A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing

Table 1 BRCA panel specifications

Panel name	Design coverage*	No. amplicons	Mean amplicon size (bp) ⁺	Amplicon size range (bp) ⁺	No. primer pools	DNA input recommendation
GeneRead V.1 BRCA1/2	97%	276	155 (104)	(58–125)	4	80 ng total, 20 ng/pool (determined by OD260)
GeneRead V.2 BRCA1/2	100%	237	153 (109)	105-200 (52–159)	4	80 ng total, 20 ng/pool (determined by OD260)
Ion AmpliSeq BRCA1/2	100%	167	197 (145)	126-298 (71–242)	3	30 ng total, 10 ng/pool (determined by qPCR)

*The minimal target regions for 100% design coverage for this assessment was defined as 100% coverage of the BRCA1 and BRCA2 CDS (NM_007294.3 and NM_00059.3), respectively, plus at least the 2 bp flanking each coding exon (canonical splice sites). The lengths of these features across the two genes were calculated as 5,680 bp and 10,361 bp for BRCA1 and BRCA2, respectively (total 16,041 bp). Although the designs in many cases covered sequence further into intronic regions, these were not considered in this calculation.
⁺Amplicon size ranges are quoted as length with primer sequences, where known, and length without primer sequences in parentheses.

ISSN: 1472-6890