Clinical Application of a Custom AmpliSeq Library and Ion Torrent PGM Sequencing to Comprehensive Mutation Screening for Deafness Genes

Abstract

Background: Congenital hearing loss is one of the most common sensory disorders, with 50-70% of cases attributable to genetic causes. Although recent advances in the identification of deafness genes have resulted in more accurate molecular diagnosis, leading to the better determination of suitable clinical interventions, difficulties remain with regard to clinical applications due to the extreme genetic heterogeneity of deafness. Aim: Toward more effective genetic testing, we adopted Massively Parallel DNA Sequencing (MPS) of target genes using an Ion PGMÔ system and an Ion AmpliSeqÔ panel to diagnose common mutations responsible for deafness and discover rare causative gene mutations. Before its clinical application, we investigated the accuracy of MPS-based genetic testing. Results: We compared the results of Invader assay-based genetic screening, the accuracy of which has already been verified in previous studies, with those of MPS-based genetic testing for a large population of Japanese deafness patients and revealed that over 99.98% of the results were the same for each genetic testing system. Conclusion: The Ion Personal Genome Machine system had sufficient uniformity and accuracy for application to the clinical diagnosis of common causative mutations and efficiently identified rare causative mutations and/or mutation candidates.

Deafness Gene Variations in a 1120 Nonsyndromic Hearing Loss Cohort

“…13,17 The sequence data were mapped to the human genome sequence (build GRCh37/hg19) with the Torrent Mapping Alignment Program. 13,17 The sequence data were mapped to the human genome sequence (build GRCh37/hg19) with the Torrent Mapping Alignment Program.…”

“…11,13,16,17 In the current study, on the basis of our PCR-based technologies in combination with MPS, 13,17 we increased the number of patients (1120 cases of nonsyndromic hearing loss) to establish a database for clinical molecular diagnosis and to confirm the molecular epidemiology of deafness. Hybridization-based capture is commonly used for genomic target enrichment, but for clinical application, polymerase chain reaction (PCR)-based technologies in combination with MPS have also been proposed.…”

Evaluation of the Ion Torrent Personal Genome Machine for Gene-Targeted Studies Using Amplicons of the Nitrogenase Gene nifH

“…A few studies used PGM for medical diagnosis with marker genes (15)(16)(17). No studies have (i) used ecofunctional genes to evaluate Ion Torrent PGM error profiles with the Sequencing 400 kit (400-bp kit) or the recently released Hi-Q Sequencing kit (Hi-Q kit) or (ii) directly compared the PGM data with those obtained with another sequencing platform.…”

Simple and efficient germline copy number variant visualization method for the Ion AmpliSeq™ custom panel

“…We thank the participants of the Deafness Gene Study Consortium for providing samples and clinical information (Nishio et al., ). This study was grant aided by a Health and Labor Sciences Research Grant for Research on Rare and Intractable diseases and Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labor and Welfare of Japan (S.U.…”

“…Ion AmpliSeq™ is a representative method of multiplex PCR‐based target enrichment widely used in the genetic analysis of germline and somatic mutations. The advantages of Ion AmpliSeq™ are the low DNA input volume, high assay success rate, and relatively easy workflow (Nishio, Hayashi, Watanabe, & Usami, ; Tsongalis et al., ). The data obtained from Ion AmpliSeq™ differ in nature from those obtained from hybridization capture‐based target genome enrichment.…”