Table 1.
High-risk hereditary breast cancer genes analyzed on the BRCAPlus panel.
Figure 1.
Schematic diagram of BRCAplus assay workflow.
Figure 2.
Redundant primer tiling design limits allele drop-out.
A) A typical primer design with one to two amplicons covering region of interest. A polymorphism under the primer of amplicon one would result in allele drop-out and a false negative. B) BRCAplus tiling primer design has overlapping amplicons designed over region of interest. The same polymorphism in amplicon one would not result in false negative due to amplicon redundancy of amplicon two.
Figure 3.
Primer sequence trimming increases detection sensitivity.
Two heterozygous causative variants A) BRCA1 c.3671_3672insCTTC and B) BRCA2 c.2918C>A that were missed without primer trimming were detected with the function enabled in the pipeline. Both calls were confirmed with Sanger sequencing.
Figure 4.
Microarray has exon level resolution for deletion and duplication variant detection.
A sample harboring a 2 Kb exon 13 deletion in BRCA2 was detected using the custom designed microarray.
Table 2.
Validation of BRCAplus deleterious variant detection on previously characterized samples.
Figure 5.
Average depth of coverage for each exon on the test from 3,000 representative samples.
The red line indicates the 50X coverage threshold in which any region with insufficient coverage would be Sanger sequenced.
Figure 6.
Heterozygous read ratio vs. read coverage.
To identify the profile of false positives, sequencing coverage was plotted against the heterozygous read ratios using Sanger sequencing confirmed NGS variants. Green circle, Sanger confirmed variants. Red triangle, Sanger cleared false positive.
Figure 7.
Distribution of pathogenic mutations reported out in first 3,000 patient samples referred for BRCAplus testing.
Figure 8.
Causative heterozygous mutations, which had previously gone undetected, identified with BRCAplus assay.
A) Mosaic pathogenic mutation c.5583delA in BRCA2 was detected at allele ratio of 14.9% and confirmed by Sanger sequencing. B) A heterozygous splice site mutation c.1909+1 G>A in BRCA2, was detected by BRCAplus assay.