title: Evaluation of an amplicon-based next-generation sequencing panel for detection of BRCA1 and BRCA2 genetic variants.
abstract: The recent advances in the next-generation sequencing (NGS) technology have enabled fast, accurate, and cost-effective genetic testing. Here, we evaluated the performance of a targeted NGS panel for BRCA1/2 sequencing and confirmed its applicability in routine clinical diagnostics. We tested samples from 88 patients using the TruSeq custom panel (Illumina Inc, USA) and a MiSeq sequencer (Illumina) and compared the results to the outcomes of conventional Sanger sequencing. All 1015 sequence variations identified by Sanger sequencing were detected by NGS, except for one missense variant that might have been missed due to a rare mutation on a primer-binding site. One deletion variation, c.1909 + 12delT of BRCA2, was falsely called in all samples due to a homopolymer error. In addition, seven different single-nucleotide substitutions with low variant frequencies (range: 16.2-33.3 %) were falsely called by NGS. In a separate batch, 10 different false-positive variations were found in five samples. The overall sensitivity and positive predictive value of NGS were estimated to be 99.9 and 87.5 %, respectively. The false-positive results could be excluded by setting quality and alternative allele ratio filters and/or by visual inspection using the IGV software. Targeted NGS panel for BRCA1 and BRCA2 showed an excellent agreement with Sanger sequencing results. We therefore conclude that this NGS panel can be used for routine diagnostic method in a clinical genetic laboratory.
authors.affiliation: Department of Laboratory Medicine, Hallym University College of Medicine, Seoul, Korea.
authors.forename: Saeam
authors.initials: S
authors.lastname: Shin
authors.affiliation: Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea.
authors.forename: In Sik
authors.initials: IS
authors.lastname: Hwang
authors.affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, Korea. LEE.ST@yuhs.ac.
authors.forename: Seung-Tae
authors.identifier: 0000-0003-1047-1415
authors.initials: ST
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, Korea. CJR0606@yuhs.ac.
authors.forename: Jong Rak
authors.initials: JR
authors.lastname: Choi
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D055088:Early Detection of Cancer; D005069:Evaluation Studies as Topic; D005189:False Positive Reactions; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D009154:Mutation; D011237:Predictive Value of Tests; D056910:Republic of Korea; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human
keywords: BRCA1; BRCA2; MiSeq; Next-generation sequencing; Targeted sequencing; TruSeq