Breast Cancer Research and Treatment

, Volume 158, Issue 3, pp 433–440 | Cite as

Evaluation of an amplicon-based next-generation sequencing panel for detection of BRCA1 and BRCA2 genetic variants

  • Saeam Shin
  • In Sik Hwang
  • Seung-Tae Lee
  • Jong Rak Choi
Preclinical study


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.


Next-generation sequencing BRCA1 BRCA2 TruSeq MiSeq Targeted sequencing 



This study was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2043879), and by a grant from the National Research Foundation of Korea (NRF-2015R1C1A2A01055967).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Laboratory MedicineHallym University College of MedicineSeoulKorea
  2. 2.Brain Korea 21 PLUS Project for Medical ScienceYonsei UniversitySeoulKorea
  3. 3.Department of Laboratory MedicineYonsei University College of MedicineSeoulKorea

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