Output

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

Pipeline overview

The pipeline is built using Nextflow and processes data using the final output of the pipeline should include the following files:

• all_features_merged_resolved.mzTab - the community standard format for sharing mass spectrometry search results
• all_features_merged_resolved.csv - aggregate csv report, containing all information about peptide identification and quantification results
• found_neoepitopes.csv - a csv listing all neoepitopes found in the mass spectrometry search, independant of binding predictions
• vcf_neoepitopes.csv - a csv listing all theoretically possible neoepitope sequences from the variants specified in the vcf
• _vcf.fasta - the fasta database including mutated proteins used for the database search
• class_1/2_binding_predictions - a folder containing the respective binding predictions of all detected peptides and all theoretically possible neoepitope sequences
• Intermediate_resuls - a folder containing all intermediate results from the steps in the pipeline (unfiltered and filtered PSMs, aligned mzMLs, features, etc. ..)
• Intermediate_results/mhcquant_file_order.txt - a txt file listing the order of files used for annotating intensitites in the mzTab output
• Documentation - a folder containing summarized reports of the pipeline execution
• pipeline_info - a folder containing detailed reports on computational runtimes and workflow steps

mzTab

The output mzTab contains many columns annotating the most important information - here are a few outpointed:

PEP   sequence   accession   best_search_engine_score[1]   retention_time   charge   mass_to_charge   peptide_abundance_study_variable[1]

Most important to know that in this format we annotated the q-value of each peptide identification in the best_seach_engine_score[1] column and peptide quantities in the peptide_abundance_study_variable columns.

mzTab is a light-weight format to report mass spectrometry search results. It provides all important information about identified peptide hits and is compatible with the PRIDE Archive - proteomics data repository:

Griss, J. et al. The mzTab Data Exchange Format: Communicating Mass-spectrometry-based Proteomics and Metabolomics Experimental Results to a Wider Audience. Mol Cell Proteomics 13, 2765-2775 (2014)

csv

The csv output file is a table containing all information extracted from a database search throughout the pipeline. See the OpenMS or PSI documentation for more information about annotated scores and format.

#MAP    id      filename        label   size

MAP contains information about the different mzML files that were provided initially

#RUN    run_id  score_type      score_direction date_time       search_engine_version   parameters

RUN contains information about the search that was performed on each run

#PROTEIN        score   rank    accession       protein_description     coverage        sequence

PROTEIN contains infomration about the protein ids corresponding to the peptides that were detected (No protein inference was performed)

#UNASSIGNEDPEPTIDE      rt      mz      score   rank    sequence        charge  aa_before       aa_after        score_type      search_identifier       accessions      FFId_category   feature_id      file_origin     map_index       spectrum_reference      COMET:IonFrac   COMET:deltCn    COMET:deltLCn   COMET:lnExpect  COMET:lnNumSP   COMET:lnRankSP  MS:1001491      MS:1001492      MS:1001493      MS:1002252      MS:1002253      MS:1002254      MS:1002255      MS:1002256      MS:1002257      MS:1002258      MS:1002259      num_matched_peptides    protein_references      target_decoy

UNASSIGNEDPEPTIDE contains information about PSMs that were identified but couldn’t be quantified to a precursor feature on MS Level 1.

#CONSENSUS      rt_cf   mz_cf   intensity_cf    charge_cf       width_cf        quality_cf      rt_0    mz_0    intensity_0     charge_0        width_0 rt_1    mz_1    intensity_1     charge_1        width_1 rt_2    mz_2    intensity_2     charge_2        width_2 rt_3    mz_3    intensity_3     charge_3        width_3

CONSENSUS contains information about precursor features that were identified in multiple runs (eg. run 1-3 in this case)

#PEPTIDE        rt      mz      score   rank    sequence        charge  aa_before       aa_after        score_type      search_identifier       accessions      FFId_category   fea

PEPTIDE contains information about peptide hits that were identified and correspond to the consensus features described one row above.

found_neoepitopes

csv file listing detected neoepitope sequences:

peptide sequence   geneID

vcf_neoepitopes

csv file listing theoretically possible neoepitope sequences:

Sequence        Antigen ID       Variants

class_1/2_binding_predictions

The prediction outputs are comma separated table (csv) for each allele, listing each peptide sequence and its corresponding predicted affinity scores:

peptide   allele   prediction   prediction_low   prediction_high   prediction_percentile