Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing

Abstract

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second‐Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high‐quality, full‐length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene‐locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD‐powered CYP2D6 gene‐variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy‐number variation.

Added Value of Clinical Sequencing: WGS-Based Profiling of Pharmacogenes

“…The most efficient and straight forward method for variant phasing, however, is long-read sequencing, using e.g., PacBio (pacb.com) or Oxford Nanopore Technologies (nanoporetech.com). It has been shown that targeted long-read sequencing of the CYP2D6 gene enables phasing and accurate calling of SNVs, indels, and SVs [30][31][32], which could be further evaluated using long-read WGS or hybrid assemblies of short and long reads.…”

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].…”

Nanopore sequencing of the pharmacogene CYP2D6 allows simultaneous haplotyping and detection of duplications

“…The regions in between these 285 two strings are notoriously difficult to sequence, resulting in a gap in most PharmVar catalouged 286 alleles or subvariants. Variation in the number of A's within 2943-2964 has been reported 287 previously (36), however this region has not been included as part of the haplotype definition 288 (6). Although nanopore sequencing was not able to reliably detect variation in the poly A string, 289 the regions adjacent to the strings were sequenced accurately in this study, including the 290 detection of variants such as 2966A>G, which are still ambiguous in many star (*) alleles.…”

“…The advantages of long amplicon sequencing of CYP2D6 have been recently reported by two 269 papers, using the PacBio single molecule real-time (SMRT) sequencing platform (35,36). 270…”