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>.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 (optionally) cleavage site, stability predictions, and reference proteome similarity metrics 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

• 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.

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. NA if the VCF entry doesn’t contain TSL information.
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 position of the start of the mutation within the epitope sequence. 0 if the start of the mutation is before the epitope
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 Score Method	Prediction algorithm with the lowest mutant ic50 binding affinity for this epitope
Best MT Score	Lowest ic50 binding affinity of all prediction algorithms used
Corresponding WT Score	ic50 binding affinity of the wildtype epitope. NA if there is no WT Epitope Seq.
Corresponding Fold Change	Corresponding WT Score / Best MT 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 Score	Median ic50 binding affinity of the mutant epitope across all prediction algorithms used
Median WT 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 Score / Median MT 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 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
Index	A unique idenitifer for this variant-transcript combination
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. It presents the best-scoring (lowest binding affinity) 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.

Column Name	Description
HLA Alleles (multiple) (T/F)	For each HLA allele in the run, did the mutation result in an epitope that bound well to the HLA allele? (with median mutant binding affinity < 1000).
Gene	The Ensembl gene name of the affected gene
AA_change	The amino acid change for the mutation
Num_Transcript	The number of transcripts for this mutation that resulted in at least one well-binding peptide (median mutant binding affinity < 1000).
Peptide	The best-binding mutant epitope sequence (lowest median mutant binding affinity)
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)
Num_Peptides	The number of unique well-binding peptides for this mutation.
ic50_MT	Median ic50 binding affinity of the best-binding mutant epitope across all prediction algorithms used
ic50_WT	Median ic50 binding affinity of the corresponding wildtype epitope across all prediction algorithms used.
percentile_MT	Median binding affinity percentile rank of the best-binding mutant epitope across all prediction algorithms used (those that provide percentile output)
percentile_WT	Median 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.
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.

The pVACseq Aggregate Report Tiers

To bin a variant in a tier, the best binding epitope is evaluated as follows:

Tier	Citeria
NoExpr	Mutant allele is not expressed
LowExpr	Mutant allele has low expression (TPM * RNA_VAF < 1)
Subclonal	Likely not in the founding clone of the tumor (DNA_VAF > max(DNA_VAF)/2)
Anchor	Mutation is at an anchor residue in the shown peptide, and the WT allele has good binding (WT ic50 <1000)
Poor	Fails two or more of the above criteria
Relaxed	Passes the above criteria, has decent MT binding (ic50 < 1000)
Pass	Passes the above criteria, has strong MT binding (ic50 < 1000) and strong expression (TPM * RNA_VAF > 3)