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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: 1R43HG009461-01A1
Agency Tracking Number: R43HG009461
Amount: $269,718.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: NHGRI
Solicitation Number: PA16-302
Solicitation Year: 2016
Award Year: 2017
Award Start Date (Proposal Award Date): 2017-05-01
Award End Date (Contract End Date): 2019-04-30
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 an improved method RealSeq DC to prepare libraries of
cell free DNA cfDNA for next generation sequencing NGS cfDNAs which are found in blood and in other
bodily fluids represent promising minimally invasive liquid biopsy samples for human cancer and prenatal
diagnosis of fetal genetic diseases cfDNAs comprise highly fragmented double stranded DNA fragments
to bp in length having single strand nicks as well as andapos and andapos end overhangs They are normally present
at low concentration in biofluids In cancer patients concentration of cfDNA and level of fragmentation are
positively correlated with tumor weight with the majority of cfDNA fragments being shorter than 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 and prognosis and for
evaluating tumor progression and response to treatment Next generation sequencing NGS has great
potential to assess these parameters However due to the high level of DNA fragmentation in cancer derived
cfDNAs they cannot be efficiently incorporated into DNA Seq libraries and therefore are under detected by
conventional double stranded methods of sequencing library preparation To overcome this problem we
propose an advanced method that uses short ssDNA fragments prepared by denaturation of cfDNA for ligation
with a single combo adapter followed by circularization of the ligation product and direct PCR amplification
rather than rolling circle amplification RCA of the circular templates This method produces monomer
amplicons each containing a single cfDNA sequence insert flanked by standard Illumina andapos and andapos adapter
sequences The method minimizes the formation of amplicons comprising empty adapter dimers In Phase I
we plan to develop enzymatic steps specific for ssDNAs demonstrate the feasibility of the RealSeq DC
approach proof of concept and its superiority in sequencing DNA fragments of nt the size range which
is typical for cancer specific cfDNA over the two currently available methods of DNA Seq library preparation
cfDNA isolated from plasma samples from breast cancer patients will be assayed for this comparison In
Phase II we will further streamline the RealSeq DC protocol for commercialization extend the protocol to
identify methylated nucleotides test its reproducibility and minimize the required cfDNA input We also will
increase the number of samples studied as well as the range of physiological states and diseases with which
these samples are associated to evaluate the full potential of this approach and identify any limitations 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 and fetal abnormalities including identification
of specific genetic defects that may be addressable by targeted therapies

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

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