Evaluation of the Ion Torrent PGM sequencing workflow for the routine rapid detection of BRCA1 and BRCA2 germline mutations

Purpose: Conventional methods used to identify BRCA1/2 germline mutations in hereditary cancers are time-consuming and expensive, due to the large size of the genes. The recent introduction of next generation sequencing (NGS) benchtop platforms is a great promise, which is rapidly revolutionizing genetic screening in diagnostic and clinical applications. We recently transferred our methodology for routine BRCA1/2 mutation screening (denaturing High Performance Liquid Chromatography plus Sanger sequencing) to the Ion Torrent PGM platform with the Ion Ampliseq BRCA1 and BRCA2 panel and tested the performance of the system.

Methods: We first validated the NGS approach in a cohort of 33 patients who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we tested 29 newly diagnosed and uncharacterized patients by NGS, and Sanger sequencing was used to confirm results from the NGS platform.

Results: In the validation cohort, all previously identified single nucleotide variants, insertions and deletions (also composed of multiple bases and within complex homopolymeric stretches) were identified by NGS in their correct zygosity status except for variants in a complex multinucleotide region within intron 7 of BRCA1 gene. NGS approach was further able to identify previously undetected variants. In the prospective cohort, almost all (99.3%) called variants were confirmed by Sanger. In both cohorts, in addition to the false positive (31) and false negative (110) results in the intron 7 of BRCA1 gene, the NGS method detected 10 false positives, that were solved by Sanger.

Conclusions: The Ion Torrent PGM NGS approach in BRCA1/2 germline mutation identification is highly sensitive, easy to use, faster and cheaper than traditional approaches. Therefore, according to other recently published works, we highly recommend this system for routine diagnostic testing on BRCA1/2 genes, along with Sanger confirmation of the called variants, and support the usefulness of the approach also in other routine genetic analysis.