Advanced method for preparing cell-free DNA sequencing libraries

制备游离DNA测序文库的先进方法

• 批准号: 10082266
• 负责人: SERGEI A KAZAKOV
• 金额: $ 82.62万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082266
• 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 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; 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; genetic profiling; improved; indexing; interest; liquid biopsy; malignant breast neoplasm; methylation pattern; minimally invasive; molecular diagnostics; next generation sequencing; noninvasive diagnosis; novel; outcome forecast; repaired; treatment optimization; treatment response; tumor; tumor DNA

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.

拟议的研究的目的是开发一种新颖的高级方法来准备无细胞的库 用于下一代测序（NGS）的循环DNA（CFDNA）。在血液中发现的CFDNA，大多数其他 代表有希望的，微创诊断的生物流体（“液体活检”），CFDNA水平为 癌症患者的血浆和血清中升高。虽然，即使存在癌症，大多数 CFDNA源自非肿瘤细胞，可以通过特征改变在CFDNA中识别肿瘤DNA 在碎片尺寸分布以及遗传和表观遗传谱中。这种循环的肿瘤DNA（CTDNA）更多 比健康个体的CFDNA降级，片段大于100 bp。分析CfDNA的肿瘤特异性特征，例如DNA的量，其碎片水平， 突变和甲基化残留物的存在可用于癌症诊断，对 治疗和预后。由于ctDNA中的单链刻度的频率很高，因此短ctDNA片段 不能有效地将其通过常规的非定义CFDNA制备的测序库中 DNA-seq方法。为了克服这个问题，在第一阶段开发了一种新颖的专有方法，称为 无hasl-fre-fre-seq，用于制备可以从ssDNA（和变性dsDNA）的测序库来制备可以 有效捕获20至50 nt尺寸范围内的超短cfDNA以及更长的DNA片段。我们也是 证明血浆样品中超短cfDNA片段的比例可以提供鲁棒 健康供体和乳腺癌患者之间的歧视。在第二阶段，我们将优化无hasl- SEQ协议和套件，用于商业生存能力。我们将将其性能与替代方法的性能进行比较， 包括已发布的“实验室酿造”协议，以记录我们技术的优势。使用一群 具有匹配临床信息的等离子体样本，我们将验证其区分健康的能力 供体和乳腺癌患者基于肿瘤特异性ctDNA碎片模式，突变特征 和甲基化模式。 II阶段完成后，我们计划将无hasl-free-seq商业化 通过销售图书馆准备套件的销售以及与既定的 试剂和分子诊断公司以及对开发感兴趣的制药公司 伴侣诊断。