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A Comprehensive Assay for CFTR Mutational Analysis Using Next‐Generation Sequencing
Author(s) -
Abou Tayoun Ahmad N.,
Tunkey Christopher D.,
Tafe Laura J.,
Levandowsky Elizabeth,
Spangler Jessica,
Dindinger Matthew,
Ross Tristen,
Shah Minita,
McLaughlin Stephen,
Lee Clarence C.,
Harkins Timothy T.,
Raia Gabrielle,
Amos Christopher I.,
Wells Wendy A.,
Tsongalis Gregory J.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.874.1
Subject(s) - ion semiconductor sequencing , genotyping , amplicon , genetics , dna sequencing , biology , computational biology , gene , genotype , polymerase chain reaction
Background Cystic fibrosis (CF) is a life‐threatening genetic disorder with over 1000 causative mutations in the CFTR gene detected to date. Current CF genotyping assays target a limited number of mutations and risk missing deleterious ones, both known and novel. Here, we evaluate the performance of a CFTR assay using PCR target enrichment and next‐generation sequencing. Methods 68 DNA samples were blindly sequenced on the Ion Torrent Personal Genome Machine (PGM). The CFTR gene was targeted using a custom AmpliSeqTM panel, which consists of 72 amplicons covering all exons and splice sites. Base calls, read alignments, and variant calling were performed using the Torrent SuiteTM software. Results By correctly genotyping 66/68 (97%) of the cases (3/3 normal, 41/42 carriers, 22/23 CF), the first iteration of our assay demonstrates a high sensitivity of calling various pathogenic mutations in CFTR. There was 100% reproducibility of mutation detection between independent runs. The average coverage was greater than 2380 per base pair when 12 samples were pooled on a 318 chip, thus allowing the use of different PGM chips to scale to the desired amount of multiplexing and depth of coverage. Conclusions This assay offers clinical laboratories a comprehensive, scalable and cost‐effective means of CFTR genetic testing.