高通量测序样本准备方法及其在孕妇外周血微量循环核酸研究中的应用

Circulating nucleic acid are getting a lot of attentions in cancer research and prenatal diagnosis. It must be more significant to detect circulating nucleic acid using the next generation sequencing technology which could made its application in early diagnosis, staging, monitoring and prognosis more comprehensive and reliable. In this present proposal, we propose a low input, quick and effective next generation sequencing sample preparation method which set library preparartion and sequencing template amplification in one on the surface of micro-beads. The circulating nucleic acid extraction method was also modified and improved. Circulating DNA/RNA/miRNA in and out of the cMVs (Circulating Microvesicles) from pregnant women in their different gestation stages and abnormal status were sequenced and analyzed. We attempt to explain the basic characteristics such as fragment sizes, distribution and existing rules of circulating DNA using bioinformatics method based on these sequencing data, as well as the whole regulating relations between miRNA and RNA or DNA under certain pregnancy. This provides new way and research platform for circulating nucleic acid and non-invasive prenatal diagnosis. The project has wide application in founctional genomics, molecular geneitics and biomedical science.

近年来循环核酸在肿瘤、产前筛查等领域得到了极大的关注。利用高通量测序手段在全基因组水平上对循环核酸进行系统的研究分析对于其在疾病的早期诊断、分期、监测、预后判断等方面更好的应用意义重大。本项目结合高通量测序技术平台提出了一种低样本输入量、快速、高效的集文库制备与模板扩增为一体的测序样本准备方案，同时优化改良血浆中循环核酸的提取方法，结合生物信息分析平台，对不同妊娠时期和几种异常妊娠状态下孕妇外周血浆中cMVs（Circulating Microvesicles）内外的循环DNA/RNA/miRNA进行全基因组范围的差异表达分析。用以阐述循环DNA的大小、分布及存在规律等基础生物学特性，并试图对miRNA与mRNA/DNA之间的相互调控关系及其与妊娠等的关联进行更加全面的解释，为循环核酸用于无创性产前筛查与诊断提供新的思路和研究平台。本课题研究在功能基因组学，分子遗传学及生物医学领域。

循环核酸随着当前生物技术的发展在临床研究中备受关注，本课题结合高通量测序技术文库制备的问题提出了一种快速高效的微量循环核酸样本准备技术，并且在孕妇外周血液游离循环核酸和肺癌血浆miRNA的研究中进行了应用。实现了微量循环核酸的高通量测序分析与验证，在正常和异常妊娠孕妇外周血液中分别对其中的循环DNA/RNA以及exosome中DNA和RNA进行了表征和分析。首先寻找其在整个孕期的存在规律和表达变化情况，随后重点对常见的几种异常妊娠（先兆子痫、妊娠糖尿病、巨大儿等）进行了系统的研究，找到了一些特异性的循环核酸分子，进一步分析显示其与这些疾病的发生发展存在密切关联，具有很大的作为早期诊断标志物的潜力。同时我们还对肺癌病人的外周血液循环miRNA进行了系统的研究，通过与肿瘤组织、癌旁组织和正常组织中这些miRNA的差异表达情况进行比较分析，为肺癌的分型、分期和发病机制的研究奠定了基础。