CYP2D6 Caller

The first step of CYP2D6 calling is to determine the combined copy number of CYP2D6 and CYP2D7. Reads aligned to regions in either CYP2D6 or CYP2D7 are counted. The counts in each region are corrected for GC-bias, and then normalized to a diploid baseline. The GC-bias correction and normalization factors are determined from read counts in 3000 preselected 2 kb regions across the genome. The combined CYP2D6 and CYP2D7 copy number is then calculated from the average sequencing depth across the CYP2D6 and CYP2D7 regions.

The CYP2D6-derived copy number is calculated at 117 predefined differentiating sites across the CYP2D6 gene. The differentiating sites are selected at positions with sequence differences in CYP2D6 and CYP2D7 where calling the CYP2D6-derived copy number shows an accuracy of greater than 98% based on sequencing data from the 1000 Genomes Project.

For each differentiating site, CYP2D6-specific and CYP2D7-specific alleles are counted in reads mapping to either CYP2D6 or the homologous region in CYP2D7. The CYP2D6-derived copy number is then calculated from the two gene-specific allele counts using the total CYP2D6 and CYP2D7 copy number calculated from the previous step.

The CYP2D6-derived copy number along the CYP2D6 gene is used to identify known population structural variants (SVs), including whole gene deletions and duplications as well as certain gene conversions and gene fusions. The following fusion variants are detected:

In addition to the exon 9 fusion breakpoints, exon 9 can participate in CYP2D7 gene conversion resulting in an embedded CYP2D7 sequence instead of a true hybrid. The structural variant caller also detects exon 9 gene conversions. Because only changes in CYP2D6-derived copy number yield structural variant calls, there might be rare cases where two hybrid copies result in no structural variant calls. For example, when both *36 and *13 with fusion breakpoint in exon 9 are present. However, the structural variant caller is capable of detecting multiple copies of the same fusion type (eg, *36x2) or cases where both an exon 9 gene conversion copy and an exon 9 2D6-2D7 hybrid are present.

118 small variants that define various star alleles are detected from the read alignments. 96 of these variants are in unique (nonhomologous) regions of CYP2D6 with high mapping quality. Only reads mapping to CYP2D6 are used for calling variants in nonhomologous regions. The other 22 variants occur in homologous regions of CYP2D6 where reads mapping to either CYP2D6 or CYP2D7 are used for variant calling.

For each variant, reads containing either the variant allele or the nonvariant alleles are counted. A binomial model that incorporates the sequencing errors is then used to determine the most likely variant copy number (0 for nonvariant). A strand bias filter is applied for certain noisy variants that tend to have false positive calls.

Samples with poor sequencing quality or greater than five copies of CYP2D6 will have allele counts with higher variance. This elevated variance increases the chance that the most likely variant copy number is wrong. To handle these cases, the small variant caller also indicates alternate, less likely variant copy numbers.

The called SVs and small variant genotypes are matched against the definitions of 128 different star alleles. This might result in different sets of star alleles matching the called variant genotypes, such as with *1, *46 and *43, *45 where both sets of star alleles contain the same 4 small variants. When the small variant caller emits alternate, less likely variant copy numbers in addition to the most likely variant copy numbers this might result in different sets of star alleles being identified, since these alternate sets of variant copy numbers are also matched to the star allele definitions. The number of matched star alleles must match the number of CYP2D6-derived gene copies determined from previous steps. When there are fewer than two CYP2D6-derived gene copies, then one or more *5 deletion haplotypes are included in the output set of star alleles. If all variant genotypes cannot be matched to a set of star alleles, the CYP2D6 Caller returns a no call during the genotyping step with filter value No_call.

Given a possible set of star alleles, the genotyping step attempts to identify the two likely haplotypes that contain all star alleles in the set. The deletion haplotype (*5) is considered as a possible haplotype during this process. The likelihood of any given genotype is determined from a table of population frequencies determined from the 1000 Genomes Project and the genotype with the highest population frequency is selected. When two or more possible genotypes are identified with similar population frequencies, then all genotypes are emitted. This results in a call with filter value More_than_one_possible_genotype.

The CYP2D6 Caller prints out its calls in the targeted callers output file, <prefix>.targeted.json (that also aggregates calls from other targeted callers). An example of this file with the CYP2D6 caller set is as follows:

{
    "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",
    "cyp2d6": {
    "genotype": "*17/*2",
    "genotypeFilter": "PASS",
    "pharmcatDescription": "An individual carrying two normal function alleles",
    "pharmcatMetabolismStatus": "Normal Metabolizer"
  },
}

For CYP2D6 caller, the fields are defined as follows.

When the option --targeted-enable-legacy-output=true is set, the CYP2D6 Caller also generates a <output-file-prefix>.cyp2d6.tsv file in the output directory. The output file contains the following tab-delimited fields with no header line:

Each CYP2D6 genotype contains two haplotypes separated by a slash (eg, *1/*2). Each haplotype consists of one or more star alleles separated by a plus sign (eg, *10+*36). When a haplotype contains more than one copy of the same star allele, that star allele only appears once and is followed by a multiplication sign, and then the number of copies (eg, *1x2 for two copies of *1).

Output File Examples

The following are example output files:

NA18632 *10+*36x2/*52 PASS

HG01190 *4+*68/*5 PASS

NA17244 *2/*4x2+*13+*83;*2/*4+*4.013+*13+*39 More_than_one_possible_genotype

NA19908 *1/*46;*43/*45 More_than_one_possible_genotype

NA18611 None No_call