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

wupengomics/PASS

PASS