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Comparative Study
. 2013 Nov;15(6):796-809.
doi: 10.1016/j.jmoldx.2013.07.004. Epub 2013 Oct 4.

A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations

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Comparative Study

A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations

Ian E Bosdet et al. J Mol Diagn. .
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Erratum in

  • J Mol Diagn. 2014 May;16(3):378

Abstract

Individuals who inherit mutations in BRCA1 or BRCA2 are predisposed to breast and ovarian cancers. However, identifying mutations in these large genes by conventional dideoxy sequencing in a clinical testing laboratory is both time consuming and costly, and similar challenges exist for other large genes, or sets of genes, with relevance in the clinical setting. Second-generation sequencing technologies have the potential to improve the efficiency and throughput of clinical diagnostic sequencing, once clinically validated methods become available. We have developed a method for detection of variants based on automated small-amplicon PCR followed by sample pooling and sequencing with a second-generation instrument. To demonstrate the suitability of this method for clinical diagnostic sequencing, we analyzed the coding exons and the intron-exon boundaries of BRCA1 and BRCA2 in 91 hereditary breast cancer patient samples. Our method generated high-quality sequence coverage across all targeted regions, with median coverage greater than 4000-fold for each sample in pools of 24. Sensitive and specific automated variant detection, without false-positive or false-negative results, was accomplished with a standard software pipeline using bwa for sequence alignment and samtools for variant detection. We experimentally derived a minimum threshold of 100-fold sequence depth for confident variant detection. The results demonstrate that this method is suitable for sensitive, automatable, high-throughput sequence variant detection in the clinical laboratory.

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