Open AccessValidation of a semiconductor next‐generation sequencing assay for the clinical genetic screening of CFTR
Author(s) -
Trujillano Daniel,
Weiss Maximilian E. R.,
Köster Julia,
Papachristos Efstathios B.,
Werber Martin,
Kandaswamy Krishna Kumar,
Marais Anett,
Eichler Sabrina,
Creed Jenny,
Baysal Erol,
Jaber Iqbal Yousuf,
Mehaney Dina Ahmed,
Farra Chantal,
Rolfs Arndt
Publication year - 2015
Publication title -
molecular genetics and genomic medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.765
H-Index - 29
ISSN - 2324-9269
DOI - 10.1002/mgg3.149
Subject(s) - sanger sequencing , ion semiconductor sequencing , dna sequencing , genetics , cystic fibrosis , biology , genetic testing , gene , molecular diagnostics , mutation , computational biology , bioinformatics
Genetic testing for cystic fibrosis and CFTR ‐related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next‐generation sequencing ( NGS ) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq ™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI : 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI : 100% to 100%), a positive predictive value of 100% (95% CI : 100% to 100%), and a negative predictive value of 99.99% (95% CI : 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly‐thymidine‐guanine and poly‐thymidine (poly‐ TG ‐T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.