Advanced method for preparing cell-free DNA sequencing libraries

这项研究的目的是开发一种新的，先进的方法来制备无细胞的文库， 循环DNA（cfDNA）用于下一代测序（NGS）。在血液和大多数其他组织中发现的cfDNA 生物流体代表了癌症的有前景的微创诊断（“液体活检”），并且cfDNA水平是 在癌症患者的血浆和血清中升高。虽然，即使癌症存在，大多数 cfDNA来源于非肿瘤细胞，可以通过特征性改变在cfDNA内鉴定肿瘤DNA。 片段大小分布以及遗传和表观遗传特征。这种循环肿瘤DNA（ctDNA）比 与来自健康个体的cfDNAs相比，cfDNAs降解，其中相当大一部分片段短于100 BP. cfDNA的肿瘤特异性特征的分析，例如DNA的量，其片段化水平， 以及突变和甲基化残基的存在，可用于癌症诊断， 治疗和预后。由于ctDNA中单链切口的高频率，短ctDNA片段 不能有效地掺入通过常规方法从非变性cfDNA制备的测序文库中， DNA-Seq方法。为了克服这个问题，在第一阶段，我们开发了一种新颖的专有方法， 用于从ssDNA（和变性dsDNA）制备测序文库的无HASL-Seq，其可以 有效地捕获沿着在20至50 nt大小范围内的超短cfDNA以及较长的DNA片段。我们也 结果表明，血浆样品中超短cfDNA片段的比例可以提供稳健的结果。 健康捐赠者和乳腺癌患者之间的歧视。在第二阶段，我们将优化无HASL- 用于商业可行性的Seq方案和试剂盒。我们将比较其性能与替代方法， 包括已发布的“lab-brew”协议，以记录我们技术的优势。使用一组 血浆样本与匹配的临床信息，我们将验证其区分健康 基于肿瘤特异性ctDNA片段化模式、突变特征 和甲基化模式。在第二阶段完成后，我们计划将HASL-free-Seq商业化。 通过销售图书馆准备工具包以及对外许可，并与已建立的 试剂和分子诊断公司以及对开发感兴趣的制药公司 伴随诊断。

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.