Recipe for generating proteotypic peptides for example database

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Notes creating proteotypic peptide information - based on Human 2009-04 modified to include changes needed to show (almost) all peptides. Updated based on 2009-08 mouse build, see specific build README below for additional info and examples. Root directory for this is at /net/db/projects/PeptideAtlas/species


1. Set up reference database file 


- *Set up add a couple dirs to your PATH, bash syntax 
- export PATH=/regis/sbeams/bin/:/package/genome/tmhmm_sigp_wrapper/:/net/db/projects/PeptideAtlas/species/bin/:$PATH
  • Make processing dir, cd there, and assemble source data.

cd /net/db/projects/PeptideAtlas/species mkdir organism mkdir date

  • Get database file to work on - see Mouse build instrux below if this needs to be assembled.

cd /net/db/projects/PeptideAtlas/species/organim/date cp reference_db.fasta .

  • Assuming accessions are correct, filter decoys and trim long proteins longer than 8999 AA (which choke Peptide Sieve)

processFasta.pl -f reference_db.fasta -r 'DECOY_' -e -o reference_db_no-decoys.fasta

  • Break files into bite-sized chunks!

split_fasta.pl --entries 10000 --filename_root input_split reference_db_no-decoys.fasta


2. Run predictor algorithms.

    • Run PDP, then merge into one results file.
  • symlink binaries.

ln -s /net/db/src/DetectabilityPredictor/Standalone/PeptideDetectabilityPredictor ln -s /net/db/src/DetectabilityPredictor/Standalone/stand.bin

  • These scripts automate the searching, and are located in

/bin/net/db/projects/PeptideAtlas/species/bin run_PDP.csh runs predictor on each sub-file

  • Once the run is complete...

mk_PDP.csh merges results files into results.PDP

  • Run Peptide Sieve

ln -s /net/db/projects/PeptideAtlas/ExternalData/proteotypic/bin/PepSieve_20080530/peptideSieve_080527/peptideSieve/bin/PeptideSieve . ln -s /net/db/projects/PeptideAtlas/ExternalData/proteotypic/bin/PepSieve_20080530/peptideSieve_080527/peptideSieve/properties.txt .

Run PeptideDetectabilityPredictor, then merge into one results file These scripts automate the searching, and are located in /bin/net/db/projects/PeptideAtlas/species/bin run_PS.csh runs predictor on each sub-file

Once the run is complete... mk_PS.csh merges results files into results.PS


Merge the results from the two prediction engines. /regis/sbeams/bin/mergeProteotypicScores.pl -f reference_db_no-decoys.fasta -p results.PS -d results.PDP -o proteotypic_merged.tsv

Sort with ENS entries first for mapping, since the same peptides from proteins without mapping can then borrow the mapping info. For yeast use the -y flag, other non-ENS organism flags may be needed. sortEnsFirst.pl proteotypic_merged.tsv > proteotypic_merged-sorted.tsv

Finally, calculate genome mappings. If calc script is invoked with no args, it will output a usage stmt that includes current (2009-09) ENS mapping options ( -d species_core_52_37e ) nohup calculateNGenomeMappings.pl -f reference_db_no-decoys.fasta -p proteotypic_merged-sorted.tsv -o proteotypic_merged-sorted-mapped.tsv -d species_core_52_37e &


End general notes section


This section outlines the steps taken to process the new mouse reference database 2009-08.

Add /regis/sbeams/bin to PATH export PATH=/regis/sbeams/bin/:$PATH

1: Fetch up-to-date data sources, do some light processing.

