Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6

Abstract

The CYP2D6 enzyme metabolizes ~25% of common medications, yet homologous pseudogenes and copy-number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single-molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run non-reference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement (CYP2D6*36+*41). Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.

Clinical Implementation of Pharmacogenetic Testing in a Hospital of the Spanish National Health System: Strategy and Experience Over 3 Years

“…Our designed custom genomic tool PharmArray (based on the TaqMan OpenArray) was of great help to this aim. Although tests based on next‐generation sequencing (NGS) technologies have been reported in the literature, we found that our platform results are much more suitable for the clinical approach in terms of simplicity of analysis, turnaround times, and affordable costs in our NHS. This genotyping tool allowed us the implementation of a semipreemptive strategy with eight preestablished protocols agreed upon with various clinical services in risk populations.…”

Pharmacogenomic Biomarkers for Improved Drug Therapy—Recent Progress and Future Developments

“…The long reads allow for accurate variant calling as well as phasing of multiple heterozygous variants whose genomic location might be several kilobases apart. As such, SMRT provides an excellent technology for the sequencing of complex CYP loci and, using CYP2D6 as an example, has been demonstrated to allow the simultaneous detection of SNVs and CNVs in multiplexed samples [122,123]. In addition to genomic sequencing, SMRT allows direct decoding of epigenetic marks [124].…”

A Rigorous Interlaboratory Examination of the Need to Confirm NGS-Detected Variants with an Orthogonal Method in Clinical Genetic Testing

“…Lab 1 validated [23] the PacBio circular consensus sequencing method [24] specifically for use in confirmation. This method provides high accuracy [25] and has been successfully applied in other clinical genetic tests [26]. Lab 2 used only Sanger confirmation.…”