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PASS

  • Description: a Proteomics Alternative Splicing Screening pipeline.

  • Version: 1.1.0

  • Install

    • unzip PASS-master.zip
    • chmod a+x *
    • export PATH=/PASS_install_path/:$PATH
  • System requirements

    • Tophat2
      • wget tophat2-*.tar.gz
      • tar -zxvf tophat2-*.tar.gz
      • export PATH=/topaht2_install_path/:$PATH
    • Cufflinks
      • wget cufflinks-*.tar.gz
      • tar -zxvf cufflinks-*.tar.gz
      • export PATH=/cufflinks_install_path/:$PATH
    • MSGF+
      • wget MSGFPlus_v20190228.zip
      • unzip MSGFPlus_v20190228.zip
  • processRNASEQ:

    • Description: Align RNA-Seq reads to the reference genome and reconstruct transcripts.

    • Usage: processRNASEQ [options] -g <genome> -f <.gtf> -r <.fastq>

      -g    Genome bowtie2 index name.
      -f    Gene annotation file, .gtf format.
      -r    File names for sequencing reads, .fastq format.
            - Compressed files (.fastq.gz) are also supported.
            - Paired-end files separated by commas.
      -t    Path to tophat, eg. /home/user/bin/tophat
            - By default, we try to search tophat in system PATH.
      -c    Path to cufflinks, eg. /home/user/bin/cufflinks
            - By default, we try to search cufflinks in system PATH. 
      -p    Number of used threads. [Default: 12]
      -o    Output folder. [Default: ./PASS_out]
      -h    Help message.
      
    • Example: processRNASEQ -g path_genomeandbowtie2index/genome.test -f exampleData/genes.test.gtf -r exampleData/Sample_R1.fastq.gz,exampleData/Sample_R2.fastq.gz

  • getORF:

    • Description: Protein sequences translation.

    • Usage: getORF [options] -f <.gtf> -g <genome.fa>

      -f    File name of gene annotation, .gtf format.
            - Recommend cufflinks to generate this file. 
      -g    Reference genome file name, fasta format.
      -o    Output folder. [Default: ./PASS_out]
      -h    Help message.
      
    • Example: getORF -f exampleData/transcripts.gtf -g exampleData/genome.test.fa

    • Output:

      • transcripts.longestorf.gtf
      • transcript.longestorf.fa
      • protein.longestorf.fa
  • searchMS:

    • Description: Search MS file against protein sequence database.

    • Usage: searchMS [options] -s <MSGF_path> -m <example.mzML> -f <protein.fa>

      -s    Path to MSGFPlus.jar. eg. ~/software/MSGF.
      -m    MS/MS file. 
            - Support file formats including .mzML, .mzXML, .mgf, .ms2, .pkl and _dta.txt
            - Spectral should be centroided.
      -f    Protein sequences
      -p    Number of used threads. [Default: 12]
      -t    Modification file name.
      -o    Output folder. [Default: ./PASS_out]
      -h    Help message.
      
    • Example: searchMS -s ~/software/MSGF -m exampleData/example.mzML -f exampleData/protein.longestorf.fa

    • Output:

      • PSM.tab
  • generateSAM:

    • Description: Convert peptide spectal matches to alignment file.

    • Usage: generateSAM [options] -m <PSM> -f <.gtf> -t <transcript.fa> -p <protein.fa>

      -m    Peptide spectral matches.
      -f    File name of gene annotation, .gtf format.
      -t    File name of transcript sequences, .fa format.
      -p    File name of protein sequences, .fa format.
      -o    Output folder. [Default: ./PASS_out]
      -h    Help message.
      
    • Example: generateSAM -m exampleData/PSM.tab -f exampleData/transcripts.longestorf.gtf -t exampleData/transcript.longestorf.fa -p exampleData/protein.longestorf.fa

    • Output:

      • PSM.sam
  • screenAS:

    • Description: Detect AS events from annotation and alignment file.

    • Note: This function code is sourced from MATS.

    • Usage: screenAS [options] -s <PSM.sam> -g <genes.gtf>

      -s    Sam format file generated by proteome identification.
      -g    Gene annotation file, .gtf format.
      -o    Output folder. [Default: ./PASS_out]
      -h    Help message.
      
    • Example: screenAS -s exampleData/PSM.sam -g exampleData/transcripts.longestorf.gtf

    • Output

      • summary.txt
      • PASS.SE.txt
      • PASS.RI.txt
      • PASS.MXE.txt
      • PASS.A5SS.txt
      • PASS.A3SS.txt
      • PASS.AFE.txt
      • PASS.ALE.txt
  • PASS:

    • Description: All-in-one command.

    • Usage: PASS [options] -g <genome> -f <genes.gtf> -r <reads.fastq> -s <MSGFPlus.jar> -m <example.mzML>

      -g    Genome bowtie2 index name.
      -f    Gene annotation file, .gtf format.
      -r    File names for sequencing reads, .fastq format.
              - Compressed files (.fastq.gz) are also supported.
              - Paired-end files separated by commas.
      -t    Path to tophat, eg. /home/user/bin/tophat
        - By default, we try to search tophat in system PATH.
      -c    Path to cufflinks, eg. /home/user/bin/cufflinks
        - By default, we try to search cufflinks in system PATH. 
      -p    Number of used threads. [Default: 12]
      -s    Path to MSGFPlus.jar. eg. ~/software/MSGF.
      -m    MS/MS file. 
              - Support file formats including .mzML, .mzXML, .mgf, .ms2, .pkl and _dta.txt
              - Spectra should be centroided.
      -d    Modification file name.
      -o    Output folder. [Default: ./PASS_out]
      -h    Help message.
      
    • Example: PASS -g path_genomeandbowtie2index/genome.test -f exampleData/genes.test.gtf -r exampleData/Sample_R1.fastq.gz,exampleData/Sample_R2.fastq.gz -s ~/software/MSGF -m exampleData/example.mzML -p 4

    • Output:

      • summary.txt
      • PASS.SE.txt
      • PASS.RI.txt
      • PASS.MXE.txt
      • PASS.A5SS.txt
      • PASS.A3SS.txt
      • PASS.AFE.txt
      • PASS.ALE.txt
  • Contact:

    Peng Wu; wupeng1@ihcams.ac.cn

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