【作者】 李巧玲；

【导师】 杜红丽； 章文蔚；

【作者基本信息】 华南理工大学 ， 生物工程， 2015， 硕士

【摘要】 液体活检,即通过血液或者体液检测肿瘤循环DNA(circulating tumor DNA,ctDNA),获得肿瘤基因突变的信息,具有非侵入性、取样方便、可持续监测的特点,是肿瘤个体化治疗的重要工具。由于ctDNA具有的含量一般低于1%,并且肿瘤呈现高度异质性,而新一代测序技术存在约1%的错误率,使突变信息难以被准确检测到。双重测序法利用文库片段两端的随机标签组合去除测序错误,可以将测序准确度提高10000倍以上。因此本文利用6个已知突变频率的尿液来验证双重测序法结合芯片捕获技术的灵敏度,并用1例膀胱癌手术前后的尿液样本来证明该方法在肿瘤液体活检的可行性。本文探讨研究了双重测序法结合芯片捕获技术的灵敏度,以及方法在真实样本检测中的可行性。方法的灵敏度:在正常人尿液提取的DNA中加入1%,0.1%,0.01%的胃癌823细胞系DNA(拥有90个已知的突变位点)作为模拟样本,通过双重测序法和包含胃癌细胞系DNA 90个突变的芯片来建库捕获,检测混合样品里的突变位点。检测膀胱癌突变的可行性:用双重测序法结合芯片捕获技术对1例膀胱癌患者术前和术后的尿液DNA,一个正常人的尿液DNA进行检测,比较3个样品的基因突变情况。灵敏度测试的结果显示,在3个梯度的突变率中均能检测出突变,测序深度达到8000x时,含1%突变的样本的真实突变检出率大于66%,准确率大于93%。该方法用于检测膀胱癌突变时,芯片捕获率约60%,在病人术前、术后和正常人的尿液DNA中分别找到了39个,1个和4个突变位点。本研究说明,双重测序法是一个能够检测到0.01%低频突变的方法,结合芯片捕获技术,可特异性地检测膀胱癌尿液样本中疾病相关的基因突变位点。双重测序结合芯片捕获技术检测低频突变,是一个有发展潜力并且比较新的方法,需要更多的样本和数据支持,让它可以更好更广泛地应有。更多还原

【Abstract】 Liquid biopsy, which can detect the circulation tumor DNA(ctDNA) in blood of other body fluid, make us discover the mutation in patients timely and repeatable, that is very important in the individualized therapy. The ctDNA is usually low in body fluid, and the tumor is highly heterogeneous. So we cannot get accurate information about the low rate mutation sites by the normal next-generation sequencing technology which error rate is about 1%. The advent of Duplex Sequencing is an ultra-sensitive sequencing methodology capable of increasing the accuracy by 1000 times. Therefore, six urine samples with known mutation frequency were used to verify the reliability of the methods of Duplex Sequencing combining Region Target Capture technology, and samples from bladder cancer patients before and after surgery were used to prove the feasibility of this method in liquid biopsy.Sensibility of methods: Added 1%, 0.1%, 0.01% 823 gastric cancer cell line DNA(90 known mutations) into the normal urine as the analog samples, detected the mutations using a combination of Duplex Sequencing with cancer mutation target capture.Feasibility of detecting bladder cancer mutation: Used Duplex Sequencing method combined Region Target Capture sequencing to detect DNA mutation in urine from one bladder cancer patients before and after operation, one normal urine, compared the gene mutations of three samples.The results of the test of sensibility showed that mutations can be detected in the three gradients with 4000 x sequencing depth and the higher the depth we got, the more mutations we found. When it reached 8000 x, detection rate of real mutations is more than 66% in the samples containing 1% mutation, the accuracy rate is more than 93%. In the feasibility test, capture rate was about 60%; 39, 1 and 4 mutations were detected in patients before and after surgery, and normal controls respectively.In this research, Duplex sequencing is a precise approach to detect the low-frequency mutation, and is capable of binding to capture chip to catch the urine-related gene mutations. Further work has to be focused on the library construction process, data analysis, and application.更多还原