Development and Validation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Variants for the Clinical Laboratory

Abstract

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.

Gene Copy Number Estimation from Targeted Next-Generation Sequencing of Prostate Cancer Biopsies: Analytic Validation and Clinical Qualification

“…Our method reliably estimates gene copy number changes from amplicon-based targeted NGS with the acquired results being comparable with widely used assays, including exome sequencing, aCGH, digital droplet PCR, and FISH (17). Critically, the data processing time for this panel-based CNA estimation is substantially shorter of that for exome sequencing and can be easily completed even with a personal computer.…”

NovelBRCA1andBRCA2Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas

“…Several next‐generation sequencing (NGS) approaches to determine the mutation status of BRCA1 and BRCA2 have been developed, but most approaches were validated using high‐quality DNA (i.e., blood‐derived DNA) [Feliubadalo et al., 2013; Hirotsu et al., 2015; Strom et al., 2015]. Therefore, these approaches can successfully be implemented in a diagnostic setting to screen for germline defects in BRCA1 and BRCA2 using blood‐derived DNA [D'Argenio et al., 2015; Trujillano et al., 2015], but cannot be used to sequence low quality and highly fragmented DNA derived from FFPE tumor blocks.…”

Next-generation sequencing: advances and applications in cancer diagnosis

“…Despite several findings on HBC with NGS, a recent study demonstrated that with regard to the two most common platforms, neither the Illumina MiSeq sequencer with the supplied MiSeq Reporter software nor the Life Technologies Ion Torrent Personal Genome Machine (Ion PGM) with the supplied Torrent Suite software were completely suitable for clinical laboratory sequencing of BRCA1 or BRCA2 10. The inability of the MiSeq system is that it fails to detect insertions and deletions larger than nine base pairs.…”