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Advanced method for preparing cell-free DNA sequencing libraries

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 2R44HG009461-02A1
Agency Tracking Number: R44HG009461
Amount: $1,611,692.00
Phase: Phase II
Program: SBIR
Solicitation Topic Code: 172
Solicitation Number: PA19-272
Solicitation Year: 2019
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-08-01
Award End Date (Contract End Date): 2022-07-31
Small Business Information
Santa Cruz, CA 95060-5790
United States
DUNS: 013494781
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (831) 426-7700
Business Contact
Phone: (831) 426-7700
Research Institution

The goal of the proposed research is to develop a novel, advanced method for preparing libraries of cell-free
circulating DNA (cfDNA) for next-generation sequencing (NGS). cfDNAs found in blood and in most other
biofluids represent promising, minimally invasive diagnostics (“liquid biopsy”) for cancer, and cfDNA levels are
elevated in the plasma and serum of cancer patients. Although, even when cancer is present, the majority of
cfDNAs are derived from non-tumor cells, tumor DNA can be identified within cfDNA by characteristic alterations
in fragment size distribution and in genetic and epigenetic profiles. This circulating tumor DNA (ctDNA) is more
degraded than is cfDNAs from healthy individuals, with a substantial fraction of fragments being shorter than 100
bp. Analysis of tumor-specific characteristics of cfDNA, such as the amount of DNA, its level of fragmentation,
and the presence of mutations and methylated residues, can be utilized for cancer diagnosis, response to
treatment and prognosis. Due to the high frequency of single-strand nicks in ctDNA, short ctDNA fragments
cannot be efficiently incorporated into sequencing libraries prepared from non-denatured cfDNA by conventional
DNA-Seq methods. To overcome this problem, in Phase I we developed a novel proprietary method called
HASL-free-Seq for the preparation of sequencing libraries from ssDNA (and denatured dsDNA) that can
efficiently capture ultrashort cfDNAs in the 20 to 50 nt size range along with longer DNA fragments. We also
demonstrated that the proportion of ultrashort cfDNA fragments in plasma samples could provide robust
discrimination between healthy donor and breast cancer patients. In Phase II, we will optimize the HASL-free-
Seq protocol and kit for commercial viability. We will compare its performance with that of alternative methods,
including published “lab-brew” protocols, to document the advantages of our technology. Using a cohort of
plasma samples with matching clinical information, we will validate its ability to discriminate between healthy
donor and breast cancer patients based on tumor-specific ctDNA fragmentation patterns, mutation signatures
and methylation patterns. Upon the completion of Phase II, we plan to commercialize the HASL-free-Seq
technology through sales of library preparation kits as well as out-licensing and in partnership with established
reagent and molecular diagnostic companies as well as pharmaceutical companies interested in development
of companion diagnostics.Cell-free DNAs (cfDNAs), which are found in blood and in most other bodily fluids, represent promising minimally
invasive “liquid biopsy” samples for human cancer and prenatal diagnosis of fetal genetic diseases. Although
next-generation sequencing has great potential for analysis of cfDNA for cancer diagnosis, prognosis and
treatment optimization, limitations of conventional methods under-detect the short, cancer-specific cfDNA
fragments. The novel, improved method of preparing samples for sequencing proposed here is likely to improve
the prospects of early, noninvasive diagnosis of cancer.

* Information listed above is at the time of submission. *

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