Frequently Asked Questions

What type of variants does pVAC-Seq support?

pVAC-Seq makes predictions for all transcripts of a variant that were annotated as missense_variant, inframe_insertions, inframe_deletion, or frameshift_variant by VEP as long as the transcript was not also annotated as start_lost. In addition, pVAC-Seq only includes variants that were called as homozygous or heterozygous variant. Variants that were not called are skipped.

My pVAC-Seq command has been running for a long time. Why is that?

The rate-limiting factor in running pVAC-Seq is the number of calls that are made to the IEDB software for binding score predictions.


It is generally faster to make IEDB calls using a local install of IEDB than using the IEDB web API. It is, therefore, recommended to use a local IEDB install for any in-depth analysis.

There are a number of factors that determine the number of IEDB calls to be made:

  • Number of variants in your VCF

    pVAC-Seq will make predictions for each missense, inframe indel, and frameshift variant in your VCF.

    Speedup suggestion: Split the VCF into smaller subsets and process each one individually, in parallel.

  • Number of transcripts for each variant

    pVAC-Seq will make predictions for each transcript of a supported variant individually. The number of transcripts for each variant depends on how VEP was run when the VCF was annotated.

    Speedup suggestion: Use the --pick option when running VEP to annotate each variant with the top transcript only.

  • The --fasta-size parameter value

    pVAC-Seq takes an input VCF and creates a wildtype and a mutant fasta for each transcript. The number of fasta entries that get submitted to IEDB at a time is limited by the --fasta-size parameter in order to reduce the load on the IEDB servers. The smaller the fasta-size, the more calls have to be made to IEDB.

    Speedup suggestion: When using a local IEDB install, increase the size of this parameter.

  • Number of prediction algorithms, epitope lengths, and HLA-alleles

    One call to IEDB is made for each combination of these parameters for each chunk of fasta sequences. That means, for example, when 7 prediction algorithms, 4 epitope lengths, and 6 HLA-alleles are chosen, 7*4*6=168 calls to IEDB have to be made for each chunk of fastas.

    Speedup suggestion: Reduce the number of prediction algorithms, epitope lengths, and/or HLA-alleles to the ones that will be the most meaningful for your analysis. For example, the NetMHCcons method is already a consensus method between NetMHC, NetMHCpan, and PickPocket. If NetMHCcons is chosen, you may want to omit the underlying prediction methods. Likewise, if you want to run NetMHC, NetMHCpan, and PickPocket individually, you may want to skip NetMHCcons.

  • --downstream-sequence-length parameter value

    This parameter determines how many amino acids of the downstream sequence after a frameshift mutation will be included in the wildtype fasta sequence. The shorter the downstream sequence length, the lower the number of epitopes that IEDB needs to make binding predictions for.

    Speedup suggestion: Reduce the value of this parameter.

My pVAC-Seq output file does not contain entries for all of the alleles I chose. Why is that?

There could be a few reasons why the pVAC-Seq output does not contain predictions for alleles:

  • The alleles you picked might’ve not been compatible with the prediction algorithm and/or epitope lengths chosen. In that case no calls for that allele would’ve been made and a status message would’ve printed to the screen.
  • It could be that all epitope predictions for some alleles got filtered out. You can check the <sample_name>.combined.parsed.tsv file to see all called epitopes before filtering.

Why are some values in the WT Epitope Seq column NA ?

Not all mutant epitope sequences will have a corresponding wildtype epitope sequence. This occurs when the mutant epitope sequence is novel and a comparison is therefore not meaningful:

  • An epitope in the downstream portion of a frameshift might not have a corresponding wildtype epitope at the same position at all. The epitope is completely novel.
  • An epitope that overlaps an inframe indel or multinucleotide polymorphism (MNP) might have a large number of amino acids that are different from the wildtype epitope at the corresponding position. If less than half of the amino acids between the mutant epitope sequence and the corresponding wildtype sequence match, the corresponding wildtype sequence in the report is set to NA.

What filters are applied during a pVAC-Seq run?

By default we filter the neoepitopes on their binding score. If bam-readcount files and/or cufflinks files are provided we also filter on the depth, VAF, and FPKM. In addition, candidates where the mutant epitope sequence is the same as the wildtype epitope sequence will also be filtered out.

How can I see all of the candidate epitopes without any filters applied?

The <sample_name>.combined.parsed.tsv will contain all of the epitopes predicted before filters are applied.

Why have some of my epitopes been filtered out even though the Best MT Score is below 500?

By default, the binding filter will be applied to the Median MT Score column. This is the median score value among all chosen prediction algorithms. The Best MT Score column shows the lowest score among all chosen prediction algorithms. To change this behavior and apply the binding filter to the Best MT Score column you may set the --top-score-metric parameter to lowest.

Why are entries with NA in the VAF and depth columns not filtered?

We do not filter out NA entries for depth and VAF since there is not enough information to determine whether the cutoff has been met one way or another.

Why don’t some of my epitopes have score predictions for certain prediction methods?

Not all prediction methods support all epitope lengths or all alleles. To see a list of supported alleles for a prediction method you may use the pvacseq valid_alleles command. For more details on each algorithm refer to the IEDB MHC Class I and Class II documentation.

How do I use StringTie instead of Cufflinks for transcript/gene abundance estimates?

You may also provide FPKM values from other sources, including StringTie, by creating cufflinks-formatted input files.

For transcript FPKM: a tab-separated file with a tracking_id column containing Ensembl transcript IDs and a FPKM column containing FPKM values.

For gene FPKM: a tab-separated file with a tracking_id column containing Ensembl gene IDs, a locus column describing the region within the gene, and a FPKM column containing FPKM values. In the pVAC-Seq pipeline the FPKM values will be summed for all loci of a gene. You may also provide already summed FPKM values. In that case you will still need to provide a locus column but the values in that column can be empty.

How is pVAC-Seq licensed?

pVAC-Seq is licensed under NPOSL-3.0.

How do I cite pVAC-Seq?

Jasreet Hundal, Beatriz M. Carreno, Allegra A. Petti, Gerald P. Linette, Obi L. Griffith, Elaine R. Mardis, and Malachi Griffith. pVAC-Seq: A genome-guided in silico approach to identifying tumor neoantigens. Genome Medicine. 2016, 8:11, DOI: 10.1186/s13073-016-0264-5. PMID: 26825632.