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.