Filtering Commands¶

pVACsplice currently offers four filters: a binding filter, a coverage filter, a transcript support level filter, and a top score filter.

These filters are always run automatically as part of the pVACsplice pipeline using default cutoffs.

All filters can also be run manually on the filtered.tsv file to narrow the results down further, or they can be run on the all_epitopes.tsv file to apply different filtering thresholds.

The binding filter is used to remove neoantigen candidates that do not meet desired peptide:MHC binding criteria. The coverage filter is used to remove variants that do not meet desired read count and VAF criteria (in normal DNA and tumor DNA/RNA). The transcript support level filter is used to remove variant annotations based on low quality transcript annotations. The top score filter is used to select the most promising peptide candidate for each variant. Multiple candidate peptides from a single somatic variant can be caused by multiple peptide lengths, registers, HLA alleles, and transcript annotations.

Further details on each of these filters is provided below.

Note

The default values for filtering thresholds are suggestions only. While they are based on review of the literature and consultation with our clinical and immunology colleagues, your specific use case will determine the appropriate values.

Binding Filter¶

usage: pvacsplice binding_filter [-h] [-b BINDING_THRESHOLD]
                                 [-p PERCENTILE_THRESHOLD]
                                 [-m {lowest,median}] [--exclude-NAs] [-a]
                                 input_file output_file

Filter variants processed by IEDB by binding score.

positional arguments:
  input_file            The all_epitopes.tsv or filtered.tsv pVACseq report
                        file to filter.
  output_file           Output .tsv file containing list of filtered epitopes
                        based on binding affinity.

optional arguments:
  -h, --help            show this help message and exit
  -b BINDING_THRESHOLD, --binding-threshold BINDING_THRESHOLD
                        Report only epitopes where the mutant allele has ic50
                        binding scores below this value. (default: 500)
  -p PERCENTILE_THRESHOLD, --percentile-threshold PERCENTILE_THRESHOLD
                        Report only epitopes where the mutant allele has a
                        percentile rank below this value. (default: None)
  -m {lowest,median}, --top-score-metric {lowest,median}
                        The ic50 scoring metric to use when filtering epitopes
                        by binding-threshold or minimum fold change. lowest:
                        Use the Best MT IC50 Score, Corresponding Fold Change,
                        and Best MT Percentile (i.e. use the lowest MT ic50
                        binding score, orresponding fold change of all chosen
                        prediction methods, and lowest MT percentile). median:
                        Use the Median MT IC50 Score, Median Fold Change, and
                        Median MT Percentile i.e. use the median MT ic50
                        binding score, fold change, and MT percentile of all
                        chosen prediction methods). (default: median)
  --exclude-NAs         Exclude NA values from the filtered output. (default:
                        False)
  -a, --allele-specific-binding-thresholds
                        Use allele-specific binding thresholds. To print the
                        allele-specific binding thresholds run `pvacsplice
                        allele_specific_cutoffs`. If an allele does not have a
                        special threshold value, the `--binding-threshold`
                        value will be used. (default: False)

The binding filter removes variants that don’t pass the chosen binding threshold. The user can chose whether to apply this filter to the lowest or the median binding affinity score by setting the --top-score-metric flag. The lowest binding affinity score is recorded in the Best MT IC50 Score column and represents the lowest ic50 score of all prediction algorithms that were picked during the previous pVACseq run. The median binding affinity score is recorded in the Median MT IC50 Score column and corresponds to the median ic50 score of all prediction algorithms used to create the report. Be default, the binding filter runs on the median binding affinity.

When the --allele-specific-binding-thresholds flag is set, binding cutoffs specific to each prediction’s HLA allele are used instead of the value set via the --binding-threshold parameters. For HLA alleles where no allele-specific binding threshold is available, the binding threshold is used as a fallback. Alleles with allele-specific threshold as well as the value of those thresholds can be printed by executing the pvacsplice allele_specific_cutoffs command.

In addition to being able to filter on the IC50 score columns, the binding filter also offers the ability to filter on the percentile score using the --percentile-threshold parameter. When the --top-score-metric is set to lowest, this threshold is applied to the Best MT Percentile column. When it is set to median, the threshold is applied to the Median MT Percentile column.

By default, entries with NA values will be included in the output. This behavior can be turned off by using the --exclude-NAs flag.

