Abstract LB-412: TargetRich cancer gene panels: targeted next generation sequencing in cancer samples

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Crucial to the adoption of next-generation sequencing within clinical cancer research is the ability to deeply sequence hundreds to thousands of targeted genomic regions across a large number of patient samples. Several targeting (PCR) and capture (hybridization) methods have been developed for next-generation sequencing, however these methods have limited utility with cancer patient specimens due to drawbacks such as large input DNA quantity requirements, wasted off-target sequencing, high cost of reagents, low sample throughput, specialized equipment, and inflexible content design. We developed TargetRich cancer gene panels employing Nested PatchPCR, which enables the targeted sequencing of hundreds of genomic regions in large numbers of patient samples to fully and efficiently utilize next-generation sequencers. Nested PatchPCR required only 250 ng genomic DNA, and was compatible with the fragmented DNA obtained from formalin fixed paraffin embedded samples (FFPE). No special equipment was needed, only a standard PCR machine. Patient samples were processed in parallel in a single 96-well plate. For each patient sample, we simultaneously amplified hundreds of exons from genes that are frequently mutated in cancer. We utilized standard library construction and barcoding methods to sequence targeted genes from 96 patient samples on a single Illumina GAIIx flowcell. The TargetRich cancer gene panels were highly specific; 95% of sequencing reads aligned to the targeted regions. The high percentage of on-target sequence data and focused content, allowed us to achieve high coverage across the loci (average of 400X read depth) for the sensitive detection of cancer mutations. We have created two cancer panels: CR (157 regions covering exons from 10 genes) and CS (760 regions covering exons from 62 genes) for use on the Illumina MiSeq, GAIIx & HiSeq platforms. Additionally, a CR panel for the Ion Torrent PGM has been developed with platform-specific primers added during the Nested PatchPCR reaction, allowing direct sequencing of products without time-consuming library construction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-412. doi:1538-7445.AM2012-LB-412