
#        "suppa.py joinFiles -f tpm -i {input[0]} {input[1]} -o {output}"

rule PSI_Con1:
    input:
        "AlternativeSplicing/localAS/allevents.ioe",
        {S1R1}
    output:
        "AlternativeSplicing/localAS/Condition1"
    shell:
        "suppa.py psiPerEvent --ioe-file {input[0]} --expression-file {input[1]}.tpm -o {output}"

# rule PSI_Con2:
#     input:
#         "AlternativeSplicing/localAS/allevents.ioe",
#         {S2R1}
#     output:
#         "AlternativeSplicing/localAS/Condition2"
#     shell：
#         "suppa.py psiPerEvent --ioe-file {input[0]} --expression-file {input[1]}.tpm -o {output}"

#rule diffsplice:
#    input:
#        "AlternativeSplicing/localAS/allevents.ioe",
#        "AlternativeSplicing/localAS/Condition1",
#        "AlternativeSplicing/localAS/Condition2",
#        "AlternativeSplicing/localAS/Condition1",
#        "AlternativeSplicing/localAS/Condition2"
#    output:
#        "AlternativeSplicing/localAS/Con1vs2"
#    shell： 
#        "suppa.py diffSplice --method empirical --input {input[0]} --psi {input[1]}.psi {input[2]}.psi --tpm {input[3]}.tpm {input[4]}.tpm --area 1000 --lower-bound 0.05 -gc -o {output}"

#rule cluster:
#    input:
#        "AlternativeSplicing/localAS/Con1vs2"
#    output:
#        "AlternativeSplicing/localAS/cluster"
#    shell:
#        "suppa.py clusterEvents --dpsi {input}.dpsi --psivec {input}.psivec --sig-threshold 0.1 --eps 0.1 --min-pts 20 --groups 1-2,3-4 -o <output-file>" 

#rule trans_gE:
#    input:
#        {ref}
#    output:
#        "AlternativeSplicing/transcript"
#    shell:
#        "mkdir AlternativeSplicing/transcript | suppa.py generateEvents -i {input} -o {output}/local -f ioe -e {SE,SS,MX,RI,FL}"

4. configForModifAS.yaml

ref: /home/.../PlasmoDB-66_PvivaxP01_AnnotatedTranscripts.fasta
S1R1: /home/.../FASTQ

5. Commands ran:

$ mamba activate FdR
$ snakemake -s dRNAas_single.smk -c 2

Thanks for your help to our project. I will check ChildIOException error as soon as possible, maybe it is due to the edit for request below. But I was also aware that '''S1R1: /home/.../FASTQ'''
The S1R1 needs expression file (TPM generate from former process), not fastq files