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Output Files

The pVACseq pipeline will write its results in separate folders depending on which prediction algorithms were chosen:

  • MHC_Class_I: for MHC class I prediction algorithms

  • MHC_Class_II: for MHC class II prediction algorithms

  • combined: If both MHC class I and MHC class II prediction algorithms were run, this folder combines the neoepitope predictions from both

Each folder will contain the same list of output files (listed in the order created):

File Name

Description

<sample_name>.tsv

An intermediate file with variant, transcript, coverage, vaf, and expression information parsed from the input files.

<sample_name>.tsv_<chunks> (multiple)

The above file but split into smaller chunks for easier processing with IEDB.

<sample_name>.fasta

A fasta file with mutant and wildtype peptide subsequences for all processable variant-transcript combinations.

<sample_name>.net_chop.fa

A fasta file with mutant and wildtype peptide subsequences specific for use in running the net_chop tool.

<sample_name>.all_epitopes.tsv

A list of all predicted epitopes and their binding affinity scores, with additional variant information from the <sample_name>.tsv. Only epitopes resulting from supported variants (missense, inframe indels, and frameshifts) are included. If the --pass-only flag is set, variants that have a FILTER set in the VCF are excluded.

<sample_name>.filtered.tsv

The above file after applying all filters, with (optionally) cleavage site, stability predictions, and reference proteome similarity metrics added.

<sample_name>.all_epitopes.aggregated.tsv

An aggregated version of the all_epitopes.tsv file that gives information about the best epitope for each mutation in an easy-to-read format. Not generated when running with elution algorithms only.

<sample_name>.all_epitopes.aggregated.tsv.reference_matches (optional)

A file outlining details of reference proteome matches

<sample_name>.all_epitopes.aggregated.metrics.json

A JSON file with detailed information about the predicted epitopes, formatted for pVACview. This file, in combination with the aggregated.tsv file, is required to visualize your results in pVACview. Not generated when running with elution algorithms only.

ui.R, app.R, server.R, styling.R, anchor_and_helper_functions.R

pVACview R Shiny application files. Not generated when running with elution algorithms only.

www (directory)

Directory containing image files for pVACview. Not generated when running with elution algorithms only.

Filters applied to the filtered.tsv file

The filtered.tsv file is the all_epitopes file with the following filters applied (in order):

  • Binding Filter

  • Coverage Filter

  • Transcript Support Level Filter

  • Top Score Filter

Please see the Standalone Filter Commands documentation for more information on each individual filter. The standalone filter commands may be useful to reproduce the filtering or to chose different filtering thresholds.

Prediction Algorithms Supporting Percentile Information

pVACseq outputs binding affinity percentile rank information when provided by a chosen prediction algorithm. The following prediction algorithms calculate a percentile rank:

  • MHCflurry

  • NetMHC

  • NetMHCcons

  • NetMHCpan

  • NetMHCIIpan

  • NNalign

  • PickPocket

  • SMM

  • SMMPMBEC

  • SMMalign

The following prediction algorithms do not provide a percentile rank:

  • MHCnuggets

Prediction Algorithms Supporting Elution Scores

  • MHCflurryEL

  • NetMHCpanEL

  • NetMHCIIpanEL

  • BigMHC_EL

Prediction Algorithms Supporting Immunogenicity Scores

  • BigMHC_IM

  • DeepImmuno

Please note that when running pVACseq with only elution or immunogenicity algorithms, no aggregate report and pVACview files are created.

all_epitopes.tsv and filtered.tsv Report Columns

Column Name

Description

Chromosome

The chromosome of this variant

Start

The start position of this variant in the zero-based, half-open coordinate system

Stop

The stop position of this variant in the zero-based, half-open coordinate system

Reference

The reference allele

Variant

The alt allele

Transcript

The Ensembl ID of the affected transcript

Transcript Support Level

The transcript support level (TSL) of the affected transcript. Not Supported if the VCF entry doesn’t contain TSL information.

Transcript Length

The protein sequence length of the affected transcript

Biotype

The biotype of the affected transcript

Ensembl Gene ID

The Ensembl ID of the affected gene

Variant Type

The type of variant. missense for missense mutations, inframe_ins for inframe insertions, inframe_del for inframe deletions, and FS for frameshift variants

Mutation

The amnio acid change of this mutation

Protein Position

The protein position of the mutation

Gene Name

The Ensembl gene name of the affected gene

HGVSc

The HGVS coding sequence variant name

HGVSp

The HGVS protein sequence variant name

HLA Allele

The HLA allele for this prediction

Peptide Length

The peptide length of the epitope

Sub-peptide Position

The one-based position of the epitope within the protein sequence used to make the prediction

Mutation Position

The one-based positional range (inclusive) of the mutation within the epitope sequence. If the mutation is a deletion, the amino acids flanking the deletion are recorded. Note that in the case of ambiguous amino acid changes, this reflects the change that is left-aligned, starting from the first changed amino acid; this may differ from the Mutation column.

