Abstract 3566: A robust and rapid targeted sequencing technology for iterative multiple genomic features in cancer

Abstract

Abstract Targeted resequencing has enabled researchers and clinicians to analyze specific genomic regions of interest from cancer. For example, deep sequencing of cancer genes has allowed for the sensitive detection of rare somatic variants as well as oncogenic rearrangements that are otherwise difficult to observe with shotgun resequencing. Deep targeted resequencing of these regions can potentially provide prognostic information as well as guide treatment regimens. Current targeted resequencing methods, however, suffer from poor on-target performance, slow capture rates on the order of days, and significant manual effort. Existing commercial kits are extremely restricted for targeting custom regions of interest. Furthermore, libraries prepared for targeted resequencing purposes generally cannot be reused, thus limiting the number of replicates and follow-up experiments that can be performed for cancer samples. We present APEX (attachment-based porous extension) capture, a platform that rapidly and robustly generates targeted DNA fragments for next-generation sequencing on the Illumina platform in as little as two hours. It is based on the principle that chemical reactions are rapidly enhanced in high surface-to-area ratio microenvironments when one reaction component is surface-bound. As a proof of concept, we covalently tethered next-generation sequencing compatible DNA libraries to a silica membrane found in conventional spin columns, performed target-specific primer extension, and eluted them for next-generation sequencing. Through serial dilutions of cytomegalovirus (CMV) and human DNA admixtures, we demonstrate CMV target enrichment down to single genome equivalents. In addition, using a tiling primer strategy we were able to robustly capture and resequence entire bacterial genomes. Because our targeted resequencing technology preserves the original sample library, we can sequentially target different regions of interest in a series of guided experiments. We performed multi-tiered analyses of colon biopsies, where we deep resequence cancer genes as well as survey fro cancer-related pathogens such as bacterial genomes that are co-purified in the sample. APEX capture therefore enables researchers to aggressively and rapidly characterize cancer genomic material, where large-scale targeting panels are followed by precise discriminatory ones that can guide diagnosis and treatment. Citation Format: Billy Lau, Anna Cushing, Hanlee Ji. A robust and rapid targeted sequencing technology for iterative multiple genomic features in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2014-3566