A comprehensive assay for CFTR mutational analysis using next-generation sequencing.

Abstract:

BACKGROUND:Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS:We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform. RESULTS:The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS:With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Abou Tayoun AN,Tunkey CD,Pugh TJ,Ross T,Shah M,Lee CC,Harkins TT,Wells WA,Tafe LJ,Amos CI,Tsongalis GJ

doi

10.1373/clinchem.2013.206466

subject

Has Abstract

pub_date

2013-10-01 00:00:00

pages

1481-8

issue

10

eissn

0009-9147

issn

1530-8561

pii

clinchem.2013.206466

journal_volume

59

pub_type

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