Exome & General Genetics
The clinical implementation of new target enrichment methods and next-generation sequencing (NGS) technology has rapidly transformed genetic testing. Diagnostic labs can now offer a wide variety of large comprehensive multi-gene panels or even full exome sequencing to help clinicians diagnose and treat patients. The unmatched sensitivity, accuracy and throughput of NGS compared to traditional Sanger sequencing make it an ideal technology not only for panels but also high volume single gene assays. Here we describe the validation and performance of an NGS based assay for sequencing the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The custom designed assay utilizes TruSeq Custom Amplicon (TSCA) target enrichment and modified bioinformatics pipeline to identify different classes of mutations, including small deletions and insertions. Validation of the test with 151 previously characterized CFTR variants resulted in 100% accuracy. Test specificity of 99.99% was determined by analyzing Sanger sequencing confirmation data from the first 2,000 samples. In addition, the assay was able to detect variants missed by previous testing due to allele-dropout. The transition of the CFTR sequencing assay from Sanger sequencing to a custom NGS based test has not only increased the sensitivity and reliability of the assay but also cut the turn-around-time in half, allowing clinicians to diagnose and make treatment decisions quicker.