MT Epitope Seq

The mutant epitope sequence

WT Epitope Seq

The wildtype (reference) epitope sequence at the same position in the full protein sequence. NA if there is no wildtype sequence at this position or if more than half of the amino acids of the mutant epitope are mutated

Best MT IC50 Score Method

Prediction algorithm with the lowest mutant ic50 binding affinity for this epitope

Best MT IC50 Score

Lowest ic50 binding affinity of all prediction algorithms used

Corresponding WT IC50 Score

ic50 binding affinity of the wildtype epitope. NA if there is no WT Epitope Seq.

Corresponding Fold Change

Corresponding WT IC50 Score / Best MT IC50 Score. NA if there is no WT Epitope Seq.

Best MT Percentile Method

Prediction algorithm with the lowest binding affinity percentile rank for this epitope

Best MT Percentile

Lowest percentile rank of this epitope’s ic50 binding affinity of all prediction algorithms used (those that provide percentile output)

Corresponding WT Percentile

binding affinity percentile rank of the wildtype epitope. NA if there is no WT Epitope Seq.

Tumor DNA Depth

Tumor DNA depth at this position. NA if VCF entry does not contain tumor DNA readcount annotation.

Tumor DNA VAF

Tumor DNA variant allele frequency (VAF) at this position. NA if VCF entry does not contain tumor DNA readcount annotation.

Tumor RNA Depth

Tumor RNA depth at this position. NA if VCF entry does not contain tumor RNA readcount annotation.

Tumor RNA VAF

Tumor RNA variant allele frequency (VAF) at this position. NA if VCF entry does not contain tumor RNA readcount annotation.

Normal Depth

Normal DNA depth at this position. NA if VCF entry does not contain normal DNA readcount annotation.

Normal VAF

Normal DNA variant allele frequency (VAF) at this position. NA if VCF entry does not contain normal DNA readcount annotation.

Gene Expression

Gene expression value for the annotated gene containing the variant. NA if VCF entry does not contain gene expression annotation.

Transcript Expression

Transcript expression value for the annotated transcript containing the variant. NA if VCF entry does not contain transcript expression annotation.

Median MT IC50 Score

Median ic50 binding affinity of the mutant epitope across all prediction algorithms used

Median WT IC50 Score

Median ic50 binding affinity of the wildtype epitope across all prediction algorithms used. NA if there is no WT Epitope Seq.

Median Fold Change

Median WT IC50 Score / Median MT IC50 Score. NA if there is no WT Epitope Seq.

Median MT Percentile

Median binding affinity percentile rank of the mutant epitope across all prediction algorithms (those that provide percentile output)

Median WT Percentile

Median binding affinity percentile rank of the wildtype epitope across all prediction algorithms used (those that provide percentile output) NA if there is no WT Epitope Seq.

Individual Prediction Algorithm WT and MT IC50 Scores and Percentiles (multiple)

ic50 binding affintity and percentile ranks for the MT Epitope Seq and WT Eptiope Seq for the individual prediction algorithms used

MHCflurryEL WT and MT Processing Score and Presentation Score and Percentile (optional)

MHCflurry elution processing score and presentation score and percentiles for the MT Epitope Seq and WT Epitiope Seq if the run included MHCflurryEL as one of the prediction algorithms

Index

A unique idenitifer for this variant-transcript combination

Problematic Positions (optional)

A list of positions in the MT Epitope Seq that match the problematic amino acids defined by the --problematic-amino-acids parameter

cterm_7mer_gravy_score

Mean hydropathy of last 7 residues on the C-terminus of the peptide

max_7mer_gravy_score

Max GRAVY score of any kmer in the amino acid sequence. Used to determine if there are any extremely hydrophobic regions within a longer amino acid sequence.

difficult_n_terminal_residue (T/F)

Is N-terminal amino acid a Glutamine, Glutamic acid, or Cysteine?

c_terminal_cysteine (T/F)

Is the C-terminal amino acid a Cysteine?

c_terminal_proline (T/F)

Is the C-terminal amino acid a Proline?

cysteine_count

Number of Cysteines in the amino acid sequence. Problematic because they can form disulfide bonds across distant parts of the peptide

n_terminal_asparagine (T/F)

Is the N-terminal amino acid a Asparagine?

asparagine_proline_bond_count

Number of Asparagine-Proline bonds. Problematic because they can spontaneously cleave the peptide

Best Cleavage Position (optional)

Position of the highest predicted cleavage score

Best Cleavage Score (optional)

Highest predicted cleavage score

Cleavage Sites (optional)

