3rd generation and linked read sequencing technologies from PacBio, Oxford Nanopore, and 10X Genomics have begun revolutionizing an array of genomics applications, including de novo sequencing, phasing, and structural variant analysis, by offering the ability to sequence long stretches of DNA spanning hundreds of kilobases to megabases in length. However, these capabilities are predicated on the ability to efficiently manipulate high molecular weight (HMW) DNA. Not only must HMW DNA be extracted, it is equally important that it is not damaged or lost during subsequent library preparation. Sequencing library preparation typically requires 3 - 4 size selection purification steps to cleanup enzymatic reactions and to ensure that only properly prepared library molecules of the desired insert size are sequenced. In Phase I, we demonstrated that Nanobind Size Select can enhance long-read sequencing read lengths while dramatically speeding library preparation and improving library yields. The proposed Nanobind Size Select technology is the only method that can offer the speed of magnetic particles with the high cutoffs of gel purification. Rather than millions of individual microparticles, Nanobind uses a solid magnetic disk (1 - 6 mm dia.) layered with a high density of micro- and nanostructured silica. The unique disk format protects DNA from shear damage resulting in greater numbers of very long reads (>100 kb). It also achieved a 10X wider range of cutoffs (100 bp ? 10 kb) than any magnetic particle or column method. At the same time, Nanobind?s high purification efficiency results in up to 19X less adapter dimer contamination, 5X higher recovery of HMW DNA, and 8.5X faster process time. In Phase II, we will create a single, unified kit capable of size selection purification of high MW gDNA libraries, fragmented gDNA libraries, and PCR amplicon libraries. This kit will address a broader range of 3rd generation sequencing applications while allowing future extension to short-read NGS applications as well. First, we will develop Nanobind Size Select chemistries for HMW gDNA libraries that have even higher size selection cutoffs and require lower input concentrations than in Phase I. Then, we will optimize the chemistries to purify fragmented DNA and PCR amplicon libraries. Finally, we validate Nanobind Size Select by comparing against existing commercial products using long-read read sequencing.

Public Health Relevance

Over the next decade, next generation sequencing is expected to have tremendous impact on human health through new diagnostic tests, better therapeutic targeting, and even improved crop yields. New purification methods for sequencing library preparation have the potential to reduce sequencing cost by reducing sample and reagent requirements, speeding processing time, and improving overall sequencing data quality.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1)
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Krepkiy, Dmitriy
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Circulomics, Inc.
United States
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