CYP2D6 is a widely expressed human xenobiotic metabolizing enzyme, best known for its role in the hepatic phase I metabolism of up to 25% of prescribed medications, which is also expressed in other organs including the brain, where its potential role in physiology and mental health traits and disorders is under further investigation. Owing to the presence of homologous pseudogenes in the CYP2D locus and transposable repeat elements in the intergenic regions, the gene encoding the CYP2D6 enzyme, CYP2D6, is one of the most hypervariable known human genes - with more than 140 core haplotypes. Haplotypes include structural variants, with a subtype of these known as fusion genes comprising part of CYP2D6 and part of its adjacent pseudogene, CYP2D7. The fusion genes are particularly challenging to identify. The CYP2D6 enzyme activity corresponding to some of these fusion genes is known, while for others it is unknown. The most recent (high fidelity, or HiFi) version of single molecule real-time (SMRT) long-read sequencing can cover whole CYP2D6 haplotypes in a single continuous sequence read, ideal for structural variant detection. In addition, the accuracy of base calling has increased to a level sufficient for accurate characterization of single nucleotide variants. As new CYP2D6 haplotypes are continuously being discovered, and likely many more remain to be identified in populations that are relatively understudied to date, a method of characterization that employs sequencing with at least this degree of accuracy is required. The aim of the work reported herein was to develop an efficient and accurate HiFi SMRT amplicon-based method capable of detecting the full range of CYP2D6 haplotypes including fusion genes. We report proof-of-concept for 20 amplicons, aligned to fusion gene haplotypes, with prior cross-validation data. Amplicons with CYP2D6-D7 fusion genes aligned to *36, *63, *68, and *4 (*4-like; *4N, or *4.013) hybrid haplotypes. Amplicons with CYP2D7-D6 fusion genes aligned to the *13 subhaplotypes predicted (e.g., *13F, *13A2). Data analysis was efficient, and further method development indicates that this technique could suffice for the characterization of the full range of CYP2D6 haplotypes. Although included in drug labelling by regulatory bodies (the U.S. Food and Drug Administration, the European Medicines Agency, the Pharmaceuticals and Medical Devices Agency) and prescribing recommendations by consortia (Clinical Pharmacogenetics Implementation Consortium and the Dutch Pharmacogenetics Working Group), the identification of CYP2D6 variants is not yet routine in clinical practice. The HiFi sequencing method reported herein is suitable for high throughput, efficient, identification of the full range of known CYP2D6 haplotypes and novel haplotypes, and can be completed in a week or less. Moreover, the method that we have developed could be extended to other complex loci and to other species in a multiplexed high throughput assay.

Pharmacogenomics aims to use the genetic information of an individual to personalize drug prescribing. There is evidence that pharmacogenomic testing before prescription may prevent adverse drug reactions, increase efficacy, and reduce cost of treatment.CYP2D6is a key pharmacogene of relevance to multiple therapeutic areas. Indeed, there are prescribing guidelines available for medications based on CYP2D6 enzyme activity as deduced fromCYP2D6genetic data. The Agena MassARRAY system is a cost-effective method of detecting genetic variation that has been clinically applied to other genes. However, its clinical application toCYP2D6has to date been limited by weaknesses such as the inability to determine which haplotype was present in more than one copy for individuals with more than two copies of theCYP2D6gene. We report application of a new protocol forCYP2D6haplotype phasing of data generated from the Agena MassARRAY system. For samples with more than two copies of theCYP2D6gene for which the prior consensus data specified which one was present in more than one copy, our protocol was able to conductCYP2D6haplotype phasing resulting in 100% concordance with the prior data. In addition, for three reference samples known to have more than two copies ofCYP2D6but for which the exact number ofCYP2D6genes was unknown, our protocol was able to resolve the number for two out of the three of these, and estimate the likely number for the third.