Haplotyping pharmacogenes using TLA combined with Illumina or Nanopore sequencing

The currently used pharmacogenetic genotyping assays offer limited haplotype information, which can potentially cause specific functional effects to be missed. This study tested if Targeted Locus Amplification (TLA) combined with Illumina or Nanopore sequencing can offer an advantage in terms of accurate phasing. The TLA method selectively amplifies and sequences entire genes based on crosslinking DNA in close physical proximity. This way, DNA fragments that were initially further apart in the genome are ligated into one molecule, making it possible to sequence distant variants within one short read. In this study, four pharmacogenes, CYP2D6, CYP2C19, CYP1A2, and BRCA1, were sequenced after enrichment using different primer pairs. Only 24% or 38% of the nucleotides mapped on target when using Illumina or Nanopore sequencing respectively. With an average depth of more than 1000X for the regions of interest, none of the genes were entirely covered with either sequencing method. For three of the four genes, this resulted in less than half of the variants that were phased correctly compared to the reference. Only the Nanopore dataset with the optimized primer pair for CYP2D6 resulted in the correct haplotype. Therefore, the TLA workflow cannot be considered a suitable alternative for reliably genotyping and phasing phar-macogenes.