二代测序技术（Next-generation Sequencing）在生命科学领域和临床诊断等各方面发挥的作用日益显著。在此基础上发展起来的靶向富集技术（Target Enrichment）改善了全基因组测序（Whole Genome Sequencing, WGS）的价格高和部分无效测序等缺点，只针对外显子等研究者感兴趣的区域进行测序研究。我们对现在普遍使用的液相杂交捕获技术进行了杂交添加剂的测试，以提高目标区域捕获效率。同时还利用全外显子测序(Whole Exome Sequencing, WES)，以健康人的多种组织为样本，绘制了健康组织中人体细胞突变图谱。针对线粒体开创了操作简单的、富集特异性高的方法。液相杂交捕获是利用与目标基因互补的RNA作为探针杂交捕获目标DNA库的技术。DNA和RNA杂交效率与杂交环境相关。因此，测试了在杂交液中加入适当的化学试剂，包括甜菜碱（Betaine）、碘克沙醇（Iodixanol）、二甲基亚砜（DMSO）。初步验证中，每种试剂分别测试了三种浓度，随着浓度增加，只有DMSO显示出了增强富集的效果。其中碘克沙醇和甜菜碱的富集特异性在实验组与对照组没有差异。DMSO的最大添加量为16.9%，特异性可达到65%左右。我们将靶向外显子测序技术应用于微量样品的健康人体细胞突变情况的研究中。其中我对Blocker的改造弥补了这项应用中商业试剂盒中合适Blocker的空缺。本研究展示了人形态健康的组织普遍存在体细胞突变的积累，并通过突变进行了遗传分析发现了克隆扩增。此项研究描绘了来自同一个体多器官的体细胞突变图谱，为了解健康细胞到癌细胞的变化过程提供了新的角度。线粒体基因组（mtDNA）独立于核基因组以外。基于这个特点，开发了一种利用细胞裂解液裂解细胞释放线粒体的方法进行mtDNA的富集。细胞或者血液经低温裂解释放线粒体，离心后用商业DNA提取试剂盒提取DNA，再经过Tn5转座酶建库，可得到约25%线粒体基因组。Tn5建库前，提取出的DNA 85℃预热5分钟，线粒体基因组的富集特异性可高达70%。整个流程时间仅约2个小时。
Next-generation sequencing technology (NGS) plays an increasingly significant role in the field of life sciences and clinical diagnosis. Targeted enrichment sequencing technology developed to improve the disadvantages of high price and partial invalid sequencing reads of Whole Genome Sequencing (WGS), by enriching exons and other interested regions. For targeted sequencing, we tested hybridization additives in liquid phase hybridization, to increase the target region capture efficiency. At the same time, Whole Exome Sequencing (WES) was used to draw a map of human cell mutation in various tissues of healthy people. For mitochondria, a simple and efficient method for enrichment has been created.The liquid phase hybridization capture technology uses RNA complementary to the target gene as a capture probe to hybridize with the target gene DNA library，so that the target gene can be enriched. DNA and RNA hybridization efficiency is related to the hybridization environment. Therefore, adding appropriate chemical reagents including betaine, Iodixanol, and dimethyl sulfoxide (DMSO) to the hybridization solution was tested. In the preliminary validation, three concentrations of each reagent were tested, and as the concentration increased, only DMSO showed the effect of enhancing enrichment. The enrichment specificity of iodixanol and betaine had no significant difference between the experimental group and the control group, while the enrichment effect increased by DMSO addition. The maximum addition amount of DMSO is 16.9%, and the specificity can be 65%.We apply targeted exome sequencing technology to study the mutational profile of healthy human cells of small samples. My modification to the Blocker fills the gap of a suitable Blocker in the commercial kit for this application. This study demonstrates the pervasive accumulation of somatic mutations in human morphologically healthy tissues and uncovered clonal expansion by genetic analysis of the mutations. The study maps somatic mutations from multiple organs in the same individual, providing a new perspective on the process of change from healthy cells to cancer cells.The mitochondrial genome (mtDNA) is independent of the nuclear genome. Based on this feature of mitochondrial genome, a method was developed to enrich mtDNA by lysing cell to release mitochondria. Cells or blood are lysed at low temperature to release mitochondria and then centrifuge. DNA is extracted with a commercial DNA extraction kit, and then Tn5 transposase is used to build a library, and about 25% of the mitochondrial genome can be obtained. If the DNA was preheated at 85℃ for 5 minutes before Tn5 library construction, the enrichment efficiency of mitochondrial genome could be as high as 70%. The entire process takes only about two hours.
