Star Allele Caller

For genes CACNA1S, CFTR, CYP2C19, CYP2C9, CYP3A5, CYP4F2, IFNL3, RYR1, NUDT15, SLCO1B1, TPMT, UGT1A1, VKORC1, DPYD, G6PD, MT-RNR1, ABCG2 the allele definitions are sourced from PharmGKB. Because BCHE does not have defined star alleles, the Star Allele Caller checks if a sample is positive for any of the variants that are reported.

For genes CYP2C19, CYP2C9, CYP3A5, CYP4F2, NUDT15, SLCO1B1, DPYD, the definitions are sourced from PharmVAR. For the remaining hg19/GRCh37 genes, ie, ABCG2, CACNA1S, IFNL3, F5 and VKORC1 - the allele definitions have been lifted from their corresponding definitions for hg38, which are sourced from PharmGKB.

The Star Allele Caller has the following features.

The Star Allele Caller can accept as input, different forms of sequence data such as FASTQs files, BAM/CRAM files or gVCF/VCF files. In the simplest case, the caller takes a DRAGEN gVCF file as input. The following is an example of the command line for this use case.

Contrary to a variant-only VCF file, a DRAGEN gVCF file contains the genotypes for all positions in a genome. Although the gVCF format is the preferred format for the caller, it can also accept a standard variant-only VCF file as input. The command line for that case will be the same as above, with the VCF file passed instead of a gVCF file. Also, the CNV-VCF file is optional - in this case the Star Allele Caller will not call star alleles that are detected through CNV analysis.

The following is an example of this use case, with only a variant only VCF file as input.

The small variant VCF/gVCF and CNV-VCF files should meet the following specifications.

VCF/gVCF files can be substituted with a compressed GZ file (ie <file_name>.vcf.gz or <file_name>.gvcf.gz). If a BAM or FASTQ file is passed as input, then the preferred command to use for Star Allele Caller is the option --enable-pgx which turns on all necessary components that the Star Allele Caller needs. The human reference needs to be passed as a command line option. The Star Allele Caller detects the reference version (ie, hg19, GRCh37 or hg38) and accordingly reads in the correct allele definitions.

Note that the directory passed with the -r option points to the directory containing the DRAGEN hash table for a reference, not the raw reference FASTA. DRAGEN team can provide these hash tables or they may be built using the following command (where hg38.fa is a reference FASTA).

Once the hash tables are generated, the path <directory_for_ref_hashtables> can then be passed with the -r parameter. If the BAM file is not aligned to the available hg38 reference, automatic remapping can be done by setting the option --enable-map-align true. If the user has a BAM file that is pre-aligned to a custom hg38 reference, and wishes to use this reference for the Star Allele Caller, then the user needs to create a DRAGEN reference hash table, which can be built using the above command.

For passing a FASTQ file as input, additional options, --RGID and --RGSM need to be set in the command line. An example of the command line for this use case as follows.

The setting --enable-pgx turns on other PGx callers such as CYP2D6, CYP2B6, and HLA - in addition to the Star Allele Caller. Note that to run the HLA caller, the passed reference must contain anchored_hla, a specific subdirectory with HLA-specific reference files. If a user wants to ONLY turn on the Star Allele Caller and not the other PGx callers, then in addition to enabling the Star Allele Caller, the variant caller also needs to be enabled. Optionally, the CNV caller should also be preferably enabled for analyzing CNV star alleles. An example of the command line for this use case is as follows.

Additional options with the variant caller such as --vc-forcegt-vcf should not be used when the Star Allele Caller is enabled.

Following completion of the DRAGEN Star Allele Caller run, the following four output files are produced.

{
  "dragenVersion": "4.2.0-724-gb600fcef",
  "sample": "NA19374",
  "pharmcatMetabolismStatusResourceUrl": "https://github.com/PharmGKB/PharmCAT/blob/aeecfe5f787e95dfb31ede62884e287affef45b3/src/main/resources/org/pharmgkb/pharmcat/definition/gene_phenotypes.json",
  "star_allele": {
    "calls": [
      {
        "gene": "DPYD",
        "lastUpdate": "10/13/2021",
        "alleleDefinitionsUrl": "https://www.pharmgkb.org/page/dpydRefMaterials",
        "genotype": "./.",
        "pharmcatDescription": null,
        "pharmcatMetabolismStatus": null,
        "variants": "chr1:97515839:T:C,<NON_REF>:0/1:49:70:PASS;chr1:97883329:A:G,<NON_REF>:1/1:65:68:PASS;chr1:97573881:C:T,<NON_REF>:0/1:50:73:PASS",
        "variantStarAllelesFound": "c.1218G>A:c.1627A>G(*5):c.85T>C(*9A)",
        "minGQ": "49",
        "missingGenotypes": "",
        "filteredGenotypes": ""
      }
    ]
  }

The fields in the json file are as follows.

Each Star allele genotype contains one or two haplotypes (a haplotype for chrM gene MT-RNR1 and chrX gene G6PD for male samples, and a diplotype for all other genes) separated by a slash (eg *1/*2). Each haplotype is a pre-defined star allele and the definitions can be found under the allele definitions URL. Note that there may be some variance in star allele definitions and notations based on the resource and when it was last updated. The Star Allele Caller follows the PharmGKB definitions and notations exactly. When the Star Allele Caller cannot identify an optimal genotype for a gene, a no-call (./. or .) is made. In certain cases, more than one genotype is optimally satisfied, in that case all satisfied genotypes are listed, separated by a colon (eg *1/*2:*3/*4)

Additionally, a json file, <prefix>.star_allele.json is also produced when this option is set that contains the genotype calls for just the Star Allele Caller (contrary to the main output file, <prefix>.targeted.json that aggregates calls from all targeted callers). The format of this json file is the same as the json format described above.