Optional Downstream Analysis Tools¶
Generate Protein Fasta¶
usage: pvacseq generate_protein_fasta [-h] [--input-tsv INPUT_TSV]
[-p PHASED_PROXIMAL_VARIANTS_VCF]
[--pass-only] [--mutant-only]
[-d DOWNSTREAM_SEQUENCE_LENGTH]
[-s SAMPLE_NAME]
input_vcf flanking_sequence_length
output_file
Generate an annotated fasta file from a VCF with protein sequences of
mutations and matching wildtypes
positional arguments:
input_vcf A VEP-annotated single- or multi-sample VCF containing
genotype, transcript, Wildtype protein sequence, and
Downstream protein sequence information.The VCF may be
gzipped (requires tabix index).
flanking_sequence_length
Number of amino acids to add on each side of the
mutation when creating the FASTA.
output_file The output fasta file.
optional arguments:
-h, --help show this help message and exit
--input-tsv INPUT_TSV
A pVACseq all_epitopes or filtered TSV file with
epitopes to use for subsetting the input VCF to
peptides of interest. Only the peptide sequences for
the epitopes in the TSV will be used when creating the
FASTA. (default: None)
-p PHASED_PROXIMAL_VARIANTS_VCF, --phased-proximal-variants-vcf PHASED_PROXIMAL_VARIANTS_VCF
A VCF with phased proximal variant information to
incorporate into the predicted fasta sequences. Must
be gzipped and tabix indexed. (default: None)
--pass-only Only process VCF entries with a PASS status. (default:
False)
--mutant-only Only output mutant peptide sequences (default: False)
-d DOWNSTREAM_SEQUENCE_LENGTH, --downstream-sequence-length DOWNSTREAM_SEQUENCE_LENGTH
Cap to limit the downstream sequence length for
frameshifts when creating the fasta file. Use 'full'
to include the full downstream sequence. (default:
1000)
-s SAMPLE_NAME, --sample-name SAMPLE_NAME
The name of the sample being processed. Required when
processing a multi-sample VCF and must be a sample ID
in the input VCF #CHROM header line. (default: None)
This tool will extract protein sequences surrounding supported protein altering variants in an input VCF file. One use case for this tool is to help select long peptides that contain short neoepitope candidates. For example, if pvacseq was run to predict nonamers (9-mers) that are good binders and the user wishes to select long peptide (e.g. 24-mer) sequences that contain the nonamer for synthesis or encoding in a DNA vector. The protein sequence extracted will correspond to the transcript sequence used in the annotated VCF. The alteration in the VCF (e.g. a somatic missense SNV) will be centered in the protein sequence returned (if possible). If the variant is near the beginning or end of the CDS, it will be as close to center as possible while returning the desired protein sequence length. If the variant causes a frameshift, the full downstream protein sequence will be returned unless the user specifies otherwise as described above.
Generate Aggregated Report¶
usage: pvacseq generate_aggregated_report [-h] [--tumor-purity TUMOR_PURITY]
input_file output_file
Generate an aggregated report from a pVACseq .all_epitopes.tsv report file.
positional arguments:
input_file A pVACseq .all_epitopes.tsv report file
output_file The file path to write the aggregated report tsv to
optional arguments:
-h, --help show this help message and exit
--tumor-purity TUMOR_PURITY
Value between 0 and 1 indicating the fraction of tumor
cells in the tumor sample. Information is used during
aggregate report creation for a simple estimation of
whether variants are subclonal or clonal based on VAF.
If not provided, purity is estimated directly from the
VAFs. (default: None)
This tool produces an aggregated version of the all_epitopes TSV. It finds the best-scoring (lowest binding affinity) epitope for each variant, and outputs additional binding affinity, expression, and coverage information for that epitope. It also gives information about the total number of well-scoring epitopes for each variant, the number of transcripts covered by those epitopes, 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. For a full definition of these tiers, see the pVACseq output file documentation.
