Building Peptide Atlas
From SPCTools
Here is how I built a human urine PeptideAtlas in December 2008.
Start with one or more PeptideProphet output files (pepXML) for each experiment in each project.
A project is a set of related experiments. For example, a project may study proteins found in normal and diseased liver, and may include 4 experiments: tissue from two normal patients and from two diseased patients. The pepXML files should be created by searching the spectra with a database search engine such as SEQUEST, X!Tandem, or SpectraST, then validating the hits using PeptideProphet. If you are going to combine search results using iProphet (see below), iProphet should not be run on each set of search results individually; to avoid running iProphet you will need to run xinteract manually because scripts such as runtandemsearch automatically run iProphet after PeptideProphet.
Searching databases containing decoys is recommended to provide a reference point for evaluating the FDR (false discovery rate) of the final Atlas. As of fall 2008, spectral libraries containing decoys are available for SpectraST searching.
It is helpful when referencing files using wildcards if the pepXML files all reside at the same level in the directory tree. If you have to move files to achieve this, adjust the paths within them using
$ /sbeams/bin/updateAllPaths.pl *.xml *.xls *.shtml.
Copy build recipe and follow it
The unix commands needed to do each step below are given in mimas.systemsbiology.net:/net/db/projects/PeptideAtlas/pipeline/recipes/master_recipe.notes. Copy this file to <your_build>_recipe.notes, follow along, and edit as needed for your build. Most stuff takes place via mimas (a.k.a. db) at /net/db/projects/PeptideAtlas.
Register projects and experiments using SBEAMS interface.
- Go to db.systemsbiology.net.
- Login to SBEAMS.
- Click tab "My Projects" or "Accessible Projects" and click "Add new project" at bottom.
- Fill out fields. Owner of project should be the experimenter who created the data. Project tag should match name of subdirectory in /sbeams/archive/<project_owner> that contains the data.
- To register experiments, go to SBEAMS Home > Proteomics > Manage Data > Experiments
Run iProphet and ProteinProphet
If you ran multiple search engines for each experiment, combine per experiment using iProphet.
Create a directory, parallel to the search results directories, named iProphet. Then, for example,
$ iProphet ../{XTK,SPC,SEQ}*/interact-prob.pep.xml interact-combined.pep.xml
Be sure that the input pepXML files were not already processed by iProphet -- you don't want to run iProphet twice. Caution: if you ran an automated post-processing script such as runtandemsearch (which calls finishtandemsearch), iProphet may already have been run automatically. Conversely, if you are using only one search engine and did not run iProphet immediately after running ProteinProphet, run it now.
The resulting pepXML files will be used to generate final peptide probabilities in the "gather all peptides" step of the Atlas build process below.
Combine all pepXML files for project using iProphet, then run ProteinProphet
First create a directory for your project in your data area, for ample disk space. Run on regis9 for ample memory.
$ ssh regis9
$ cd /regis/data3/tfarrah/search
$ mkdir HsUrine; cd HsUrine; mkdir MultipleExps; cd MultipleExps; mkdir iProphet; cd iProphet
$ iProphet /regis/sbeams/archive/{phaller,youngah}/*Urine*/*/{XTK,SPC,SEQ}*/interact-prob.pep.xml
$ ProteinProphet interact-combined.pep.xml interact-combined.prot.xml UNMAPPED NORMPROTLEN PROTLEN MININDEP0.2 IPROPHET > & ProteinProphet.out
Combining all pepXML files may not be feasible with many and/or large files. In that case, you will need to run iProphet on the experiments in batches, then run ProteinProphet on all the resulting pepXML files combined. Consult David S. for advice.
The purpose of the ProteinProphet run is to adjust the probabilities of all the peptides according to NSP (number of sibling peptides). The adjusted probabilities are not used directly, but are used to generate a multiplicative factor for each peptide which is then applied to the iProphet probability for each observation of that peptide. In particular, it is important to note that, as of January 2009, ProteinProphet protein probabilities are not displayed or used in any way in the PeptideAtlas. This is because these probabilities are, for large datasets, overly optimistic.
Obtain search batch IDs for each experiment.
Run PeptideAtlas build "pipeline".
Scripts can be found in /net/db/projects/PeptideAtlas/pipeline/run_scripts. Each script ultimately calls /net/db/projects/PeptideAtlas/pipeline/run_scripts/run_Master_current.csh, and this is where the meat of the pipeline resides.
