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

摘要: Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian breast cancer. These genes can also be involved the development non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found some these cancers. Since patients with BRCA may benefit from treatment poly ADP ribose polymerase inhibitors, it is important able test for changes routinely available tumour samples. Such samples typically formalin-fixed paraffin-embedded (FFPE) tissue, where extracted DNA tends highly fragmented and limited quantity, making analysis large such challenging. This made more difficult evident only part sample, due presence normal tissue. We examined feasibility analysing FFPE tissue identify significant BRCA1/ using next generation sequencing methods that were sensitive enough detect low level mutations, multiplexed reduce amount required had short amplicon design. The utility two GeneRead DNAseq Targeted Exon Enrichment Panels different designs targeting exons, Ion AmpliSeq community panel, followed by library preparation adaptor ligation TruSeq PCR-Free HT Sample Preparation Kit NGS on MiSeq investigated. Using method, we successfully analysed over 76% samples, >95% coverage coding regions mean average read depth >1000-fold. All identified confirmed possible Sanger replication eliminate risk false positive results artefacts within material. Admixture experiments demonstrated could detected if present >10% sample. A sample subset was evaluated achieving >99% sufficient proportion Detection fixed feasible, performed prospectively facilitate optimum decisions cancer patients.