IPI - version 3.62 (mouse 3.62 56733) wget ftp://ftp.ebi.ac.uk/pub/databases/IPI/current/ipi.MOUSE.fasta.gz wget ftp://ftp.ebi.ac.uk/pub/databases/IPI/current/README -O readme.ipi gunzip ipi.MOUSE.fasta.gz Fix IPI fasta accession line, forces seqs to one line. processFasta.pl -f ipi.MOUSE.fasta -i -v -o mouse_ipi_fixed-acc.fasta

Ensembl ftp://ftp.ensembl.org/pub/current_fasta/mus_musculus/pep/ wget ftp://ftp.ensembl.org/pub/current_fasta/mus_musculus/pep/Mus_musculus.NCBIM37.55.pep.all.fa.gz gunzip Mus_musculus.NCBIM37.55.pep.all.fa.gz wget ftp://ftp.ensembl.org/pub/current_fasta/mus_musculus/pep/README -O readme.ens Streamline the sequence to a single line processFasta.pl -f Mus_musculus.NCBIM37.55.pep.all.fa -v -o mouse_ensembl.fasta

Swiss Prot Fetch and then filter with processFasta, extracting MOUSE entries, fixing accession, main sp wget ftp://ftp.ebi.ac.uk/pub/databases/uniprot/knowledgebase/uniprot_sprot.fasta.gz processFasta.pl -f uniprot_sprot.fasta -v -s -r '_MOUSE' -o mouse_sprot_main.fasta

isoforms file wget ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot_varsplic.fasta.gz /regis/sbeams/bin/extractFasta.pl -f uniprot_sprot_varsplic.fasta -r '_MOUSE' -s -o swiss-prot_varsplice_mouse.fasta processFasta.pl -f uniprot_sprot_varsplic.fasta -v -s -r '_MOUSE' -o mouse_sprot_isoforms.fasta

concatenate - use processFasta to eliminate redundancy... processFasta.pl -f mouse_sprot_main.fasta -f mouse_sprot_isoforms.fasta -m -o mouse_sprot_merged.fasta -v

cRAP cp /regis/dbase/users/sbeams/cRAP/crap.fasta . processFasta.pl -f crap.fasta -s -o crap_clean.fasta

Decoys from original search/reference database (not common?) cp /net/db/projects/PeptideAtlas/pipeline/output/Mouse_2008-12_Ens47_P0.9/DATA_FILES/Mus_musculus.fasta ./Original_Mouse_reference_db.fasta processFasta.pl -f Original_Mouse_reference_db.fasta -r 'DECOY_' -v -o mouse_ipi_decoys.fasta


Concatenate all together! cat mouse_ensembl.fasta mouse_sprot_merged.fasta mouse_ipi_fixed-acc.fasta mouse_ipi_decoys.fasta crap_clean.fasta > mouse_reference_2009-08.fasta

Count unique/redundant seqs by 'merging' file to itself! processFasta.pl -f mouse_reference_2009-08.fasta -m -v -o mouse_reference_non-redundant_2009-08.fasta

total_files => 1 total_seqs => 133420 unique => 76830 redundant => 56590

Finally, for the proteotypic peptide stuff, remove DECOY seqs and trim any long (> 9000) sequences. processFasta.pl -f mouse_reference_2009-08.fasta -t 8999 -r DECOY_ -e -v -o mouse_reference_trimmed_no-decoy.fasta

2: Run the proteotypic scripts. kin => 3238189 no_pdp_prot => 488 no_ps_prot => 819640 orphan => 956 pdp_nan => 3086 pdp_no => 10123 pdp_ok => 3229022 prots => 121195 psieve_cterm_no => 17555 psieve_cterm_ok => 44824 psieve_has_first => 73870 psieve_has_last => 62903 psieve_no => 1091825 psieve_nterm_no => 31861 psieve_nterm_ok => 41755 psieve_ok => 2011325 

3239145 merged_proteotypic.tsv

wc: wc: No such file or directory 

3239145 total

mergeProteotypicScores.pl -f mouse_reference_nodecoys_2009-08.fasta -p results.PS -d results.PDP -o proteotypic_merged.tsv sortEnsFirst.pl proteotypic_merged.tsv > proteotypic_merged-sorted.tsv

nohup calculateNGenomeMappings.pl -f mouse_reference_nodecoys_2009-08.fasta -p proteotypic_merged-sorted.tsv -o proteotypic_merged-sorted-mapped.tsv -d mus_musculus_core_52_37e &

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