Long‐read HiFi sequencing of NUDT15 : Phased full‐gene haplotyping and pharmacogenomic allele discovery

Abstract

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.−121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

Allele-specific polymerase chain reaction can determine the diplotype of NUDT15 variants in patients with childhood acute lymphoblastic Leukemia

“…Tsujimoto et al defined the diplotypes of 14 patients harboring the two variants (i.e., c.415C > T and c.55_56insGAGTCG) using droplet digital PCR (ddPCR); however, they could not identify any patients with NUDT15 *3/*6 24 . Recently, although the robustness of a long-read HiFi amplicon sequencing assay has been reported for phased full gene characterization and the discovery of pharmacogenomic allele 25 , the method may not conveniently elucidate the phasing of NUDT15 in daily clinical practice due to its higher cost and unavailability in most laboratories in Taiwan.…”

“…However, if c.415C > T and the c.55_56insGAGTCG are identified simultaneously, AS-PCR with LNA primers can be used to determine the diplotype, which will be cost-effective (approximately 20 USD per sample) and fast (TAT within 5 working days). Importantly, the process is lesser labor-intensive than ddPCR or NGS-based methods 16 , 24 , 25 .…”