Abstract 4851: A single-tube protocol for next gen library construction increases complexity and simplifies parallel sample handling

Abstract

Abstract With the advent of second-generation sequencing technologies, exome- and transcriptome- and whole genome sequencing are tools of choice in studying cancer to detect alterations in the genome correlating with tumorigenesis. It is essential to compare genomic alterations in cancer with matched normal DNA from the same individual largely due to incomplete knowledge of the “normal” variations. There have been concerted efforts for targeted sequencing of cancer-related exomes in a high throughput manner. However, the needs to process multiple samples simultaneously while maintaining the complexity of libraries have not been addressed adequately. Fragment library generation for single- and paired-end reads is typically performed by a series of enzymatic steps following gDNA shearing by ultrasonication. These may include end-repair, A-tailing, and ligation, each followed by bead- or column-based purification. The clean-ups are necessary to prevent enzyme activity carryover from one step to another. We have developed a single-tube method to take sheared, bead size-selected DNA through end-repair, A-tailing, and ligation without intermittent purifications. The A-tailing is done by a thermophilic polymerase at a temperature sufficient to denature the end-repair enzymes. Ligation is performed by adding ligase and adaptors after cooling the reaction. A bead- or column-based purification is then performed after the ligation reaction to produce template for amplification. With the removal of the clean-up steps, the workflow is simplified, allowing faster processing of samples with less hands-on time. We also reduce loss of sample associated with the purifications, increasing the complexity of the output library or enabling a lower sample input concentration. Our method is well suited for labs which are producing multiple libraries each week but have not scaled up to full automation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2011-4851