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	Description
<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.

all_epitopes.tsv and filtered.tsv Report Columns

Column Name	Description
Mutation	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 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 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
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