title: Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening of CFTR.
abstract: 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.
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Trujillano
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Maximilian E R
authors.initials: ME
authors.lastname: Weiss
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Köster
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Efstathios B
authors.initials: EB
authors.lastname: Papachristos
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Werber
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Krishna Kumar
authors.initials: KK
authors.lastname: Kandaswamy
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Anett
authors.initials: A
authors.lastname: Marais
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Eichler
authors.affiliation: Centogene AG Rostock, Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Creed
authors.affiliation: Pathology and Genetics Department, Dubai Genetic and Thalassemia Center, Dubai Health Authority Dubai, United Arab Emirates.
authors.forename: Erol
authors.initials: E
authors.lastname: Baysal
authors.affiliation: Pathology and Genetics Department, Dubai Genetic and Thalassemia Center, Dubai Health Authority Dubai, United Arab Emirates.
authors.forename: Iqbal Yousuf
authors.initials: IY
authors.lastname: Jaber
authors.affiliation: Clinical and Chemical Pathology Department, Faculty of Medicine, Cairo University Cairo, Egypt.
authors.forename: Dina Ahmed
authors.initials: DA
authors.lastname: Mehaney
authors.affiliation: Department of Pathology & Laboratory Medicine, American University of Beirut Medical Center Beirut, Lebanon.
authors.forename: Chantal
authors.initials: C
authors.lastname: Farra
authors.affiliation: Centogene AG Rostock, Germany ; Albrecht-Kossel-Institute for Neuroregeneration, Medical University Rostock Rostock, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Rolfs
keywords: CFTR; Ion Torrent; cystic fibrosis; molecular diagnostics; next-generation sequencing; validation