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 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 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>.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.

all_epitopes.tsv and filtered.tsv Report Columns

In order to keep the outputs consistent, pVACfuse uses the same output columns as pVACseq but some of the values will be NA if a column doesn’t apply to pVACfuse.

Column Name	Description
Chromosome	The chromosome of the 5p and 3p portion of the fusion, separated by ” / “
Start	The start position of the 5p and 3p portion of the fusion, separated by ” / “
Stop	The stop position of the 5p and 3p portion of the fusion, separated by ” / “
Reference	fusion
Variant	fusion
Transcript	The Ensembl IDs of the affected transcripts
Transcript Support Level	NA
Ensembl Gene ID	NA
Variant Type	The type of fusion. inframe_fusion for inframe fusions, frameshift_fusion for frameshift fusions
Mutation	NA
Protein Position	The position of the fusion in the fusion protein sequence
Gene Name	The Ensembl gene names of the affected genes
HGVSc	NA
HGVSp	NA
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	NA
MT Epitope Seq	Mutant epitope sequence
WT Epitope Seq	NA
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	NA
Corresponding Fold Change	NA
Tumor DNA Depth	NA
Tumor DNA VAF	NA
Tumor RNA Depth	NA
Tumor RNA VAF	NA
Normal Depth	NA
Normal VAF	NA
Gene Expression	NA
Transcript Expression	NA
Median MT Score	Median ic50 binding affinity of the mutant epitope of all prediction algorithms used
Median WT Score	NA
Median Fold Change	NA
Individual Prediction Algorithm WT and MT Scores (multiple)	ic50 scores for the MT Epitope Seq and WT 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

filtered.condensed.ranked.tsv Report Columns

Column Name	Description
Gene Name	The Ensembl gene names of the affected genes
Mutation	NA
Protein Position	The position of the fusion in the fusion protein sequence
HGVSc	NA
HGVSp	NA
HLA Allele	The HLA allele for this prediction.
Mutation Position	NA
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	NA
Median Fold Change	NA
Best MT Score	Lowest ic50 binding affinity of all prediction algorithms used
Corresponding WT Score	NA
Corresponding Fold Change	NA
Tumor DNA Depth	NA
Tumor DNA VAF	NA
Tumor RNA Depth	NA
Tumor RNA VAF	NA
Gene Expression	NA
Rank	A priority rank for the neoepitope (best = 1).

The pVACfuse Neoeptiope Priority Rank

The underlying formula for calculating the pVACfuse rank is the same as it is for The pVACseq Neoeptiope Priority Rank. However, since only the binding affinity is available for fusion predictions, the pVACfuse simply ranks the neoeptiopes according to their 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.