Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations

Abstract

This study describes how the new massive parallel sequencing technology can be implemented in a diagnostic setting for the breast cancer susceptibility genes (BRCA1 and BRCA2). The throughput was maximized by increasing uniformity in coverage, obtained by a multiplex approach, which outperformed pooling of singleplex PCRs. We evaluated the sensitivity by analysis of 133 distinct sequence variants; three (2%) deletions or duplications in homopolymers of greater than or equal to seven nucleotides remained undetected, illustrating a limitation of pyrosequencing. Furthermore, other limitations like nonrandom sequencing errors, pseudogene amplification, and failure to detect multiexon deletions are thoroughly described. Our workflow illustrates the potential of massive parallel sequencing of large genes in a diagnostic setting, which is of great importance to meet the increasing expectations of genetic testing. Implementation of this approach will hopefully lead to a strong reduction in turnaround times. As a consequence a wider spectrum of at risk women will be able to benefit from therapeutic interventions and prophylactic interventions.

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

“…This result outperforms the homogeneity previously reported by other groups describing next-generation BRCA testing using either longrange PCR, 4 primer-specific direct capture for single-molecule sequencing, 13 or in-house single/multiplex PCR. 12,14 It is also important to note that all of the MIDs used in the present study showed similar coverage results. Overall, this commercial assay allows the generation of a robust library for all the patients under study, maximizing the number of samples analyzed in a run.…”

“…The main disadvantage of pyrosequencing relative to other NGS technologies is the accuracy of length determination in homopolymers. 12,17,21 In pyrosequencing, the light-intensity signal observed in each cycle is proportional to the actual number of incorporated nucleotides, which is the base for homopolymer length calling. The accuracy of this method decreases with homopolymer length, which may eventually generate artefactual insertions and deletions in long homopolymers.…”

“…The systematic application of a set of Next-generation sequencing and BRCAs diagnosis L Feliubadaló et al evaluated filters is needed. 12 Ours is a four-filter approach: three run automatically and a fourth filter generates a list of variants that require visual examination or Sanger confirmation. Visual examination took about 3 h per run per revisor, and both revisions provided concordant results.…”

“…The fourth filter was able to remove a substantial proportion of the FPs without losing any TP when compared with other series. 12 The use of the commercial homopolymer kit was paramount for correctly reading sequences containing homopolymer stretches, which often require visual inspection and/or Sanger sequencing. Nevertheless, further development of tools for analysis of HP regions in NGS is needed to improve performance and to reduce the number of results requiring visual inspection.…”

“…10,11 The development of cost-effective BRCA mutation detection workflows will not only benefit the genetic counseling process for patients with HBOCS but will also enhance the process of selecting patients for personalized treatments, as could be the case of PARP inhibitors, for example. Mutation analyses of BRCA1 and BRCA2 using NGS have been already performed for high-capacity NGS platforms, such as the 454 FLX (Roche), 12 the Helicos (Heliscope), 13 the Genome Analyzer (Illumina) 4 and, very recently, the GS Junior instrument. 14 Most of these studies used large-capacity platforms that generally exceed the demand of most mid-sized genetic testing laboratories and whose approaches are difficult to translate to benchtop next-generation sequencers.…”