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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 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 score for each neoepitope candidate added.

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 name
HGVSp The HGVS protein sequence 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 in the protein sequence used to make the prediction
Mutation Position The one-based position of the start of the mutation in the epitope. 0 if the start of the mutation is before the epitope
MT Epitope Seq Mutant epitope sequence
WT Epitope Seq 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 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.
Normal DNA Depth Normal DNA depth at this position. NA if VCF entry does not contain normal DNA readcount annotation.
Normal DNA VAF Normal DNA variant allele frequency at this position. NA if VCF entry does not contain normal DNA readcount annotation.
Gene Expression Gene expression value at this position. NA if VCF entry does not contain gene expression annotation.
Transcript Expression Transcript expression value at this position. NA if VCF entry does not contain transcript expression annotation.
Median MT Score Median ic50 binding affinity of the mutant epitope of all prediction algorithms used
Median WT Score Median ic50 binding affinity of the wildtype epitope of 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
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 Half Life (optional) The stability half life of the MT Epitope Seq
Stability Rank (optional) The % rank stability of the MT Epitope Seq
NetMHCstab allele (optional) Nearest neighbor to the HLA Allele. Used for NetMHCstab prediction
pVACseq ouput file columns illustration

filtered.condensed.ranked.tsv Report Columns

Column Name Description
Gene Name The Ensembl gene name of the affected gene.
Mutation The amnio 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.
MT Epitope Seq Mutant epitope sequence.
MT IC50 If --top-score-metric is set to lowest, this corresponds to the Best MT Score in the full report. If --top-score-metric is set to median this corresponds to the Median MT Score in the full report.
WT IC50 If --top-score-metric is set to lowest, this corresponds to the Corresponding WT Score in the full report. If --top-score-metric is set to median this corresponds to the Median WT Score in the full report.
Fold Change If --top-score-metric is set to lowest, this corresponds to the Corresponding Fold Change in the full report. If --top-score-metric is set to median this corresponds to the Median Fold Change in the full report.
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.
Score A priority score for the neoepitope. The higher the score, the better the neoepitope.

The pVACseq Neoeptiope Priority Score

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

  • B = MT IC50 binding affinity, with the lowest being the best.
  • F = Fold Change between MT and WT alleles, with the highest being the best.
  • M = Mutant allele expression, calculated as (Gene Expression * Tumor RNA VAF), with the highest being the best.
  • D = Tumor DNA VAF, with the highest being the best.

The Score is calculated from the above ranks with the following formula: B + F + (M * 2) + (D / 2)