Rapid and cost-effective high-throughput sequencing for identification of germline mutations of BRCA1 and BRCA2

Genetic testing for breast cancer predisposing genes, brca1 and brca2, can take advantage for early identification of carriers with pathogenic germline mutations. however, conventional approaches based on sanger sequencing are laborious and expensive. next-generation sequencing technology has a great impact on investigation of medical genomics and now applied clinical genetics. we provide a protocol based on a pool and capture method followed by high-throughput sequencing, which realizes a rapid, high-quality, high-accuracy and low-cost testing for mutations in brca1 and brca2 by using small amounts of input dna. custom capture probes were designed for 195 kb regions encompassing the entire brca1 and brca2. dna libraries of 96 samples with distinct indices were pooled before hybridizing to the capture probes, which largely reduced labor and cost. the captured library was run on the illumina miseq sequencer. we applied the method to 384 japanese individuals including 11 patients with breast cancer whose mutation statuses had been determined by standard clinical testing and 373 individuals from a general population. 99.99% of coding exons and their 20 bp flanking regions were covered with a minimum of 20 reads and the average depth was 179.5, supporting confident variant detection. the sequencing method rendered concordant results for 11 patients with breast cancer compared with the standard clinical testing including nine mutations in eight patients. among 373 individuals from the general population, novel stop gain and frameshift deletion in brca2 were identified, which led to truncated protein and were most likely to be pathogenic. the result suggests the importance of a large-scale population-wide screening for carriers of mutations in these genes.