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.