The next-generation sequencing technology and application

As one of the key technologies in biomedical research, DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years. This is largely due to the advent of newer generations of sequencing platforms, offering ever-faster and cheaper ways to analyze sequences. In our previous review, we looked into technical characteristics of the next-generation sequencers and provided prospective insights into their future development. In this article, we present a brief overview of the advantages and shortcomings of key commercially available platforms with a focus on their suitability for a broad range of applications.     

高通量转录组测序技术及其在鳞翅目昆虫上的应用

转录组是指组织或者细胞在某一特定状态下转录出来的所有RNA的集合。高通量第2代测序技术使转录组学的研究模式发生了巨大的改变,所衍生出的转录组测序迅速成为研究非模式生物的先进技术。转录组测序能够在整体水平上探究细胞内基因表达的种类和数量,揭示在特定条件下机体生理生化发生过程以及其中的分子机理。本文简要阐述了转录组测序技术的基本概念、技术流程与原理,详细介绍了转录组测序在解决鳞翅目昆虫的分类、毒理、发育、与寄主互作以及非编码RNA调控等问题上做出的贡献,并对该技术现存的困难进行了系统的阐述并对其未来的发展趋势作出了简要的预测与剖析。     

深度突变扫描技术在蛋白研究中的应用

作为细胞结构与功能的中心参与者,蛋白质一直是生命科学研究的中心主题。分析蛋白质序列变异对其结构、功能的影响,是研究蛋白的重要手段之一。近年一种称为深度突变扫描(deep mutational scanning,DMS)的技术被广泛应用于蛋白研究领域,其通过高丰度DNA文库在蛋白特定区域平行引入成千上万种突变,经筛选后,利用高通量测序为每一种突变打分,从而揭示序列与功能之间的相关性。深度突变扫描以其高通量、快速简易、节省人工等特点,已经成为蛋白质功能研究以及蛋白工程改造的一种重要方法,目前已在蛋白进化、抗体改造、致病突变鉴定等蛋白研究的多个领域广泛应用。本综述简要概括了深度突变扫描技术的原理,重点介绍了其在哺乳动物细胞中的应用,同时分析了目前的技术瓶颈,旨在为相关研究提供参考。     

The next-generation sequencing technology: A technology review and future perspective

As one of the most powerful tools in biomedical research, DNA sequencing not only has been improving its productivity in an exponential growth rate but also been evolving into a new layout of technological territories toward engineering and physical disciplines over the past three decades. In this technical review, we look into technical characteristics of the next-gen sequencers and provide prospective insights into their future development and applications. We envisage that some of the emerging platforms are capable of supporting the 1000 genome and1000 genome and 100 genome goals if given a few years for technical maturation. We also suggest that scientists from China should play an active role in this campaign that will have profound impact on both scientific research and societal healthcare systems.     

Next generation sequencing technology: Advances and applications

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function.     

基于三代测序技术的微生物组学研究进展

微生物在人类生活中无处不在, 过去人们对微生物的认识仅停留在单菌培养和定性研究上, 而测序技术的发展极大地促进了微生物组学的研究。越来越多的证据表明: 人体共生微生物、特别是肠道微生物与人类健康息息相关。 二代测序技术凭借其高通量、高准确率和低成本的特点, 成为微生物组学研究中的主流测序技术。但是随着研究的深入, 二代测序技术的短读长(< 450 bp)增加了后续数据分析和基因组拼接难度, 也限制了该技术在未来研究中的应用。在此背景下, 第三代测序技术应运而生。第三代测序技术又称单分子测序, 能够直接对单个DNA分子进行实时测序, 而不需要经过PCR扩增。第三代测序技术的平均读长在2-10 kb左右, 最高可以达到2.2 Mb, 实现了长序列的高通量测序。凭借其超长的测序读长、无GC偏好性等优势, 三代测序技术为微生物基因组全长测序, 组装完整可靠的基因组提供了新的方法。本文在描述三代测序的技术特点和原理的基础上, 重点介绍了三代测序技术在微生物16S/18S rRNA基因测序、单菌的基因组组装以及宏基因组中的研究应用和进展。     

Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.     

