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.