Gather all peptides and update probabilities
Step01. Calls createPipelineInput.pl, via pipeline/bin/PeptideFilesGenerator.pm. Creates "identlist file" for each pepXML in Experiments.list, and also a combined file. This is a simple text format (one line per record) file containing all the relevant pepXML and protXML info for each peptide identification with P>=threshold (usually 0.9), with peptide probabilities adjusted using the protXML probabilities as a guide. An identlist template file is also created which contains only the unadjusted pepXML info for peptides with P>=(threshold-0.4); it is cached in the same dir as each pepXML file for use in future builds.
At the core of this step is the script createPipelineInput.pl.
Files created in build directory, all but last created by createPipelineInput.pl:
- PeptideAtlasInput_concat.PAidentlist
- PeptideAtlasInput_sorted.PAidentlist
- APD_Sc_all.tsv
- APD_Sc_all.PAxml
- APD_Sc_all.fasta (created in pipeline script after call to createPipelineInput.pl)
Download latest fasta files from web for reference DB (also called biosequence set)
Step02. Calls pipeline script with --getEnsembl and executes getEnsembl(). Gets Ensembl fasta file via FTP unless stored locally. Merges in any supplemental protein file specified by calling $PIPELINE/bin/mergeEnsembleAndIPI.pl. Files created:
- <species>.pep.fa -- Ensembl file as retrieved via FTP.
- <species>.fasta
Seems to me that this step can be skipped if we are using a reference DB / biosequence set that already exists in SBEAMS -- often the case. Also seems that this step, like the SpectraST library building step, is independent of all the others.
BLAST peptides against reference DB
Step03. Calls pipeline script with --BLASTP. Script then calls matchPeptides(), which does a system call of $PIPELINE/bin/PeptidePositionLocator.pl, which seems to search the DB without calling blast and prints "N peptides with/without a match". Files created:
- peptide_mapping.tsv
Align peptides with reference proteins and calculate coordinates (start/end points of aligned region)
Step04. Calls pipeline script with --BLASTParse. Script then calls BLAST_APD_ENSEMBL(). Files created:
- APD_ensembl_hits.tsv -- one line per matched spectrum, listing PA peptide accession, coordinates, protein sequence identifier, and a bit more.
Parse the mapping results and calculate chromosomal coordinates
Step05. Calls pipeline script with --BLASTParse --getCoordinates. Calls BLAST_APD_ENSEMBL() again, which reads any previous coordinate cache file (.enscache) Files created:
- coordinate_mapping.txt
- $PIPELINE/new_cache/$dbname.enscache
Make a list of unmappable peptides
Step06. Calls pipeline script with --lostAndFound. Files needed:
- APD_<organism>_all.fasta
- APD_ensembl_hits.tsv
Files created:
- APD_ensembl_lost_queries.dat
Compile statistics on the peptides and proteins in the build
Step07. Results in /net/db/projects/PeptideAtlas/pipeline/output/HumanUrine_2008-09_Ens49/analysis/analysis.out. This file contains instructions for doing the following manually:
- creating an experiment contribution plot. A plot for the web Atlas is generated automatically, but these instructions tell you how to create a better one.
- creating an amino acid abundance plot. Untested by Terry.
Calls the following:
- /regis/sbeams/bin/Mayu/Mayu.pl
- $PIPELINE/bin/fasta_stat.pl
- $PIPELINE/bin/peptide_stats_from_step04.pl
- $SBEAMS/lib/scripts/PeptideAtlas/calcProteinStatistics.pl
- $SBEAMS/lib/scripts/PeptideAtlas/statistics/calcPeptideListStatistics.pl
- $SBEAMS/bin/protein_chance_hits.pl
Files created automatically:
In analysis directory:
- prophet_model.sts
- search_dir_stats.txt
- analysis.out
In DATA_FILES directory:
- protein2gene.txt
- duplicate_groups.txt
- duplicate_mapping.txt
- duplicate_entries.txt
- out.2tonsequences
- PPvsDECOY.dat
- experiment_contribution_summary.out
- protein_chance_hits.out
- simplereducedproteins.txt -- a rather minimal list of proteins in sample
- msruncounts.txt
Build a SpectraST library from the build
Step08. Does not depend on any previous steps and can be executed at any point in the pipeline. Files created:
- <build-name>_all.splib
- <build-name>_all.sptxt
Load the reference DB (biosequence set) if new one is needed
Define Atlas build via SBEAMS
Load data into build
Commands below entered on command line on mimas. Full usage including desired options found in recipe.
Load data
$SBEAMS/lib/scripts/PeptideAtlas/load_atlas_build.pl. January 22, 2009: using --purge and --load options together seems to reload previous build. Instead, call first with --purge, then again with --load.
Build search key
$SBEAMS/lib/scripts/PeptideAtlas/rebuildKeySearch.pl
Update empirical proteotypic scores
$SBEAMS/lib/scripts/PeptideAtlas/updateProteotypicScores.pl
