Optional Downstream Analysis Tools¶
Generate Protein Fasta¶
usage: pvacfuse generate_protein_fasta [-h] [--input-tsv INPUT_TSV]
[--aggregate-report-evaluation AGGREGATE_REPORT_EVALUATION]
[-d DOWNSTREAM_SEQUENCE_LENGTH]
input flanking_sequence_length
output_file
Generate an annotated fasta file from AGFusion or Arriba output.
positional arguments:
input An AGFusion output directory or Arriba fusion.tsv
output file.
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 pVACfuse all_epitopes, filtered, or aggregated TSV
file with epitopes to use for subsetting the input
file to peptides of interest. Only the peptide
sequences for the epitopes in the TSV will be used
when creating the FASTA. (default: None)
--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
frameshift fusion when creating the fasta file. Use
'full' to include the full downstream sequence.
(default: 1000)
This tool will extract protein sequences surrounding fusion variant in an by parsing Arriba or AGFusion
output. One use case for this tool is to help select long peptides that contain short neoepitope
candidates. For example, if pVACfuse 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 fusion position will be centered in the protein sequence returned (if possible).
If the fusion 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.
The output can be limited to only certain variants by providing
a pVACfuse filtered.tsv report file to the --input-tsv argument. Only the peptide sequences for the epitopes in the TSV
will be used when creating the FASTA.
Generate Aggregated Report¶
usage: pvacfuse generate_aggregated_report [-h] [-b BINDING_THRESHOLD]
[--allele-specific-binding-thresholds]
[--percentile-threshold PERCENTILE_THRESHOLD]
[--aggregate-inclusion-binding-threshold AGGREGATE_INCLUSION_BINDING_THRESHOLD]
[-m {lowest,median}]
[--read-support READ_SUPPORT]
[--expn-val EXPN_VAL]
input_file output_file
Generate an aggregated report from a pVACfuse .all_epitopes.tsv report file.
positional arguments:
input_file A pVACfuse .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
-b BINDING_THRESHOLD, --binding-threshold BINDING_THRESHOLD
Tier epitopes in the "Pass" tier when the mutant
allele has ic50 binding scores below this value and in
the "Relaxed" tier when the mutant allele has ic50
binding scores below double this value. (default: 500)
--allele-specific-binding-thresholds
Use allele-specific binding thresholds. To print the
allele-specific binding thresholds run `pvacfuse
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)
--read-support READ_SUPPORT
Read Support Cutoff. When failing this cutoff, sites
will be binned in a "LowReadSupport" tier. (default:
5)
--expn-val EXPN_VAL Expression Cutoff. Expression is meassured as FFPM
(fusion fragments per million total reads). When
failing this cutoff sites will be binned in the
"LowExpr" tier. (default: 0.1)
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 information for that epitope. It also gives information about the total number of well-scoring epitopes for each variant, as well as the HLA alleles that those epitopes are well-binding to. For a full overview of the output, see the pVACfuse output file documentation.
Calculate Reference Proteome Similarity¶
usage: pvacfuse 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]
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)
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 pVACfuse 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 pVACfuse output file documentation.
NetChop Predict Cleavage Sites¶
usage: pvacfuse 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 pVACfuse 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 pVACfuse run pipeline for the filtered output TSV when specified. This tool provides a way to manually run this on pVACfuse’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 pVACfuse in the output file documentation.
NetMHCStab Predict Stability¶
usage: pvacfuse 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 pVACfuse 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 pVACfuse run pipeline for the filtered output TSV when specified. This tool provides a way to manually run this on pVACfuse’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 pVACfuse in the output file documentation.
Identify Problematic Amino Acids¶
usage: pvacfuse 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 pVACfuse report file.