Long‐read HiFi sequencing of NUDT15: Phased full‐gene haplotyping and pharmacogenomic allele discovery. Issue 11 (12th September 2022)

• Record Type: Journal Article
• Authors: Scott, Erick R.; Yang, Yao; Botton, Mariana R.; Seki, Yoshinori; Hoshitsuki, Keito; Harting, John; Baybayan, Primo; Cody, Neal; Nicoletti, Paola; Moriyama, Takaya; Chakraborty, Shreyasee; Yang, Jun J.; Edelmann, Lisa; Schadt, Eric E.; Korlach, Jonas; Scott, Stuart A.
• Other Names: Scott Stuart A. guestEditor.; Wang Kai guestEditor.; Spinner Nancy B. guestEditor.
• Is Part Of: Human mutation. Volume 43:Issue 11(2022)
• Page Start: 1557
• Page End: 1566
• Publication Date: 2022-09-12
• Subjects: amplicon sequencing -- long‐read sequencing -- NUDT15 -- PacBio -- pharmacogenetics -- pharmacogenomics -- single molecule real‐time (SMRT) HiFi sequencing; Human chromosome abnormalities -- Periodicals; Mutation (Biology) -- Periodicals; 616.04205
• DOI: 10.1002/humu.24457
• Languages: English
• ISSNs: 1059-7794

Abstract

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.