Amplicon-based long-read sequencing for accurate CYP2D6 gene deletion and duplication detection using CYP2D7 as a reference gene

We developed an amplicon-based long-read sequencing approach that enables simultaneous detection of CYP2D6 single-nucleotide variants (SNVs) and copy-number variations (CNVs) in a single reaction. The SNVs of the two pseudogenes, CYP2D7 and CYP2D8, were also comprehensively characterized. CYP2D6 CNVs were inferred using CYP2D7 as a reference gene, integrating read-depth measurements with allele-frequency (AF) ratios. The entire CYP2D locus (~30.2Kb) and the three homologous genes separately (6.3-6.6Kb) were sequenced using Oxford Nanopore sequencing. Our analysis identified 21 CYP2D7 and 17 CYP2D8 unique haplotypes, and 57% of the SNVs detected in the pseudogenes had nucleotides corresponding to CYP2D6 reference nucleotides. A total of 36 variants found within the pseudogenes have also been annotated as functional variants in the CYP2D6 gene. The combined CYP2D6:CYP2D7 read-count ratio and reference-to-alternative alleles AF ratio accurately predicted CYP2D6 CNV status. This method is simple, rapid, robust, and easily adaptable for clinical implementation of pharmacogenomics.