Targeted haplotyping in pharmacogenomics using Oxford Nanopore Technologies' adaptive sampling.

Pharmacogenomics (PGx) studies the impact of interindividual genomic variation on drug response, allowing the opportunity to tailor the dosing regimen for each patient. Current targeted PGx testing platforms are mainly based on microarray, polymerase chain reaction, or short-read sequencing. Despite demonstrating great value for the identification of single nucleotide variants (SNVs) and insertion/deletions (INDELs), these assays do not permit identification of large structural variants, nor do they allow unambiguous haplotype phasing for star-allele assignment. Here, we used Oxford Nanopore Technologies' adaptive sampling to enrich a panel of 1,036 genes with well-documented PGx relevance extracted from the Pharmacogenomics Knowledge Base (PharmGKB). By evaluating concordance with existing truth sets, we demonstrate accurate variant and star-allele calling for five Genome in a Bottle reference samples. We show that up to three samples can be multiplexed on one PromethION flow cell without a significant drop in variant calling performance, resulting in 99.35% and 99.84% recall and precision for the targeted variants, respectively. This work advances the use of nanopore sequencing in clinical PGx settings.

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

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.