Nonoptical Massive Parallel DNA Sequencing ofBRCA1andBRCA2Genes in a Diagnostic Setting

Abstract

The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false-positive calls without the introduction of false-negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing-Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time- and cost-effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.

First application of next-generation sequencing in Moroccan breast/ovarian cancer families and report of a novel frameshift mutation of the BRCA1 gene

“…The advent of NGS technology has increased sequencing capacity and lowered the cost of sequencing, thus offering a powerful alternative to Sanger sequencing for genetic testing (18). In addition, the launch of benchtop sequencers with the capacity to detect mutations from a significantly greater number of samples in parallel, and in a more cost effective manner, led to more clinical laboratories adopting this technology (19,20).…”

Comprehensive massive parallel DNA sequencing strategy for the genetic diagnosis of the neuro-cardio-facio-cutaneous syndromes

“…Although MPS has the potential to offer a solution for this problem, its implementation in a diagnostic setting is complex as it requires several steps of validation. 20 This study presents the validation of a MPS-based approach for the molecular diagnosis of patients with NCFCS and a strategy for its implementation in a clinical laboratory environment. For that, a multiplex PCR-based strategy for the enrichment of the entire coding region of the 13 NCFCS disease genes was designed.…”

“…Variability in coverage requires samples to be oversequenced to achieve the minimum desired coverage per base required for variant identification (30 reads). 20 The adjustments made in the PCR and sample pooling conditions largely improve amplification uniformity and reduce the number of amplicons below the coverage threshold. Nevertheless, because of unpredictability of the missing amplicons, we observed that the initial step of amplicon coverage analysis was of utmost importance to avoid false negative results.…”

“…20 The VPP was fine-tuned using all known variants present in the training set as surrogate markers. Genetic alterations observed in exons that were not included in our original strategy (Supplementary Figure 1) were validated by Sanger sequencing.…”

“…21 Such sequencing miscalls are nonrandomly distributed, systematic (appear in more than one case and in different runs) and have a strand-specific bias pattern (occur preferentially on one DNA strand compared with the other). 20 Calls that fulfill these characteristics were excluded from the analysis. The established VPP was then applied to the validation set.…”