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

CYP2D6 is one of the most important and widely studied genes in pharmacogenetics but it is highly polymorphic, can be affected by different forms of structural variation, and has a nearby pseudogene with highly similar sequence; therefore, its accurate genotyping and structural resolution are difficult to achieve with short sequencing reads. Liau and colleagues performed long-read amplicon sequencing of CYP2D6 on the GridION platform, which enabled its high-throughput genotyping, accurate haplotyping and variant detection.

Background 
The accurate genotyping of CYP2D6 is hindered by the very polymorphic nature of the gene, high homology with its pseudogene CYP2D7, and the occurrence of structural variations. Long read sequencing offers the promise of overcoming some of these challenges, along with the advantage of straightforward variant phasing. We have established methods for sequencing and analysis of DNA amplicons containing the whole CYP2D6 gene, using the GridION nanopore sequencer.

Materials and methods 
Seven reference and 25 clinical samples covering various haplotypes including gene duplication were barcoded and sequenced over two sequencing runs. Sequenced raw reads were analyzed using a pipeline of bioinformatics tools including two mapping tools and two variant calling tools.

Results 
Using minimap2 and nanopolish (mapping and variant calling tools respectively) resulted in the most accurate variant detection. Haplotypes of all samples could be detected accurately to the subvariant level in most samples, while the remaining samples had either novel variants or variant patterns not matched to the current PharmVar CYP2D6 haplotype database. Allele duplication could also be determined by analyzing the allelic balance between the sample haplotypes.

Conclusion 
Nanopore sequencing of CYP2D6 offers a high throughput method for genotyping, accurate haplotyping, and detection of new variants and duplicated alleles.