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]
[--aggregate-report-evaluation AGGREGATE_REPORT_EVALUATION]
[-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
Frameshift 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, filtered, or aggregated 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)
--aggregate-report-evaluation AGGREGATE_REPORT_EVALUATION
When running with an aggregate report input TSV, only
include variants with this evaluation. Valid values
for this field are Accept, Reject, Pending, and
Review. Specifiy multiple values as a comma-separated
list to include multiple evaluation states. (default:
Accept)
-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. The flanking_sequence_length positional parameter
controls how many amino acids will be included on either side of the mutation.
To incorporate proximal variants in the final sequence, use the
--phased-proximal-variants-vcf argument. Please see the Creating a phased VCF of proximal variants
section of the documentation on how to create this VCF.
The output may be limited to PASS variants only by setting the --pass only
flag and to mutant sequences by setting the --mutant-only flag.
The output can be further limited to only certain variants by providing
a pVACseq report file to the --input-tsv argument. Only the peptide sequences for the epitopes in the TSV
will be used when creating the FASTA. If this argument is an aggregated TSV
file, use the --aggregate-report-evaluation parameter to only include
peptide sequences for epitopes matching the chosen Evaluation(s). This is
useful when creating a peptide fasta for vaccine ordering after using pVACview
to select vaccine candidates and exporting the results to a new TSV.
Generate Aggregated Report¶
usage: pvacseq generate_aggregated_report [-h] [--tumor-purity TUMOR_PURITY]
[-b BINDING_THRESHOLD]
[--allele-specific-binding-thresholds]
[--percentile-threshold PERCENTILE_THRESHOLD]
[--aggregate-inclusion-binding-threshold AGGREGATE_INCLUSION_BINDING_THRESHOLD]
[-m {lowest,median}]
[--trna-vaf TRNA_VAF]
[--trna-cov TRNA_COV]
[--expn-val EXPN_VAL]
[--maximum-transcript-support-level {1,2,3,4,5}]
[--allele-specific-anchors]
[--anchor-contribution-threshold ANCHOR_CONTRIBUTION_THRESHOLD]
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)
-b BINDING_THRESHOLD, --binding-threshold BINDING_THRESHOLD
Tier epitopes in the "Pass" tier when the mutant
allele has ic50 binding scores below this value.
(default: 500)
--allele-specific-binding-thresholds
Use allele-specific binding thresholds. To print the
allele-specific binding thresholds run `pvacseq
allele_specific_cutoffs`. If an allele does not have a
special threshold value, the `--binding-threshold`
value will be used. (default: False)
--percentile-threshold PERCENTILE_THRESHOLD
When set, tier epitopes in the "Pass" tier when the
mutant allele has percentile scores below this value
and in the "Relaxed" tier when the mutant allele has
percentile scores below double this value. (default:
None)
--aggregate-inclusion-binding-threshold AGGREGATE_INCLUSION_BINDING_THRESHOLD
Threshold for including epitopes when creating the
aggregate report (default: 5000)
-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 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)
--trna-vaf TRNA_VAF Tumor RNA VAF Cutoff. Used to calculate the allele
expression cutoff for tiering. (default: 0.25)
--trna-cov TRNA_COV Tumor RNA Coverage Cutoff. Used as a cutoff for
tiering. (default: 10)
--expn-val EXPN_VAL Gene and Expression cutoff. Used to calculate the
allele expression cutoff for tiering. (default: 1.0)
--maximum-transcript-support-level {1,2,3,4,5}
The threshold to use for filtering epitopes on the
Ensembl transcript support level (TSL). Transcript
support level needs to be <= this cutoff to be
included in most tiers. (default: 1)
--allele-specific-anchors
Use allele-specific anchor positions when tiering
epitopes in the aggregate report. This option is
available for 8, 9, 10, and 11mers and only for HLA-A,
B, and C alleles. If this option is not enabled or as
a fallback for unsupported lengths and alleles, the
default positions of 1, 2, epitope length - 1, and
epitope length are used. Please see
https://doi.org/10.1101/2020.12.08.416271 for more
details. (default: False)
--anchor-contribution-threshold ANCHOR_CONTRIBUTION_THRESHOLD
For determining allele-specific anchors, each position
is assigned a score based on how binding is influenced
by mutations. From these scores, the relative
contribution of each position to the overall binding
is calculated. Starting with the highest relative
contribution, positions whose scores together account
for the selected contribution threshold are assigned
as anchor locations. As a result, a higher threshold
leads to the inclusion of more positions to be
considered anchors. (default: 0.8)
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}]
[--peptide-fasta PEPTIDE_FASTA]
[-t N_THREADS]
[-m AGGREGATE_METRICS_FILE]
input_file input_fasta
output_file
Identify which epitopes in a pVACseq|pVACfuse|pVACbind report file have
matches in the reference proteome using either BLASTp or a checking directly
against a reference proteome FASTA.
