Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

Abstract

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

Figure 1

Experimental design. Our study was divided into two parts: the Training Set and the Validation Set. In the Training Set, 28 HBOCS samples, already analyzed by our current diagnostic workflow, were assessed (Supplementary Figure 1). Of this group, 23 samples contained a variety of pathogenic mutations, including challenging insertions and deletions, inside and outside homopolymeric regions, as well as a subset of non-pathogenic variants. The remaining 5 samples belonged to affected individuals from high-risk HBOCS families, in whom no pathogenic mutation had been found after applying our current multistep protocol. In total, this subset of 28 samples contained 23 unique pathogenic mutations and 213 (33 unique) non-pathogenic DNA variants (Supplementary Table 1). The Training Set was subjected to two different experiments: in experiment 1, all 28 samples were amplified using BRCA MASTR v1.2 and sequences in 4 runs; in experiment 2, 14 of the DNAs from experiment 1 were used but they were amplified with the newly released kit (v2.0) and sequenced in two runs. In parallel, homopolymeric regions of all samples were studied with the BRCA HP kit. Thanks to the Training Set experiment, we were able to define an NGS workflow for the genetic analysis of BRCA genes in the HBOCS diagnostic routine. In the Validation Set, we assessed a total of 15 HBOC samples, 14 not previously tested and the remaining 1 containing a multi-exon duplication. These samples were analyzed in parallel with our current diagnostic workflow and with the newly designed NGS workflow. In this case, experiment 3 was carried out using the most recent version (v2.1) of the BRCA MASTR kit and samples were sequenced in three runs.

Figure 2

Proposed workflow for analyzing BRCA1 and BRCA2 using NGS. A screening using the BRCA HP kit (Multiplicom) allows detection of insertions or deletions located in homopolymers of 6 bp or longer and their surroundings. Sanger sequencing confirms any aberrant pattern found. Simultaneously, DNA samples are analyzed by NGS. BRCA1 and BRCA2 coding regions and their intron–exon boundaries are amplified using the BRCA MASTR kit (Multiplicom), adding specific identifiers (MIDs) for each sample to pool them. Sequencing of the enriched regions from pooled samples is performed by using 454 Titanium chemistry in a GS Junior platform (Roche). Data generated by the sequencer are analyzed using the public software VIP and R instructions, which allows us to align all of the sequences generated, trim the surrounding regions of each amplicon (adapters, MIDs and primers) and call putative variants. After filtering the initial variants with filters 1, 2, 3 and 4, a subset (variants with null forward or reverse coverage) is selected for visual inspection of their alignment with AVA, which will discard obvious FPs. All remaining variants are confirmed by Sanger sequencing. As our aim was to integrate this approach into the diagnostic routine, this revision was performed independently by two qualified technicians to generate a common list indicating the decision for any variant under analysis. If a discrepancy arose between the two referees, the most conservative decision was adopted. Regions with low coverage (<38 × ) are also Sanger sequenced.

Figure 3

Detection of LGRs using NGS results. Bar plots of the dose of NGS amplicons after normalization. X-axis: NGS amplicons. Y-axis: Count ratio minus 1. Fragments with normalized ratios above 1.3 and below 0.7 are highlighted in black, indicating putative duplications and deletions, respectively. (a) Control sample with no alterations. (b) Sample with a duplication of the region comprising BRCA1 exons 9–24. (c) Sample with a deletion comprising BRCA2 exon 2. (d) Sample with a deletion comprising exon 20 of BRCA1.