Deep Sequencing of Target Linkage Assay-Identified Regions in Familial Breast Cancer: Methods, Analysis Pipeline and Troubleshooting

BACKGROUND: The classical candidate-gene approach has failed to identify novel breast cancer susceptibility genes. Nowadays, massive parallel sequencing technology allows the development of studies unaffordable a few years ago. However, analysis protocols are not yet sufficiently developed to extract all information from the huge amount of data obtained. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed high throughput sequencing in two regions located on chromosomes 3 and 6, recently identified by linkage studies by our group as candidate regions for harbouring breast cancer susceptibility genes. In order to enrich for the coding regions of all described genes located in both candidate regions, a hybrid-selection method on tiling microarrays was performed. CONCLUSIONS/SIGNIFICANCE: We developed an analysis pipeline based on SOAP aligner to identify candidate variants with a high real positive confirmation rate (0.89), with which we identified eight variants considered candidates for functional studies. The results suggest that the present strategy might be a valid second step for identifying high penetrance genes.