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 neoeptiope 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>.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.condensed.ranked.tsv	A condensed version of the filtered TSV with only the most important columns remaining, with a priority score for each neoepitope candidate added.

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.
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.
Individual Prediction Algorithm WT and MT Scores (multiple)	ic50 scores for the MT Epitope Seq and WT Eptiope 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

filtered.condensed.ranked.tsv Report Columns

Column Name	Description
Gene Name	The Ensembl gene name of the affected gene.
Mutation	The amino acid change of this mutation.
Protein Position	The protein position of the mutation.
HGVSc	The HGVS coding sequence name.
HGVSp	The HGVS protein sequence name.
HLA Allele	The HLA allele for this 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	Mutant epitope sequence.
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.
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.
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 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 at this position. NA if VCF entry does not contain tumor RNA readcount annotation.
Gene Expression	Gene expression value at this position. NA if VCF entry does not contain gene expression annotation.
Rank	A priority rank for the neoepitope (best = 1).

The pVACseq Neoeptiope Priority Rank

Each of the following 4 criteria are assigned a rank-ordered value (worst = 1):

• B = Rank of the mutant IC50 binding affinity, with the lowest being the best. If the --top-score-metric is set to median (default) the Median MT Score is used. If it is set to lowest the Best MT Score is used.

• F = Rank of Fold Change between MT and WT alleles, with the highest being the best.

• M = Rank of mutant allele expression, calculated as (Gene Expression * Tumor RNA VAF), with the highest being the best.

• D = Rank of Tumor DNA VAF, with the highest being the best.

A score is calculated from the above ranks with the following formula: B + F + (M * 2) + (D / 2). This score is then converted to a rank (best = 1).

Note

The pVACseq rank calculation detailed above is just one of many ways to prioritize neoeptiope candidates. The body of evidence in this area is still incomplete, and the methodology of ranking is likely to change substantially in future releases. While we have found this ranking useful, it is not a substitute for careful curation and validation efforts.