Abstract 5365: Combining enzymatic DNA fragmentation with NGS library construction results in high quality, high yield libraries

Abstract

Abstract The use of Next Generation Sequencing (NGS) data has been instrumental in advancing our understanding of human genetics, identifying the molecular events that contribute to human disease, and supporting drug development targeted towards precision medicine. Continued advancement relies on overcoming the limitations and bottlenecks associated with NGS. In this work, we have focused on NGS library preparation, where the requirement for expensive equipment and numerous steps can lead to sample loss, errors, and limited throughput. Specifically, we have developed a library construction method that integrates enzymatic DNA fragmentation into the workflow and combines fragmentation with end repair and dA-tailing in a single step. Integrating these reactions eliminates the need for costly equipment to shear DNA and reduces the number of sample transfers and losses. Adaptor ligation is also carried out in the same tube, after which a single cleanup step is performed. For low input samples, PCR amplification is performed prior to sequencing. This method is compatible with a broad range of DNA inputs and insert sizes. Libraries generated using this streamlined method with inputs ranging from 500 pg to 500 ng of intact DNA show no significant difference in coverage uniformity or sequence quality metrics, compared to libraries generated with mechanically sheared DNA. Similarly, libraries generated to contain insert sizes that range from 150bp to 1kb display no significant difference in sequence quality from each other or from those generated with mechanically sheared DNA. Finally, this streamlined method generates libraries of substantially higher yields than those generated using mechanically fragmented DNA, allowing the use of lower DNA inputs and fewer PCR cycles. The ability to generate high quality NGS libraries from intact DNA without the need for costly equipment and numerous cleanup or liquid transfer steps substantially reduces the time, cost and errors associated with library construction. In addition, these advances will enable greater use and adoption of NGS technologies in clinical and diagnostic settings. Citation Format: Fiona Stewart, Lynne Apone, Vaishnavi Panchapakesa, Karen Duggan, Timur Shtatland, Bradley Langhorst, John Murdoch, Christine Sumner, Christine Rozzi, Pingfang Liu, Keerthana Krishnan, Deyra Rodriguez, Joanna Bybee, Danielle Rivizzigno, Laurie Mazzola, Eileen Dimalanta, Theodore Davis. Combining enzymatic DNA fragmentation with NGS library construction results in high quality, high yield libraries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5365. doi:10.1158/1538-7445.AM2017-5365