Advanced method for preparing cell-free DNA sequencing libraries

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.