Nanostructured Magnetic Disks for Wide Range Size Selection in NGS Library Preparation and PCR Purification

Information

  • Research Project
  • 9892018
  • ApplicationId
    9892018
  • Core Project Number
    R44GM134994
  • Full Project Number
    5R44GM134994-03
  • Serial Number
    134994
  • FOA Number
    PA-18-574
  • Sub Project Id
  • Project Start Date
    8/18/2017 - 7 years ago
  • Project End Date
    3/31/2021 - 3 years ago
  • Program Officer Name
    KREPKIY, DMITRIY
  • Budget Start Date
    4/1/2020 - 4 years ago
  • Budget End Date
    3/31/2021 - 3 years ago
  • Fiscal Year
    2020
  • Support Year
    03
  • Suffix
  • Award Notice Date
    3/20/2020 - 4 years ago
Organizations

Nanostructured Magnetic Disks for Wide Range Size Selection in NGS Library Preparation and PCR Purification

Project Summary 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.

IC Name
NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
  • Activity
    R44
  • Administering IC
    GM
  • Application Type
    5
  • Direct Cost Amount
  • Indirect Cost Amount
  • Total Cost
    737385
  • Sub Project Total Cost
  • ARRA Funded
    False
  • CFDA Code
    859
  • Ed Inst. Type
  • Funding ICs
    NIGMS:737385\
  • Funding Mechanism
    SBIR-STTR RPGs
  • Study Section
    ZRG1
  • Study Section Name
    Special Emphasis Panel
  • Organization Name
    CIRCULOMICS, INC.
  • Organization Department
  • Organization DUNS
    830377581
  • Organization City
    Baltimore
  • Organization State
    MD
  • Organization Country
    UNITED STATES
  • Organization Zip Code
    212023183
  • Organization District
    UNITED STATES