Abstract
Background: Target enrichment coupled with next generation sequencing provide high-throughput approaches for screening several genes of interest. These approaches facilitate screening a panel of genes for mutations associated with inherited breast cancer for research, diagnostic, and genetic counseling applications.
Objective: To evaluate the performance of our custom 13 gene breast cancer panel, based on singleplex PCR, developed by WaferGen BioSystems. The panel was evaluated using patient-derived DNA samples, in terms of target enrichment efficiency, off-target enrichment, uniformity of target capture, effect of GC content of target regions on coverage depth, and concordance with validated variant calls.
Results: At least 90% of target sequence for each gene was captured at 30x or greater. We evaluated uniformity of target capture across samples by calculating the percentage of samples with at least 90% of total target captured at 100x or greater and found 92% (33/36 samples) uniformity for our panel. Off-target enrichment ranges between 7.2% and 22.3%. We found perfect concordance between our custom panel and the Qiagen human breast cancer panel for functionally annotated variant calls in high read depth shared target regions. Altogether, there was agreement between the panels for 779 variants at 41 loci. We also confirmed 10 pathogenic mutations, initially discovered by Sanger sequencing, in the appropriate samples following target enrichment using our custom WaferGen panel.
Conclusion: Our custom hereditary breast cancer panel is sensitive to the desired target genes and facilitates deep sequencing for reliable variant calling.
