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>.<MHC_I|MHC_II|Combined>.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>.<MHC_I|MHC_II|Combined>.filtered.tsv	The above file after applying all filters, with cleavage site and stability predictions added.
<sample_name>.<MHC_I|MHC_II|Combined>.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. Not generated when only running with presentation or immunogenicity algorithms.
<sample_name>.<MHC_I|MHC_II|Combined>.all_epitopes.aggregated.tsv.reference_matches (optional)	A file outlining details of reference proteome matches

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
Index	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 IC50 Score	Median IC50 binding affinity of the epitope of all prediction algorithms used
Best IC50 Score	Lowest IC50 binding affinity of all prediction algorithms used
Best IC50 Score Method	Prediction algorithm with the lowest IC50 binding affinity for this epitope
Median Percentile	Median percentile rank of the epitope of all prediction algorithms used (those that provide percentile output)
Best Percentile	Lowest percentile rank of all prediction algorithms used (those that provide percentile output)
Best Percentile Method	Prediction algorithm with the lowest percentile rank for this epitope
Median IC50 Percentile	Median binding percentile rank of the epitope of all binding prediction algorithms used (those that provide percentile output)
Best IC50 Percentile	Lowest binding percentile rank of all binding prediction algorithms used (those that provide percentile output)
Best IC50 Percentile Method	Binding prediction algorithm with the lowest binding percentile rank for this epitope
Median Immunogenicity Percentile	Median immunogenicity percentile rank of the epitope of all immunogenicity prediction algorithms used (those that provide percentile output)
Best Immunogenicity Percentile	Lowest immunogenicity percentile rank of all immunogenicity prediction algorithms used (those that provide percentile output)
Best Immunogenicity Percentile Method	Immunogenicity prediction algorithm with the lowest immunogenicity percentile rank for this epitope
Median Presentation Percentile	Median presentation percentile rank of the epitope of all presentatio prediction algorithms used (those that provide percentile output)
Best Presentation Percentile	Lowest presentation percentile rank of all presentatio prediction algorithms used (those that provide percentile output)
Best Presentation Percentile Method	Presentation prediction algorithm with the lowest presentation percentile rank for this epitope
Individual Prediction Algorithm Scores and Percentiles (multiple)	IC50 binding affinity scores, binding scores, presentation scores, processing scores, or immunogenicity scores as well as percentile ranks for the Epitope Seq for the individual prediction algorithms used. Percentile scores may be NA if not provided by the prediction algorithm.
Problematic Positions (optional)	A list of positions in the Epitope Seq that match the problematic amino acids defined by the --problematic-amino-acids parameter
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

all_epitopes.aggregated.tsv Report Columns

The all_epitopes.aggregated.tsv file is an aggregated version of the all_epitopes TSV. It shows the best-scoring epitope for each variant, and outputs binding affinity and other information for that epitope. It gives information about the total number of well-scoring epitopes for each variant 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.

Only epitopes meeting the --aggregate-inclusion-binding-threshold are included in this report (default: 5000). If the number of unique epitopes for a mutation meeting this threshold exceeds the --aggregate-inclusion-count-limit, only the n best-binding epitopes up to this limit are included (default: 15). If the Best Peptide does not meet the aggregate inclusion criteria, it will be still be counted in the Num Included Peptides.

Whether the median or the lowest binding affinity metrics are used for determining the included eptiopes, selecting the best-scoring epitope, and which values are output in the IC50 MT and %ile MT columns is controlled by the --top-score-metric parameter.

Column Name	Description
ID	A unique identifier for the variant
HLA Alleles (multiple)	For each HLA allele in the run, the number of this variant’s epitopes that bound well to the HLA allele (with median binding affinity < 1000)
Best Peptide	The best epitope sequence (see Best Peptide Criteria below for more details on how this is determined)
Allele	The Allele that the Best Peptide is binding to
Pos	The one-based position of the epitope in the protein sequence used to make the prediction
Prob Pos	A list of positions in the Best Peptide that are problematic. None if the -–problematic-pos parameter was not set during the pVACfuse run
Num Included Peptides	The number of included peptides according to the --aggregate-inclusion-binding-threshold and --aggregate-inclusion-count-limit
Num Passing Peptides	The number of included peptides for this mutation that are well-binding.
IC50 MT	Median or lowest IC50 binding affinity of the Best Peptide across all prediction algorithms used
%ile MT	Median or lowest percentile rank of the Best Peptide across all prediction algorithms used
IC50 %ile MT	Median or lowest binding percentile rank of the Best Peptide across all binding prediction algorithms used
IM %ile MT	Median or lowest immunogenicity percentile rank of the Best Peptide across all immunogenicity prediction algorithms used
Pres %ile MT	Median or lowest presentation percentile rank of the Best Peptide across all presentation prediction algorithms used
Ref Match (T/F) (optional)	Was there a match of the peptide sequence to the reference proteome?
Tier	A tier suggesting the suitability of variants for use in vaccines.
Evaluation	Column to store the evaluation of each variant. Either Accept, Reject, or Review.

Best Peptide Criteria

To determine the Best Peptide, all peptides meeting the --aggregate-inclusion-threshold and --aggregate-inclusion-count-limit (see above) for a variant are evaluated as follows:

• Pick the entries with no Problematic Positions.

• For the remaining entries, calculate a rank for all the metrics specified via the --top-score-metric2 parameter and sum them. Whether the lowest or median value is considered for each metric is controlled by the --top-score-metric parameter. Sort the remaining entries on this sum rank followed by the rank of the first --top-score-metric2 specified (to break any ties in the sum rank). Select the highest sorted entry.