Coverage Filter¶

usage: pvacsplice coverage_filter [-h] [--normal-cov NORMAL_COV]
                                  [--tdna-cov TDNA_COV] [--trna-cov TRNA_COV]
                                  [--normal-vaf NORMAL_VAF]
                                  [--tdna-vaf TDNA_VAF] [--trna-vaf TRNA_VAF]
                                  [--expn-val EXPN_VAL] [--exclude-NAs]
                                  input_file output_file

Filter variants processed by IEDB by coverage, vaf, and gene expression

positional arguments:
  input_file            The all_epitopes.tsv or filtered.tsv pVACsplice report
                        file to filter.
  output_file           Output .tsv file containing list of filtered epitopes
                        based on coverage and expression values

optional arguments:
  -h, --help            show this help message and exit
  --normal-cov NORMAL_COV
                        Normal Coverage Cutoff. Sites above this cutoff will
                        be considered. (default: 5)
  --tdna-cov TDNA_COV   Tumor DNA Coverage Cutoff. Sites above this cutoff
                        will be considered. (default: 10)
  --trna-cov TRNA_COV   Tumor RNA Coverage Cutoff. Sites above this cutoff
                        will be considered. (default: 10)
  --normal-vaf NORMAL_VAF
                        Normal VAF Cutoff in decimal format. Sites BELOW this
                        cutoff in normal will be considered. (default: 0.02)
  --tdna-vaf TDNA_VAF   Tumor DNA VAF Cutoff in decimal format. Sites above
                        this cutoff will be considered. (default: 0.25)
  --trna-vaf TRNA_VAF   Tumor RNA VAF Cutoff in decimal format. Sites above
                        this cutoff will be considered. (default: 0.25)
  --expn-val EXPN_VAL   Gene and Transcript Expression cutoff. Sites above
                        this cutoff will be considered. (default: 1.0)
  --exclude-NAs         Exclude NA values from the filtered output. (default:
                        False)

If the pVACsplice input VCF contains readcount and/or expression annotations, then the coverage filter can be run again on the filtered.tsv report file to narrow down the results even further. You can also run this filter again on the all_epitopes.tsv report file to apply different cutoffs.

The general goals of these filters are to limit variants for neoepitope prediction to those with good read support and/or remove possible sub-clonal variants. In some cases the input VCF may have already been filtered in this fashion. This filter also allows for removal of variants that do not have sufficient evidence of RNA expression.

For more details on how to prepare input VCFs that contain all of these annotations, refer to the Input File Preparation section for more information.

By default, entries with NA values will be included in the output. This behavior can be turned off by using the --exclude-NAs flag.

Transcript Support Level Filter¶

usage: pvacsplice transcript_support_level_filter [-h]
                                                  [--maximum-transcript-support-level {1,2,3,4,5}]
                                                  input_file output_file

Filter variants processed by IEDB by transcript support level

positional arguments:
  input_file            The all_epitopes.tsv or filtered.tsv pVACsplice report
                        file to filter.
  output_file           Output .tsv file containting list of of filtered
                        epitopes based on transcript support level.

optional arguments:
  -h, --help            show this help message and exit
  --maximum-transcript-support-level {1,2,3,4,5}
                        The threshold to use for filtering epitopes on the
                        transcript support level. Keep all epitopes with a
                        transcript support level <= to this cutoff. (default:
                        1)

This filter is used to eliminate variant annotations based on poorly-supported transcripts. By default, only transcripts with a transcript support level (TSL) of <=1 are kept. This threshold can be adjusted using the --maximum-transcript-support-level parameter.

By default, entries with Not Supported values will be included in the output. These occur if VEP was run without the --tsl flag or if data is aligned to GRCh37 or older.

Top Score Filter¶

usage: pvacsplice top_score_filter [-h] [-m {lowest,median}]
                                   [--maximum-transcript-support-level {1,2,3,4,5}]
                                   input_file output_file

Pick the best neoepitope for each variant

positional arguments:
  input_file            The final report .tsv file to filter.
  output_file           Output .tsv file containing only the list of the top
                        epitope per variant.

optional arguments:
  -h, --help            show this help message and exit
  -m {lowest,median}, --top-score-metric {lowest,median}
                        The ic50 scoring metric to use for filtering. lowest:
                        Use the best MT Score (i.e. the lowest MT ic50 binding
                        score of all chosen prediction methods). median: Use
                        the median MT Score (i.e. the median MT ic50 binding
                        score of all chosen prediction methods). (default:
                        median)
  --maximum-transcript-support-level {1,2,3,4,5}
                        When determining the top peptide, only consider those
                        entries that meet this threshold for the Ensembl
                        transcript support level (TSL). Transcript support
                        level needs to be <= this cutoff to be considered.
                        (default: 1)

This filter picks the top epitope for each splice site variant. The top epitope is determined by first selecting epitopes with no Problematic Positions and among those selecting the one with lowest median/best MT IC50 score for each splice site variant

By default the --top-score-metric option is set to median which will apply this filter to the Median MT IC50 Score column. If the --top-score-metric option is set to lowest, the Best MT IC50 Score column is used instead.

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