Sequencing

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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting THE IMPACT OF ENZYMATIC FRAGMENTATION AND LIMITED LIBRARY AMPLIFICATION ON DATA QUALITY FOR HUMAN WHOLE-GENOME LIBRARIES SEQUENCED ON THE ILLUMINA HISEQ X Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.07) Maryke Appel 1 , Beverley Van Rooyen 1 , Jacqueline Meyer 1 , Penny Smorenburg 1 , Lisa Cook 2 , Catrina Fronick 2 , Vincent Magrini 2 , Robert Fulton 2 1 Roche Diagnostics, 2 Washington University Illumina’s HiSeq X platform is making the “$1,000 genome” and large‐scale human whole‐genome sequencing (WGS) a reality. Robust, high‐throughput pipelines for the production of high‐quality human genomic shotgun libraries will be needed to sustain the predicted global upsurge in human WGS research. PCR‐free library construction workflows are accepted to be the gold standard for these projects, but pose several challenges—including low and variable library yields from limited amounts of input DNA, and library stability issues. In this study we optimized library construction parameters for libraries to be sequenced on the HiSeq X; using the KAPA Hyper Prep Kit (which requires Covaris shearing), or with the KAPA HyperPlus Kit with integrated enzymatic fragmentation. Different size selection and cleanup strategies were evaluated to achieve optimal library fragment sizes (350 370 bp) and yields ( 2 nM); and minimize adapter‐dimer carry‐over. To address many of the pain points associated with high‐throughput PCR‐free workflows, we performed an amplification titration (0, 2, 4 or 6 cycles) of NA12878 libraries prepared with optimized KAPA Hyper Prep and KAPA HyperPlus protocols, and sequenced these libraries to ~30X cov‐ erage (1 library per lane equivalent). The impact of enzymatic fragmentation and low levels of PCR amplification on library QC metrics, library complexity, coverage uniformity and variant call statistics—and the implications for high‐throughput library construction pipelines for human WGS research will be discussed. Products are for life science research use only, not for use in diagnostic procedures. 73
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting A ROBUST, STREAMLINED, ENZYMATIC DNA FRAGMENTATION AND NGS LIBRARY CONSTRUCTION METHOD Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.08) Fiona Stewart Lynne Apone Pingfang Liu Vaishnavi Panchapakesa Christine Sumner Christine Rozzi Deyra Rodriguez Karen Duggan Keerthana Krishnan Bradley Langhorst Joanna Bybee Laurie Mazzola Danielle Rivizzigno Barton Slatko Eileen Dimalanta Theodore Davis New England Biolabs, Inc. Sample preparation is quickly becoming a bottleneck in the Next generation sequencing (NGS) pipeline. While NGS libraries can be multiplexed, combined and sequenced together on a single lane, or chip, generating individual libraries is tedious and time consuming. In addition, the multiple steps required to construct a library provide numerous opportunities for errors and sample loss. In order to increase the throughput of library construction, reduce errors and sample loss, we have developed a robust, flexible, enzymatic fragmentation method. Fragmentation can be combined with end repair and dA‐tailing in a single step, or performed independently, to allow use in any NGS platform and optimization prior to library construction. This method is compatible with a broad range of DNA inputs and insert sizes. Libraries generated using this enzymatic fragmentation method with 5 ng and 100 ng of intact DNA show no significant difference in coverage uniformity or distribution from libraries generated with mechanically sheared DNA. Likewise, libraries generated to contain 200 bp and 900 bp inserts show no significant difference in sequence quality from each other or those generated with mechanically sheared DNA. Interestingly and importantly, enzymatic fragmentation generates libraries of substantially higher yields (2‐3 fold) than those generated using mechanically fragmented DNA. The ability to generate high quality NGS libraries from intact DNA without the need for numerous cleanup and liquid transfer steps will substantially reduce the time, cost and errors associated with library construction. In addition, these advances will permit greater use and adoption of NGS technologies into many scientific arenas. 74
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Reliable solutions for focused NGS
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Sequencing

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