Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping

Abstract

CYP2D6 is a very important pharmacogene as it is responsible for the metabolization or bioactivation of 20 to 30% of the clinically used drugs. However, despite its relatively small length of only 4.4 kb, it is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.

Haplotyping pharmacogenes using TLA combined with Illumina or Nanopore sequencing

“…Except for CYP1A2, the genes were covered for more than 95% with the viewpoints in this study. The CYP2D6 Nanopore dataset of viewpoint 4 resulted in the correct phased genotype compared to Rubben et al 25 . In the other datasets, only a minor part of the variants was called and phased correctly compared to reference sets.…”

“… 23 . Nevertheless, according to Rubben et al 25 , these two SNVs are present in the GM12878 cell line but were not included in the reference set of Krusche et al . due to the limitations of the used sequencing techniques 23 .…”

PharmVar Tutorial on CYP2D6 Structural Variation Testing and Recommendations on Reporting

“…A recent report described Cas9‐targeted nanopore DNA sequencing to detect CYP2D6 :: CYP2D7 hybrid alleles 99 . This method uses Cas9‐targeted cleavage of native genomic DNA at key sites within or flanking regions of interest and the addition of sequencing adapters at these sites.…”

Beyond assembly: the increasing flexibility of single-molecule sequencing technology

“…The optimization of these methods will lead to greater read depths and lengths, enabling applications that need ultra-high-depth sequencing such as identifying somatic mosaic variants or intratumoural heterogeneity. Further developments in combining methods, such as Cas-mediated enrichment with adaptive sampling 161 , will improve on-target rates and drive costs even lower. Targeted long reads are likely to generate new insights into the direct molecular impact of mutations and alterations as their single-molecule nature is a proxy for cellular heterogeneity in complex clinical samples.…”