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Eric van der Walt, Maryke Appel, Gavin Rush, John Foskett, Paul McEwan Kapa Biosystems, Woburn, MA, USA |
Abstract
Dramatic improvements in commercial Next Generation Sequencing (NGS) technologies have resulted in spectacular reductions in the cost-per-base of DNA sequencing. Until recently, the primary focus for innovation has been on improvements to the core sequencing technologies, with optimization of sample preparation playing a secondary role. The exponential gains in sequencing capacity have simultaneously led to higher sample throughput, placing increasing emphasis on the importance of improved library construction protocols for multiplexed sample sequencing and automation.
We have applied directed evolution and enzyme engineering to develop novel DNA polymerases that offer unique advantages for NGS library construction and quality control. We have employed these novel enzymes in optimized, automated, high throughput library construction and quality control protocols to produce high quality NGS libraries in a wide variety of applications and contexts.
We and others have previously reported on the advantages of an engineered, high-fidelity polymerase for library amplification, which enables improved library quality and sequence coverage. To extend the benefits of low bias amplification, particularly to low-input applications, we recently developed a library construction protocol that incorporates the “with-bead” strategy conceptualized by The Broad Institute of MIT and Harvard. Together with the use of ultra-pure, high quality reagents for library construction, the “with-bead” protocol results in significantly higher recoveries of adapter-ligated molecules from lower amounts of input DNA. These benefits allow for fewer cycles of amplification, thereby further reducing the risk of PCR-induced bias, error and other artifacts that can affect library quality, sequence coverage and reliable library quantification.
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