Abstract P4-12-12: Next generation sequencing-based analysis of BRCA1 and BRCA2 genes: Applicability for fast diagnostics of large samples

Abstract

Abstract Purpose BRCA1&2 gene mutation test for hereditary breast ovarian syndrome (HBOC) has been conducted mostly by Sanger sequencing. Currently, the next generation sequencing (NGS) is rapidly incorporated to the fields of cancer research and clinical diagnostics. Here we evaluated NGS-based results of BRCA gene analysis and compared with the results of Sanger sequencing for future diagnostic applications. Methods The patients (n=100) who have genetic counseling and decided to perform BRCA test were included. All coding regions of BRCA1 and BRCA2 analyzed by both of Sanger sequencing and NGS using Access Array BRCA1, BRCA2 and TP53 kit (Fluidigm, USA). Access array kit was designed to multiplex 48 samples simultaneously for 184 amplicons and the average sequencing depth per base was 6,500X using MiSeq sequencer (Illumina, USA). We developed analysis pipelines to avoid false negative results and detect all pathogenic mutations. The reads were aligned to a reference genome (NCBI human genome assembly build 37) using the BWA-MEM, then all candidate variants called with minimum filtering parameter. Results Total of 765 variants were detected by NGS and among them 616 variants (frameshift:22, nonsense:8, splicing:3, missense:290, and synonymous variants:293) were identical with the results from Sanger sequencing. When we evaluated the results of Sanger sequencing as standard methods, the mean allele frequency showed difference as 41.7% and 12.0% for true positive heterozygous variants (616) and false positive variants (149), respectively. Conclusions There was no false negative of pathogenic mutations from NGS. The BRCA mutation detection using NGS represented potential applicability in clinical diagnosis. Citation Format: Sun-Young Kong, Eunhae Cho, Junnam Lee, Myong Cheol Lim, Jahyun Jang, Boyoung Park, Kyong-Ah Yoon, Young-Ho Kim, Eun Sook Lee. Next generation sequencing-based analysis of BRCA1 and BRCA2 genes: Applicability for fast diagnostics of large samples [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-12.