The pVACbind Aggregate Report Tiers

Tiering Parameters

To tier the Best Peptide, several cutoffs can be adjusted using parameters provided to the pVACfuse run:

Parameter	Description	Default
--binding-threshold	The threshold used for filtering epitopes on the IC50 MT binding affinity.	500
--allele-specific-binding-thresholds	Instead of the hard cutoff set by the --binding-threshold, use allele-specific binding thresholds. For alleles where no allele-specific binding threshold is available, use the --binding-threshold as a fallback. To print a list of alleles that have specific binding thresholds and the value of those thresholds, run pvacfuse allele_specific_cutoffs.	False
--binding-percentile-threshold	Use this threshold to filter epitopes on the IC50 %ile MT score.	2.0
--presentation-percentile-threshold	Use this threshold to filter epitopes on the Pres %ile MT score.	2.0
--immunogenicity-percentile-threshold	Use this threshold to filter epitopes on the IM %ile MT score.	2.0
--percentile-threshold-strategy	Specify the candidate inclusion strategy. The conservative option requires a candidate to pass the binding threshold, the binding percentile threshold, the presentation percentile threshold, AND the immunogenicity percentile threshold. The exploratory option requires a candidate to pass EITHER the binding threshold, the binding percentile threshold, the presentation percentile threshold, OR the immunogenicity percentile threshold.	conservative
--run-reference-proteome-similarity	Set this flag in order to run reference proteome similarity analysis and enable RefMatch tiering. Use --blastp-path, --blastp-db, and --peptide-fasta parameters to configure your run.	False
--problematic-amino-acids	Configure this parameter in order to define amino acids problematic for the desired therapy delivery platform and enable ProbPos tiering.	None

Tiers

Given the thresholds provided above, the Best Peptide is evaluated and binned into tiers as follows:

Tier	Criteria
Pass	Best Peptide passes the scores, reference match, and problematic position criteria
PoorBinder	Best Peptide fails the binding criteria but passes the presentation, immunogenicity, reference match, and problematic position criteria
PoorImmunogenicity	Best Peptide fails the immunogenicity criteria but passes the binding, presentation, reference match, and problematic position criteria
PoorPresentation	Best Peptide fails the presentation criteria but passes the binding, immunogenicity, reference match, and problematic position criteria
RefMatch	Best Peptide fails the reference match criteria but passes the scores and problematic position criteria
ProbPros	Best Peptide fails the problematic position criteria but passes the scores and reference match criteria
Poor	Best Peptide doesn’t fit in any of the above tiers, usually if it fails two or more criteria

Criteria Details

Criteria	Description	Evaluation Logic
Binding Criteria	Pass if Best Peptide is strong binder	binding score criteria: IC50 MT < binding_threshold binding percentile score criteria: IC50 %ile MT < binding_percentile_threshold conservative --percentile-threshold-strategy: needs to pass BOTH the binding score criteria AND the binding percentile score criteria exploratory --percentile-threshold-strategy: needs to pass EITHER the binding score criteria OR the binding percentile score criteria
Presentation Criteria	Pass if the Best Peptide is presented by the MHC	Pres %ile MT < presentation_percentile_threshold
Immunogenicity Criteria	Pass if the Best Peptide is immunogenic	IM %ile MT < immunogenicity_percentile_threshold
Scores Criteria	Pass if the Best Peptide is a strong binder, presented by the MHC, and/or immunogenic	conservative --percentile-threshold-strategy: needs to pass the binding criteria, the presentation criteria, AND the immunogenicity criteria exploratory --percentile-threshold-strategy: needs to pass the binding criteria, the presentation criteria, OR the immunogenicity criteria
Reference Match Criteria	Pass if there are no reference proteome matches	Ref Match == False
Problematic Position Criteria	Best Peptide does not contain a problematic amino acid as defined by the --problematic-amino-acids parameters	Prob Pos == None

The pVACbind Aggregate Report Sorting

The aggregate report is sorted as follows:

Sort Criteria	Sort Order
Tier column	“Pass”, “PoorBinder”, “PoorImmunogenicity”, “PoorPresentation”, “RefMatch”, “ProbPos”, “Poor”
Sum of the ascending ranks of the metrics selected via the --top-score-metric2 parameter (possible values: IC50 MT, %ile MT, IC50 %ile MT, Pres %ile MT; default: IC50 MT, %ile MT).	Ascending sum rank
First metric specified in the --top-score-metric2 as a tie breaker for identical sum ranks	Ascending rank
ID column	Alphabetical

aggregated.tsv.reference_matches Report Columns

This file is only generated when the --run-reference-proteome-similarity option is chosen.

Column Name	Description (BLAST)	Description (reference fasta)
ID	A unique identifier for the variant
Epitope Seq	The mutant peptide sequence for the epitope candidate
Peptide	The peptide sequence submitted to BLAST	The peptide sequence to search for in the reference proteome
Hit ID	The BLAST alignment hit ID (reference proteome sequence ID)	The FASTA header ID of the entry where the match was made
Hit Definition	The BLAST alignment hit definition (reference proteome sequence name)	The FASTA header description of the entry where the match was made
Match Window	The substring of the Peptide that was found in the Match Sequence
Match Sequence	The BLAST match sequence	The FASTA sequence of the entry where the match was made
Match Start	The match start position of the Match Window in the Match Sequence
Match Stop	The match stop position of the Match Window in the Match Sequence