Novel enzymatic DNA fragmentation integrated into library construction

Next Generation Sequencing (NGS) is rapidly becoming a powerful tool, by providing a high resolution and global view of the cancer genome mainly through whole genome, whole exome and transcriptome sequencing. This knowledge is important to support drug development targeted towards precision medicine. Obtaining high quality sequencing data begins with optimal sample preparation. DNA fragmentation is one of the critical first steps in the construction of high quality libraries for NGS, but current fragmentation methods create a bottleneck on library preparation throughput. We have developed an efficient, one tube fragmentation system that integrates enzyme-based DNA fragmentation with end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube. This streamlined method is compatible with a wide range of DNA samples and input amounts, resulting in reproducible and reliable fragment sizes. Genomic DNA (100pg to 500ng) with various GC content, was used in the construction of libraries, that were sequenced on an Illumina platform. For low input samples, PCR amplification was performed prior to sequencing. Libraries constructed using intact genomic DNA and this novel library preparation method produced substantially higher yields than standard methods and mechanically-fragmented DNA. Enzyme-based shearing increases library yield by reducing DNA damage and sample loss. High library yields also enable PCR-free workflows from as little as 50ng starting material. This method can be used for DNA extraction and library preparation from whole blood and dried blood samples.Sequencing quality of libraries generated with inputs ranging from 100pg to 500ng DNA show similar coverage uniformity and fragment size distribution, with minimal GC bias. We have developed an enzymatic DNA fragmentation kit that enables the construction of high quality libraries from a range of sample types and inputs with minimal sequence bias, high yields and low duplication rates. This method eliminates the need for expensive equipment to fragment DNA as well as numerous cleanup and liquid transfer steps thereby reducing the time, cost and errors associated with library construction. These advances in NGS technologies, supported by powerful bioinformatics tools, promises to revolutionize cancer research, diagnosis and therapy. Citation Format: Vaishnavi Panchapakesa, Lynne Apone, Karen Duggan, Bradley Langhorst, Chen Song, Pingfang Liu, Timur Shtatland, Christine Rozzi, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Novel enzymatic DNA fragmentation integrated into library construction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3528.