Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis

Abstract

Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing.
Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method.
Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1.
Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.

Keywords

BRCA1 / BRCA2 / massively parallel sequencing / NGS / Multiplicom

References

[1] Chen S, Parmigiani G.Meta-analysis of BRCA1 and BRCA2 penetrance. J Clin Oncol 2007;25(11):1329-1333

[2] Faraoni I, Graziani G.Role of BRCA mutations in cancer treatment with poly(ADP-ribose) polymerase (PARP) inhibitors. Cancers (Basel) 2018;10(12):487

[3] González-Santiago S, Ramón Y, Cajal T, Aguirre E, et al; SEOM Hereditary Cancer Working Group. SEOM clinical guidelines in hereditary breast and ovarian cancer (2019). Clin Transl Oncol 2020;22(02):193-200

[4] Toland AE, Forman A, Couch FJ, et al; BIC Steering Committee. Clinical testing of BRCA1 and BRCA2: a worldwide snapshot of technological practices. NPJ Genom Med 2018;3:7

[5] Zook JM, Catoe D, McDaniel J, et al. Extensive sequencing of seven human genomes to characterize benchmark reference materials. Sci Data 2016;3:160025

[6] Richards S, Aziz N, Bale S, et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17(05): 405-424

[7] Castilla LH, Couch FJ, Erdos MR, et al.Mutations in the BRCA1 gene in families with early-onset breast and ovarian cancer. Nat Genet 1994;8(04):387-391

[8] Simard J, Tonin P, Durocher F, et al.Common origins of BRCA1 mutations in Canadian breast and ovarian cancer families. Nat Genet 1994;8(04):392-398

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