pVACbind logo

Output Files

The pVACbind 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



A list of all predicted epitopes and their binding affinity scores, with additional variant information from the <sample_name>.tsv.


The above file after applying all filters, with cleavage site and stability predictions added.


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

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

  • 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


  • 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 pVACfuse with only elution or immungenicity algorithms, no aggregate report is created.

all_epitopes.tsv and filtered.tsv Report Columns

Column Name



The FASTA ID of the peptide sequence the epitope belongs to

HLA Allele

The HLA allele for this prediction

Sub-peptide Position

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

Epitope Seq

The epitope sequence

Median IC50 Score

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

Best IC50 Score

Lowest ic50 binding affinity of all prediction algorithms used

Best IC50 Score Method

Prediction algorithm with the lowest ic50 binding affinity for this epitope

Median Percentile

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

Best Percentile

Lowest binding affinity percentile rank of all prediction algorithms used (those that provide percentile output)

Best Percentile Method

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

Individual Prediction Algorithm Scores and Percentiles (multiple)

ic50 binding affinity scores and percentiles for the Epitope 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


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


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?


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?


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

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 binding affinity and other information for that epitope. It gives information about the total number of well-scoring epitopes for each variant 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, %ile MT, and columns is controlled by the --top-score-metric parameter.

Column Name



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 binding affinity < 1000)

Best Peptide

The best-binding epitope sequence (lowest median binding affinity)

Prob Pos

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

Num Passing Peptides

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


Median IC50 binding affinity of the best-binding epitope across all prediction algorithms used

%ile MT

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

Ref Match (T/F) (optional)

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


Column to store the evaluation of each variant. Either Accept, Reject, or Review.

The pVACbind Aggregate Report Tiers

Tiering Parameters

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





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



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 pvacfuse allele_specific_cutoffs.



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



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




Best Peptide passes the binding criteria


Best Peptide fails the binding criteria

Criteria Details

Binding Criteria

Pass if Best Peptide is strong binder

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

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)


A unique identifier for the variant

Epitope Seq

The mutant peptide sequence for the epitope candidate


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