.. image:: ../images/pVACseq_logo_trans-bg_sm_v4b.png :align: right :alt: pVACseq logo .. _optional_downstream_analysis_tools_label: Optional Downstream Analysis Tools ================================== Generate Protein Fasta ---------------------- .. program-output:: pvacseq generate_protein_fasta -h .. .. argparse:: :module: lib.generate_protein_fasta :func: define_parser :prog: pvacseq generate_protein_fasta 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 :ref:`phased_vcf` 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 -------------------------- .. program-output:: pvacseq generate_aggregated_report -h 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 :ref:`output file documentation `. Calculate Reference Proteome Similarity --------------------------------------- .. program-output:: pvacseq calculate_reference_proteome_similarity -h 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 :ref:`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 :ref:`output file documentation `. NetChop Predict Cleavage Sites ------------------------------ .. program-output:: pvacseq net_chop -h 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 :ref:`output file documentation `. NetMHCStab Predict Stability ---------------------------- .. program-output:: pvacseq netmhc_stab -h 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 :ref:`output file documentation `. Identify Problematic Amino Acids -------------------------------- .. program-output:: pvacseq identify_problematic_amino_acids -h 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: .. list-table:: * - ``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. 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.