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Output Files

The pVACfuse 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 and transcript information parsed from the input file(s).

<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 peptide subsequences for all processable fusion combinations.

<sample_name>.net_chop.fa

A fasta file with mutant 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.

<sample_name>.filtered.tsv

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

<sample_name>.filtered.tsv.reference_matches (optional)

A file outlining details of reference proteome matches

<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.

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

pVACfuse 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

all_epitopes.tsv and filtered.tsv Report Columns

In order to keep the outputs consistent, pVACfuse uses the same output columns as pVACseq but some of the values will be NA if a column doesn’t apply to pVACfuse.

Column Name

Description

Chromosome

The chromosomes of the 5p and 3p portion of the fusion, separated by ” / “

Start

The start positions of the 5p and 3p portion of the fusion, separated by ” / “

Stop

The stop positions of the 5p and 3p portion of the fusion, separated by ” / “

Transcript

The Ensembl IDs of the affected transcripts

Gene Name

The Ensembl gene names of the affected genes

Variant Type

The type of fusion. inframe_fusion for inframe fusions, frameshift_fusion for frameshift fusions

Mutation

A unique identifier for the fusion

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

Epitope sequence

Median Score

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

Best Score

Lowest ic50 binding affinity of all prediction algorithms used

Best Score Method

Prediction algorithm with the lowest ic50 binding affinity for this epitope

Median Percentile

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

Best Percentile

Lowest percentile rank of this epitope’s ic50 binding affinity 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 WT and MT Scores and Percentiles (multiple)

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

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

Reference Match (T/F) (optional)

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

filtered.tsv.reference_matches Report Columns

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

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

Peptide

The peptide sequence submitted to BLAST

Hit ID

The BLAST alignment hit ID (reference proteome sequence ID)

Hit Definition

The BLAST alignment hit definition (reference proteome sequence name)

Query Sequence

The BLAST query sequence

Match Sequence

The BLAST match sequence

Match Start

The match start position in the matched reference proteome sequence

Match Stop

The match stop position in the matched reference proteome sequence

all_epitopes.aggregated.tsv Report Columns

The all_epitopes.aggregated.tsv file is an aggregated version of the all_epitopes TSV. Like the all_epitopes.tsv and filtered.tsv reports, in order to keep the outputs consistent, pVACfuse uses the same output columns as pVACseq for this file but some of the values will be NA if a column doesn’t apply to pVACfuse. This report presents the best-scoring (lowest binding affinity) epitope for each variant and outputs additional binding affinity for that epitope. It also 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.

Column Name

Description

ID

A unique identifier for the fusion

HLA Alleles (multiple)

For each HLA allele in the run, the number of this fusion’s epitopes that bound well to the HLA allele (with median binding affinity < 1000)

Gene

The Ensembl gene names of the affected genes

AA Change

NA

Num Passing Transcripts

NA

Best Peptide

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

Pos

NA

Num Passing Peptides

The number of unique well-binding peptides for this fusion

IC50 MT

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

IC50 WT

NA

%ile MT

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

%ile WT

NA

RNA Expr

NA

RNA VAF

NA

Allele Expr

NA

RNA Depth

NA

DNA VAF

NA

Tier

NA

Evaluation

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