# Advanced method for preparing cell-free DNA sequencing libraries

> **NIH NIH R44** · SOMAGENICS, INC. · 2021 · $785,533

## Abstract

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.

## Key facts

- **NIH application ID:** 10227236
- **Project number:** 5R44HG009461-03
- **Recipient organization:** SOMAGENICS, INC.
- **Principal Investigator:** SERGEI A KAZAKOV
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $785,533
- **Award type:** 5
- **Project period:** 2017-05-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10227236

## Citation

> US National Institutes of Health, RePORTER application 10227236, Advanced method for preparing cell-free DNA sequencing libraries (5R44HG009461-03). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10227236. Licensed CC0.

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