Advanced method for preparing cell-free DNA sequencing libraries

制备游离DNA测序文库的先进方法

基本信息

批准号：
10227236
负责人：
SERGEI A KAZAKOV
金额：
$ 78.55万
依托单位：
SOMAGENICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10227236
关键词：
AKT1 gene Aftercare Benchmarking Biopsy Specimen Blood Breast Cancer Detection Breast Cancer Patient CST6 gene Cancer Diagnostics Cancer Patient Characteristics Clinical Consumption DNA DNA Fragmentation DNA Library DNA sequencing Data Detection Development Diagnostic Discrimination Disease ERBB2 gene ESR1 gene Epigenetic Process Frequencies GSTP1 gene Genes Genetic Genetic Diseases Goals Human Human Genome Individual KRAS2 gene Length Libraries Licensing Ligation Liquid substance Malignant Neoplasms Malignant neoplasm of pancreas Methods Methylation Mutation PIK3CA gene Pattern Performance Pharmacologic Substance Phase Plasma Plasma Cells Prenatal Diagnosis Preparation Prognosis Progress Reports Protocols documentation Publications Publishing RARB gene Reaction Reagent Records Research Sales Sampling Screening for cancer Sensitivity and Specificity Site Small RNA TP53 gene Technology Testing Time Work base bisulfite cancer diagnosis cell free DNA cohort companion diagnostics cost design dimer ds-DNA early detection biomarkers fetal diagnosis improved indexing interest liquid biopsy malignant breast neoplasm methylation pattern minimally invasive molecular diagnostics next generation sequencing noninvasive diagnosis novel repaired treatment optimization treatment response tumor tumor DNA

项目摘要

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.

拟议研究的目标是开发一种新颖、先进的方法来制备无细胞文库用于血液和大多数其他物质中发现的下一代测序 (NGS) 的循环 DNA (cfDNA)。生物流体代表了一种有前景的癌症微创诊断（“液体活检”），cfDNA 水平尽管即使存在癌症，大多数癌症患者的血浆和血清中也会升高。 cfDNA 源自非肿瘤细胞，可以通过特征改变在 cfDNA 中识别肿瘤 DNA 这种循环肿瘤 DNA (ctDNA) 的片段大小分布以及遗传和表观遗传特征更加明显。比健康个体的 cfDNA 降解，大部分片段短于 100 cfDNA 的肿瘤特异性特征分析，例如 DNA 的量、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、响应由于ctDNA中单链切口频率高，ctDNA片段短。无法有效地整合到通过常规方法由非变性 cfDNA 制备的测序文库中为了克服这个问题，我们在第一阶段开发了一种名为“DNA-Seq”的新型专有方法。 HASL-free-Seq 用于从 ssDNA（和变性 dsDNA）制备测序文库，可以我们还可以有效捕获 20 至 50 nt 大小范围内的超短 cfDNA 以及较长的 DNA 片段。证明血浆样品中超短 cfDNA 片段的比例可以提供稳健的在第二阶段，我们将优化无HASL-健康捐赠者和乳腺癌患者之间的区别。我们将其性能与替代方法进行比较，包括已发布的“实验室酿造”协议，以记录我们技术的优势。血浆样本与临床信息相匹配，我们将验证其区分健康人群的能力基于肿瘤特异性 ctDNA 碎片模式、突变特征的供体和乳腺癌患者 II 期完成后，我们计划将 HASL-free-Seq 商业化。通过销售文库制备试剂盒以及对外许可以及与已建立的合作伙伴关系来获取技术试剂和分子诊断公司以及有兴趣开发的制药公司伴随诊断。