Calculate Reference Proteome Similarity¶
usage: pvacseq calculate_reference_proteome_similarity [-h]
[--match-length MATCH_LENGTH]
[--species SPECIES]
[--blastp-path BLASTP_PATH]
[--blastp-db {refseq_select_prot,refseq_protein}]
[-t N_THREADS]
input_file input_fasta
output_file
Blast peptides against the reference proteome.
positional arguments:
input_file Input filtered or all_epitopes file with predicted
epitopes.
input_fasta For pVACbind, the original input FASTA file. For
pVACseq and pVACfuse a FASTA file with mutant peptide
sequences for each variant isoform. This file can be
found in the same directory as the input
filtered/all_epitopes file. Can also be generated by
running `pvacseq|pvacfuse generate_protein_fasta`.
output_file Output TSV filename for putative neoepitopes.
optional arguments:
-h, --help show this help message and exit
--match-length MATCH_LENGTH
The desired matching epitope length. (default: 8)
--species SPECIES The species of the input file. (default: human)
--blastp-path BLASTP_PATH
Blastp installation path. (default: None)
--blastp-db {refseq_select_prot,refseq_protein}
The blastp database to use. (default:
refseq_select_prot)
-t N_THREADS, --n-threads N_THREADS
Number of threads to use for parallelizing BLAST
calls. (default: 1)
This tool will Blast peptides against the relative reference proteome and return the results in an output TSV & reference_match file pair, given a pVACseq run’s fasta and filtered/all_epitopes TSV. Typically, this can be done as part of the pVACseq run pipeline for the filtered output TSV if specified. This tool, however, provides a standalone way to run this on pVACseq’s generated filtered/all_epitopes TSV files. For instance, this may be desired if pvacseq was originally run without this specified and one wished to perform this additional step after the fact for the filtered TSV—or perhaps instead the results of this were desired for the all_epitopes TSV which does not have this step performed. For a closer look at the generated reference_match file, see the pVACseq output file documentation.
NetChop Predict Cleavage Sites¶
usage: pvacseq net_chop [-h] [--method {cterm,20s}] [--threshold THRESHOLD]
input_file input_fasta output_file
Predict cleavage sites for neoepitopes.
positional arguments:
input_file Input filtered file with predicted epitopes.
input_fasta The required fasta file.
output_file Output tsv filename for putative neoepitopes.
optional arguments:
-h, --help show this help message and exit
--method {cterm,20s} NetChop prediction method to use ("cterm" for C term
3.0, "20s" for 20S 3.0). (default: cterm)
--threshold THRESHOLD
NetChop prediction threshold. (default: 0.5)
This tool uses NetChop to predict cleavage sites for neoepitopes from a pVACseq run’s filtered/all_epitopes TSV. In its output, it adds to the TSV 3 columns: Best Cleavage Position, Best Cleavage Score, and a Cleavage Sites list. Typically this step is done in the pVACseq run pipeline for the filtered output TSV when specified. This tool provides a way to manually run this on pVACseq’s generated filtered/all_epitopes TSV files so that you can add this information when not present if desired. You can view more about these columns for pVACseq in the output file documentation.
NetMHCStab Predict Stability¶
usage: pvacseq netmhc_stab [-h] [-m {lowest,median}] input_file output_file
Add stability predictions to predicted neoepitopes.
positional arguments:
input_file Input filtered file with predicted epitopes.
output_file Output TSV filename for putative neoepitopes.
optional arguments:
-h, --help show this help message and exit
-m {lowest,median}, --top-score-metric {lowest,median}
The ic50 scoring metric to use when sorting epitopes.
lowest: Use the best MT Score and Corresponding Fold
Change (i.e. the lowest MT ic50 binding score and
corresponding fold change of all chosen prediction
methods). median: Use the median MT Score and Median
Fold Change (i.e. the median MT ic50 binding score and
fold change of all chosen prediction methods).
(default: median)
This tool uses NetMHCstabpan to add stability predictions for neoepitopes from a pVACseq run’s filtered/all_epitopes TSV. In its output, it adds to the TSV 4 columns: Predicted Stability, Half Life, Stability Rank, and NetMHCStab Allele. Typically this step is done in the pVACseq run pipeline for the filtered output TSV when specified. This tool provides a way to manually run this on pVACseq’s generated filtered/all_epitopes TSV files so that you can add this information when not present if desired. You can view more about these columns for pVACseq in the output file documentation.