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>.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

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

• 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

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 immunogenicity algorithms, no aggregate report is created.

all_epitopes.tsv and filtered.tsv Report Columns

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 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 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 Scores and Percentiles (multiple)	ic50 binding affintity and percentile ranks for the Epitope Seq for the individual prediction algorithms used
MHCflurryEL Processing Score and Presentation Score and Percentile (optional)	MHCflurry elution processing score and presentation score and percentiles for the Epitope Seq if the run included MHCflurryEL as one of the prediction algorithms
Read Support	The sum of spanning and encompassing reads over the fusion position. NA if the run was made with AGFusion data and without a --starfusion-file input.
Expression	The number of fusion-supporting RNA-seq fragments as FFPM (fusion fragments per million total reads). NA if the run was made without a --starfusion-file input.
Problematic Positions (optional)	A list of positions in the 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

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 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 and %ile MT columns is controlled by the --top-score-metric parameter.

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
Best Peptide	The best-binding epitope sequence (lowest IC50 MT score)
Best Transcript	The fusion transcripts coding for the Best Peptide
Allele	The Allele that the Best Peptide is binding to
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 fusion
IC50 MT	Median or lowest IC50 binding affinity of the best-binding epitope across all prediction algorithms used
%ile MT	Median or lowest binding affinity percentile rank of the best-binding epitope across all prediction algorithms used (those that provide percentile output)
Expr	The number of fusion-supporting RNA-seq fragments as FFPM (fusion fragments per million total reads). NA if the run was made without a --starfusion-file input.
Read Support	The sum of spanning and encompassing reads over the fusion position. NA if the run was made with AGFusion data and without a --starfusion-file input.
Tier	A tier suggesting the suitability of variants for use in vaccines.
Ref Match (T/F) (optional)	Was there a match of the peptide sequence to the reference proteome?
Evaluation	Column to store the evaluation of each fusion. Either Accept, Reject, or Review.

The pVACfuse Aggregate Report Tiers

Tiering Parameters

To tier the Best Peptide, several cutoffs can be adjusted using parameters provided to the pVACfuse 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 pvacfuse 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
--read-support	The threshold used for filtering epitopes on the Read Support.	5
--expn-val	The threshold used for filtering epitopes on the Expr.	0.1

Tiers

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

Tier	Criteria
Pass	Best Peptide passes the binding, read support, and expression criteria
LowReadSupport	Best Peptide fails the read support criteria but passes the binding and expression criteria
LowExpr	Best Peptide fails the expression criteria but passes the binding and read support criteria
Poor	Best Peptide doesn’t fit 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 strong binder	IC50 MT < binding_threshold and %ile MT < percentile_threshold (if --percentile-threshold parameter is set)
Read Support Criteria	Pass if the variant has read support	Read Support < read_support
Expression Criteria	Pass if Best Transcript is expressed	Expr < expn_val

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)
ID	A unique identifier for the fusion
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