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Triqler: TRansparent Identification-Quantification-Linked Error Rates

Method description / Citation

The, M. & Käll, L. (2019). Integrated identification and quantification error probabilities for shotgun proteomics. Molecular & Cellular Proteomics, 18 (3), 561-570.

Requirements

Python 2 or 3 installation

Packages needed:

  • numpy 1.12+
  • scipy 0.17+

Installation via pip

pip install triqler

Installation from source

git clone https://github.com/statisticalbiotechnology/triqler.git
cd triqler
pip install .

Usage

usage: python -m triqler [-h] [--out_file OUT] [--fold_change_eval F]
                 [--decoy_pattern P] [--min_samples N] [--num_threads N]
                 [--ttest] [--write_spectrum_quants]
                 [--write_protein_posteriors P_OUT]
                 [--write_group_posteriors G_OUT]
                 [--write_fold_change_posteriors F_OUT]
                 IN_FILE

positional arguments:
  IN_FILE               List of PSMs with abundances (not log transformed!)
                        and search engine score. See README for a detailed
                        description of the columns.

optional arguments:
  -h, --help            show this help message and exit
  --out_file OUT        Path to output file (writing in TSV format). N.B. if
                        more than 2 treatment groups are present, suffixes
                        will be added before the file extension. (default:
                        proteins.tsv)
  --fold_change_eval F  log2 fold change evaluation threshold. (default: 1.0)
  --decoy_pattern P     Prefix for decoy proteins. (default: decoy_)
  --min_samples N       Minimum number of samples a peptide needed to be
                        quantified in. (default: 2)
  --num_threads N       Number of threads, by default this is equal to the
                        number of CPU cores available on the device. (default:
                        8)
  --ttest               Use t-test for evaluating differential expression
                        instead of posterior probabilities. (default: False)
  --write_spectrum_quants
                        Write quantifications for consensus spectra. Only
                        works if consensus spectrum index are given in input.
                        (default: False)
  --write_protein_posteriors P_OUT
                        Write raw data of protein posteriors to the specified
                        file in TSV format. (default: )
  --write_group_posteriors G_OUT
                        Write raw data of treatment group posteriors to the
                        specified file in TSV format. (default: )
  --write_fold_change_posteriors F_OUT
                        Write raw data of fold change posteriors to the
                        specified file in TSV format. (default: )

Example

A sample file iPRG2016.tsv is provided in the example folder. You can run Triqler on this file by running the following command:

python -m triqler --fold_change_eval 0.8 example/iPRG2016.tsv

Interface

The simplest input format is a tab-separated file consisting of a header line followed by one PSM per line in the following format:

run <tab> condition <tab> charge <tab> searchScore <tab> intensity <tab> peptide     <tab> proteins
r1  <tab> 1         <tab> 2      <tab> 1.345       <tab> 21359.123 <tab> A.PEPTIDE.A <tab> proteinA <tab> proteinB
r2  <tab> 1         <tab> 2      <tab> 1.945       <tab> 24837.398 <tab> A.PEPTIDE.A <tab> proteinA <tab> proteinB
r3  <tab> 2         <tab> 2      <tab> 1.684       <tab> 25498.869 <tab> A.PEPTIDE.A <tab> proteinA <tab> proteinB
...
r1  <tab> 1         <tab> 3      <tab> 0.452       <tab> 13642.232 <tab> A.NTPEPTIDE.- <tab> decoy_proteinA

Alternatively, if you have match-between-run probabilities, a slightly more complicated input format can be used as input:

run <tab> condition <tab> charge <tab> searchScore <tab> spectrumId <tab> linkPEP <tab> featureClusterId <tab> intensity <tab> peptide     <tab> proteins
r1  <tab> 1         <tab> 2      <tab> 1.345       <tab> 3          <tab> 0.0     <tab> 1                <tab> 21359.123 <tab> A.PEPTIDE.A <tab> proteinA <tab> proteinB
r2  <tab> 1         <tab> 2      <tab> 1.345       <tab> 3          <tab> 0.021   <tab> 1                <tab> 24837.398 <tab> A.PEPTIDE.A <tab> proteinA <tab> proteinB
r3  <tab> 2         <tab> 2      <tab> 1.684       <tab> 4          <tab> 0.0     <tab> 1                <tab> 25498.869 <tab> A.PEPTIDE.A <tab> proteinA <tab> proteinB
...
r1  <tab> 1         <tab> 3      <tab> 0.452       <tab> 6568       <tab> 0.15    <tab> 9845             <tab> 13642.232 <tab> A.NTPEPTIDE.- <tab> decoy_proteinA

Some remarks:

  • For Triqler to work, it also needs decoy PSMs, preferably resulting from a search engine search with a reversed protein sequence database concatenated to the target database.
  • The intensities should not be log transformed, Triqler will do this transformation for you.
  • The search engine scores should be such that higher scores indicate a higher confidence in the PSM.
  • We recommend usage of well calibrated search engine scores, e.g. the SVM scores from Percolator.
  • Multiple proteins can be specified at the end of the line, separated by tabs. However, it should be noted that Triqler currently discards shared peptides.

The output format is a tab-separated file consisting of a header line followed by one protein per line in the following format:

q_value <tab> posterior_error_prob <tab> protein <tab> num_peptides <tab> protein_id_PEP <tab> log2_fold_change <tab> diff_exp_prob_<FC> <tab> <condition1>:<run1> <tab> <condition1>:<run2> <tab> ... <tab> <conditionM>:<runN> <tab> peptides

Some remarks:

  • The reported protein expressions are the expected value of the protein's expression in the run. They are calculated relative to the protein's mean expression and are not log transformed.
  • The reported fold change is log2 transformed and is the expected value based on the posterior distribution of the fold change.
  • If more than 2 treatment groups are present, separate files will be written out for each pairwise comparison with suffixes added before the file extension, e.g. proteins.1vs3.tsv.