positional arguments:
input_file Input filtered, all_epitopes, or aggregated report
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.tsv/all_epitopes.tsv file. Can also be
generated by running `pvacseq|pvacfuse
generate_protein_fasta`.
output_file Output TSV filename of report file with epitopes with
reference matches marked.
optional arguments:
-h, --help show this help message and exit
--match-length MATCH_LENGTH
The minimum number of consecutive amino acids that
need to match. (default: 8)
--species SPECIES The species of the data in 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)
--peptide-fasta PEPTIDE_FASTA
A reference peptide FASTA file to use for finding
reference matches instead of blastp. (default: None)
-t N_THREADS, --n-threads N_THREADS
Number of threads to use for parallelizing BLAST
calls. (default: 1)
-m AGGREGATE_METRICS_FILE, --aggregate-metrics-file AGGREGATE_METRICS_FILE
When running with the aggregate report as an input
tsv, optionally provide the metrics.json file to
update with detailed reference match data for display
in pVACview. (default: None)
This tool will find matches of the epitope candidates in the reference proteome and return the results in an output TSV & reference_match file pair. It requires the input of a pVACseq run’s fasta file in order to look up the larger peptide sequence the epitope was derived from. Any substring of that peptide sequence that matches against the reference proteome and is at least as long as the specified match length, will be considered a hit. This tool also requires the user to provide a filtered.tsv, all_epitopes.tsv or aggregated.tsv pVACseq report file as an input and any candidates in this input file will be searched for.
This tool may be either run with BLASTp using either the refseq_select_prot or refseq_protein database.
By default this option uses the BLAST API but users may independently install BLASTp. Alternatively, users
may provide a reference proteome fasta file and this tool will string match on
the entries of this fasta file directly. This approach is recommended, because
it is significantly faster than BLASTp. Reference proteome fasta files may be
downloaded from Ensembl. For example, the latest reference proteome fasta for human
can be downloaded from this
link.
For more details on 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.
Identify Problematic Amino Acids¶
usage: pvacseq identify_problematic_amino_acids [-h]
[--filter-type {soft,hard}]
input_file output_file
problematic_amino_acids
Mark problematic amino acid positions in each epitope or filter entries that have problematic amino acids.
positional arguments:
input_file Input filtered or all_epitopes file with predicted epitopes.
output_file Output .tsv file with identification of problematic amino acids or hard-filtered to remove epitopes with problematic amino acids.
problematic_amino_acids
A list of amino acids to consider as problematic. Each entry can be specified in the following format:
`amino_acid(s)`: One or more one-letter amino acid codes. Any occurrence of this amino acid string,
regardless of the position in the epitope, is problematic. When specifying more than
one amino acid, they will need to occur together in the specified order.
`amino_acid:position`: A one letter amino acid code, followed by a colon separator, followed by a positive
integer position (one-based). The occurrence of this amino acid at the position
specified is problematic., E.g. G:2 would check for a Glycine at the second position
of the epitope. The N-terminus is defined as position 1.
`amino_acid:-position`: A one letter amino acid code, followed by a colon separator, followed by a negative
integer position. The occurrence of this amino acid at the specified position from
the end of the epitope is problematic. E.g., G:-3 would check for a Glycine at the
third position from the end of the epitope. The C-terminus is defined as position -1.
optional arguments:
-h, --help show this help message and exit
--filter-type {soft,hard}, -f {soft,hard}
Set the type of filtering done. Choosing `soft` will add a new column "Problematic Positions" that lists positions in the epitope with problematic amino acids. Choosing `hard` will remove epitope entries with problematic amino acids.
This tool is used to identify positions in an epitope with an amino acid that is problematic for downstream processing, e.g. vaccine manufacturing. Since this can differ from case to case, this tool requires the user to specify which amino acid(s) to consider problematic. This can be specified in one of three formats:
|
One or more one-letter amino acid codes. Any occurrence of this amino acid string, regardless of the position in the epitope, is problematic. When specifying more than one amino acid, they will need to occur together in the specified order. |
|
A one letter amino acid code, followed by a colon separator, followed by a positive integer position (one-based). The occurrence of this amino acid at the position specified is problematic., E.g. G:2 would check for a Glycine at the second position of the epitope. The N-terminus is defined as position 1. |
|
A one letter amino acid code, followed by a colon separator, followed by a negative integer position. The occurrence of this amino acid at the specified position from the end of the epitope is problematic. E.g., G:-3 would check for a Glycine at the third position from the end of the epitope. The C-terminus is defined as position -1. |
You may specify any number of these problematic amino acid(s), in any combination, by providing them as a comma-separated list.
This tool may be used with any filtered.tsv or all_epitopes.tsv pVACseq report file.