List of all cleavage positions and their cleavage score

Predicted Stability (optional)

Stability of the pMHC-I complex

Half Life (optional)

Half-life of the pMHC-I complex

Stability Rank (optional)

The % rank stability of the pMHC-I complex

NetMHCstab allele (optional)

Nearest neighbor to the HLA Allele. Used for NetMHCstab prediction

pVACseq ouput file columns illustration

all_epitopes.aggregated.tsv Report Columns

The all_epitopes.aggregated.tsv file is an aggregated version of the all_epitopes TSV. It shows the best-scoring epitope for each variant, and outputs additional binding affinity, expression, and coverage information for that epitope. It also gives information about the total number of well-scoring epitopes for each variant, the number of transcripts covered by those epitopes, as well as the HLA alleles that those epitopes are well-binding to. Lastly, the report will bin variants into tiers that offer suggestions as to the suitability of variants for use in vaccines. Only epitopes meeting the --aggregate-inclusion-threshold are included in this report (default: 5000). Whether the median or the lowest binding affinity metrics are output in the IC50 MT, IC50 WT, %ile MT, and %ile WT columns is controlled by the --top-score-metric parameter.

Only epitopes meeting the --aggregate-inclusion-threshold are included in this report (default: 5000). Whether the median or the lowest binding affinity metrics are output in the IC50 MT, IC50 WT, %ile MT, and %ile WT columns is controlled by the --top-score-metric parameter.

Column Name

Description

ID

A unique identifier for the variant

HLA Alleles (multiple)

For each HLA allele in the run, the number of this variant’s epitopes that bound well to the HLA allele (with median/lowest mutant binding affinity < binding_threshold)

Gene

The Ensembl gene name of the affected gene

AA Change

The amino acid change for the mutation

Num Passing Transcripts

The number of transcripts for this mutation that resulted in at least one well-binding peptide (median/lowest mutant binding affinity < 500).

Best Peptide

The best-binding mutant epitope sequence (see Best Peptide Criteria below for more details on how this is determined)

Best Transcript

The best transcript of all transcripts coding for the Best Peptide (see Best Peptide Criteria below for more details on how this is determined)

TSL

The Transcript Support Level of the Best Transcript

Allele

The Allele that the Best Peptide is binding to

Pos

The one-based position of the start of the mutation within the epitope sequence. 0 if the start of the mutation is before the epitope (as can occur downstream of frameshift mutations)

Prob Pos

A list of positions in the Best Peptide that are problematic. None if the --problematic-pos parameter was not set during the pVACseq run

Num Passing Peptides

The number of unique well-binding peptides for this mutation.

IC50 MT

Median or lowest ic50 binding affinity of the best-binding mutant epitope across all prediction algorithms used

IC50 WT

Median or lowest ic50 binding affinity of the corresponding wildtype epitope across all prediction algorithms used.

%ile MT

Median or lowest binding affinity percentile rank of the best-binding mutant epitope across all prediction algorithms used (those that provide percentile output)

%ile WT

Median or lowest binding affinity percentile rank of the corresponding wildtype epitope across all prediction algorithms used (those that provide percentile output)

RNA Expr

Gene expression value for the annotated gene containing the variant.

RNA VAF

Tumor RNA variant allele frequency (VAF) at this position.

Allele Expr

RNA Expr * RNA VAF

RNA Depth

Tumor RNA depth at this position.

DNA VAF

Tumor DNA variant allele frequency (VAF) at this position.

Tier

A tier suggesting the suitability of variants for use in vaccines.

Ref Match (T/F) (optional)

Was there a match of the mutated peptide sequence to the reference proteome?

Evaluation

Column to store the evaluation of each variant when evaluating the run in pVACview. Either Accept, Reject, or Review.

Best Peptide Criteria

To determine the Best Peptide, all peptides meeting the --aggregate-inclusion-threshold are evaluated as follows:

  • Pick all entries with a variant transcript that have a protein_coding Biotype

  • Of the remaining entries, pick the ones with a variant transcript having a Transcript Support Level <= maximum_transcript_support_level

  • Of the remaining entries, pick the entries with no Problematic Positions

  • Of the remaining entries, pick the ones passing the Anchor Criteria (see Criteria Details section below)

  • Of the remaining entries, pick the one with the lowest median/best MT IC50 score, lowest Transcript Support Level, and longest transcript.

The pVACseq Aggregate Report Tiers

Tiering Parameters

To tier the Best Peptide, several cutoffs can be adjusted using arguments provided to the pVACseq run:

Parameter

Description

Default

--binding-threshold

The threshold used for filtering epitopes on the IC50 MT binding affinity.

