Commonly Asked Questions

1. What do each column of the main table mean?/ Where can I find detailed description of each column?

• HLA allele columns: Number of good binding peptides for each specific HLA-allele (note that the same peptide could be counted in multiple columns if it was predicted to be a good binder for multiple HLA alleles).

• Gene: The Ensembl gene name of the affected gene.

• AA Change: The amino acid change for the mutation. Note that FS indicates a frameshift variant.

• Num Passing Transcripts: The number of transcripts for this mutation that resulted in at least one well-binding peptide (median mutant binding affinity < 1000).

• Best Peptide: The best-binding mutant epitope sequence (lowest median mutant binding affinity).

• Pos: 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 (as can occur downstream of frameshift mutations).

• Num Passing Peptides: The number of unique well-binding peptides for this mutation.

• IC50 MT: Median ic50 binding affinity of the best-binding mutant epitope across all prediction algorithms used.

• IC50 WT: Median ic50 binding affinity of the corresponding wildtype epitope across all prediction algorithms used.

• %ile MT (percentile MT): Median binding affinity percentile rank of the best-binding mutant epitope across all prediction algorithms used (those that provide percentile output).

• %ile WT (percentile WT): Median binding affinity percentile rank of the corresponding wildtype epitope across all prediction algorithms used (those that provide percentile output).

• RNA Expr: Gene expression value for the annotated gene containing the variant.

• RNA VAF: Tumor RNA variant allele frequency (VAF) at this position.

• Allele Expr: Gene expression value * Tumor RNA VAF. This is used to approximate the expression of the variant allele.

• RNA Depth: Tumor RNA depth at this position.

• DNA VAF: Tumor DNA variant allele frequency (VAF) at this position.

• Tier: A tier suggesting the suitability of variants for use in vaccines. Please refer here for further details.

• Eval: User-selected evaluation of neoantigen candidate. Options include: Accept, Reject, Review. (Default: Pending)

  These definitions as well as further details on the coloring and bar graphs in the main table can be found in the app by either: 1) hovering over individual column names and 2) clicking on the tooltip at the top right of the main table.

2. What does the peptide table show?

• Depending on the transcript you select, the peptide table will show the MT peptide sequences from that transcript that were predicted to be a good binder (along with their respective wildtype sequences). Currently, only predictions for good binding HLA alleles are displayed for MT/WT pairs and those with poor predictions are marked in X.

• If the WT peptide sequence is unavailable in cases such as frameshift variants, these will be labelled with the mutation type followed by NA (e.g. FS-NA for frameshifts).

3. What is the violin plot in the additional information box showing?

• pVACtools allows users to select up to 8 Class I algorithms and 4 Class II algorithms when making binding predictions. In the main table as well as the peptide table, the best peptide is determined using a median value across all binding predictions from the selected algorithms. However previous study has shown how binding algorithms can vary greatly in terms of its predictions for the same set of peptides. Thus, we want to provide users with the ability to visualize the distribution of predictions across algorithms for each MT/WT pair.

• The violin plots also provide a guiding line at 500nM and 1000nM for IC50 values and 0.5% and 2% for percentile values.

4. What is the anchor plot in the additional information box showing?

• Anchor locations and its relative position can influence prioritization decisions for neoantigens as shown here and we want to provide users with the data to be able to take these considerations into account. In the anchor plot, peptide sequences (from the peptide table) are plotted and a heatmap overlays the sequences where a darker blue represents a higher probability of being an anchor location. The mutation(s) is marked in red letters.

• To the right of the additional information box, we provide a graphical guide regarding how one can take anchor information into account, more details on how to interpret this data can be found in our paper.

5. I’m getting an error for the anchor heatmap tab saying “Error:polygon edge not found”, what do I do?

• Users have occasionally ran into the problem where their anchor heatmap does not display and instead shows an error saying “polygon edge not found”. After investigation we believe this may be related to your arial font file. This stackoverflow page describes the detailed steps to resolving this issue: https://stackoverflow.com/questions/10581440/error-in-grid-calll-textbounds-as-graphicsannotxlabel-xx-xy-polygon .