500

--allele-specific-binding-thresholds

Instead of the hard cutoff set by the --binding-threshold, use allele-specific binding thresholds. For alleles where no allele-specific binding threshold is available, use the --binding-threshold as a fallback. To print a list of alleles that have specific binding thresholds and the value of those thresholds, run pvacseq allele_specific_cutoffs.

False

--percentile-threshold

When set, use this threshold to filter epitopes on the %ile MT score in addition to having to meet the binding threshold.

None

--tumor-purity

Value between 0 and 1 indicating the fraction of tumor cells in the tumor sample. Information is used for a simple estimation of whether variants are subclonal or clonal based on VAF. If not provided, purity is estimated directly from the VAFs.

None

--trna-vaf

Tumor RNA VAF Cutoff. Used to calculate the allele expression cutoff for tiering.

0.25

--trna-cov

Tumor RNA Coverage Cutoff. Used as a cutoff for tiering.

10

--expn-val

Gene and Expression cutoff. Used to calculate the allele expression cutoff for tiering.

1.0

--maximum-transcript-support-level

The threshold to use for filtering epitopes on the Ensembl transcript support level (TSL). Transcript support level needs to be <= this cutoff to be included in most tiers.

1

--allele-specific-anchors

Use allele-specific anchor positions when tiering epitopes in the aggregate report. This option is available for 8, 9, 10, and 11mers and only for HLA-A, B, and C alleles. If this option is not enabled or as a fallback for unsupported lengths and alleles, the default positions of [1, 2, epitope length - 1, and epitope length] are used. Please see https://doi.org/10.1101/2020.12.08.416271 for more details.

False

--anchor-contribution-threshold

For determining allele-specific anchors, each position is assigned a score based on how binding is influenced by mutations. From these scores, the relative contribution of each position to the overall binding is calculated. Starting with the highest relative contribution, positions whose score together account for the selected contribution threshold are assigned as anchor locations. As a result, a higher threshold leads to the inclusion of more positions to be considered anchors.

0.8

Tiers

Given the thresholds provided above, the Best Peptide is evaluated and binned into a tier as follows:

Tier

Citeria

Pass

Best Peptide passes the binding, expression, tsl, clonal, and anchor criteria

Anchor

Best Peptide fails the anchor criteria but passes the binding, expression, tsl, and clonal criteria

Subclonal

Best Peptide fails the clonal criteria but passes the binding, tsl, and anchor criteria

LowExpr

Best Peptide meets the Low Expression Criteria and passes the binding, tsl, clonal, and anchor criteria

NoExpr

Best Peptide is not expressed (RNA Expr == 0 or RNA VAF == 0)

Poor

Best Peptide doesn’t fit in any of the above tiers, usually if it fails two or more criteria or if it fails the binding criteria

Criteria Details

Binding Criteria

Pass if Best Peptide is a strong binder

IC50 MT < binding_threshold and %ile MT < percentile_threshold (if --percentile-threshold parameter is set)

Expression Criteria

Pass if Best Transcript is expressed

Allele Expr > trna_vaf * expn_val

Low Expression Criteria

Peptide has low expression or no expression but RNA VAF and coverage

(0 < Allele Expr < trna_vaf * expn_val) OR (RNA Expr == 0 AND RNA Depth > trna_cov AND RNA VAF > trna_vaf)

TSL Criteria

Pass if Best Transcript has good transcript support level

TSL <= maximum_transcript_support_level

Clonal Criteria

Best Peptide is likely in the founding clone of the tumor

DNA VAF > tumor_purity / 4

Anchor Criteria

Fail if all mutated amino acids of the Best Peptide (Pos) are at an anchor position and the WT peptide has good binding (IC50 WT < binding_threshold)

aggregated.tsv.reference_matches Report Columns

This file is only generated when the --run-reference-proteome-similarity option is chosen.

Column Name

Description (BLAST)

Description (reference fasta)

Chromosome

The chromosome of this variant

Start

The start position of this variant in the zero-based, half-open coordinate system

Stop

The stop position of this variant in the zero-based, half-open coordinate system

Reference

The reference allele

Variant

The alt allele

Transcript

The Ensembl ID of the affected transcript

MT Epitope Seq

The mutant peptide sequence for the epitope candidate

Peptide

The peptide sequence submitted to BLAST

The peptide sequence to search for in the reference proteome

Hit ID

The BLAST alignment hit ID (reference proteome sequence ID)

The FASTA header ID of the entry where the match was made

Hit Definition

The BLAST alignment hit definition (reference proteome sequence name)

The FASTA header description of the entry where the match was made

Match Window

The substring of the Peptide that was found in the Match Sequence

Match Sequence

The BLAST match sequence

The FASTA sequence of the entry where the match was made

Match Start

The match start position of the Match Window in the Match Sequence

Match Stop

The match stop position of the Match Window in the Match Sequence