实现“终极版”核苷酸测序仪的技术要素

20世纪70年代发明的第一代DNA测序技术,尽管测序通量有限,却成功地保证了“人类基因组计划”的实施;世纪之交出现的下一代(第二代) DNA测序技术经历了通量飞跃,为各种精准医学项目的实施提供了保障;目前的第三代技术,尽管通量居二代之后,但读长和单分子测序优势也让其有立足之本;第四代测序技术的基本标志是不经过cDNA (以RNA为模版合成的互补DNA),无PCR扩增,而直接测定单分子RNA序列,以及确定单分子RNA上的修饰核苷酸位点。从技术的角度看,第三、四代技术有一定技术要素共享(比如在单分子水平测定DNA序列),但是就测序对象而言,第四代应该属于“终极版”核苷酸测序仪：可以从单细胞出发,既能测定DNA序列,也可以测定RNA序列,也可以直接确定修饰核苷酸位点。因此,要实现这个“终极版”核苷酸测序仪,就要调动相关核心技术要素,而这些要素毫无疑问地会涉及物理、化学、工程学、生物学、半导体科学、计算机科学等领域的前沿技术,包括纳米科学、单分子光学、单分子拉曼光谱、单分子核磁共振、单分子酶学、人工智能等所谓的“硬科技”。其功能是从单细胞的裂解开始,经微纳结构实现组分分流后,直接导入RNA序列测定单元,定量分析细胞RNA分子的种类、数量、序列和修饰核苷酸位点的存在频率。本文系统介绍了第四代测序仪的可能技术要素,以及应用需求和新研究范式。     

基因组学技术在生物多样性保护研究中的应用

在分子生物学、细胞生物学、微生物学、遗传学等学科的推动下, 生物多样性研究从仅关注宏观表型的博物学, 迅速演化为涵盖生态系统、物种和遗传多样性等多个维度的综合性生命科学。组学技术, 尤其是DNA测序技术的更新和发展, 使获取DNA序列所需的成本大幅下降, 促进了近年来其在生物多样性研究中取得的一系列令人瞩目成就。本文将从物种水平的遗传多样性和群落水平的物种多样性两个层面总结和介绍与DNA相关的组学技术在生物多样性研究中的一些创新和应用。其中, 物种水平主要是总结单一个体的基因组和单物种多个体在时空多个维度上的群体遗传研究; 而群落水平的物种多样性层面主要总结现有的分子鉴定技术(metabarcoding, eDNA, iDNA等), 以及上述新技术在群落多样性评估、旗舰保护物种监测以及物种间相互作用关系等研究中的应用。     

空间转录物组学技术进展及其在神经科学领域中的应用

空间转录物组学是在单细胞RNA测序技术基础上实现细胞空间位置信息测定的组学技术。该技术克服了单细胞转录物组学在单细胞分离建库过程中丢失细胞在组织中空间信息的问题,可同时提供研究对象的转录物组数据信息和在组织中的空间位置信息。空间转录物组学技术对研究细胞谱系的发生过程、细胞间的调控机制和相互作用等具有重要作用,是组学技术研究的重要发展方向和热点。近年来,空间转录物组学技术发展迅速,新的检测方法不断产生,检测灵敏度、分辨率和检测通量等技术指标不断提升。本文根据获取空间信息的原理不同,将较为常用的空间转录物组学技术进行了分类,总结了各类方法的检测原理、代表性技术手段及其相应的技术指标。随后,从脑细胞类型区分与细胞层图谱构建、神经系统相关疾病特征分析与标志物研究两个方面举例论述了空间转录物组学技术在神经科学中的应用。最后,对空间转录物组学技术目前存在的问题进行了总结,并对其未来的发展方向进行了展望。     

高通量测序技术在肉类掺假检测中的应用进展

肉类掺假现象普遍存在,导致严重的公共卫生风险和侵犯宗教信仰行为.快速、有效、准确和可靠的检测技术是有效监管肉类掺假的关键手段.近年来,基于高通量测序的DNA宏条形码技术发展迅速,具有高通量、高精度和速度快等特点,并且可以实现复杂样品中多个物种的同时检测,因而在肉类及其制品的掺假检测方面具有明显的优势.文中介绍了近20年...     

高通量测序技术及其在表观遗传学上的应用

DNA测序技术是现代生命科学研究的重要工具之一,而高通量测序技术在全基因组的研究中发挥着越来越重要的作用。简要回溯DNA测序技术的产生与发展,着重从PCR扩增测序和单分子测序两个方面全面描述了高通量测序中众多代表性的技术及直接测序技术,并从DNA甲基化、组蛋白修饰、非编码RNA调控等方面阐述了高通量测序技术在表观遗传学上的运用。     

Applications of sequencing technology in clinical microbial infection

Infectious diseases are a type of disease caused by pathogenic microorganisms. Although the discovery of antibiotics changed the treatment of infectious diseases and reduced the mortality of bacterial infections, resistant bacterial strains have emerged. Anti‐infective therapy based on aetiological evidence is the gold standard for clinical treatment, but the time lag and low positive culture rate of traditional methods of pathogen diagnosis leads to relative difficulty in obtaining the evidence of pathogens. Compared with traditional methods of pathogenic diagnosis, next‐generation and third‐generation sequencing technologies have many advantages in the detection of pathogenic microorganisms. In this review, we mainly introduce recent progress in research on pathogenic diagnostic technology and the applications of sequencing technology in the diagnosis of pathogenic microorganisms. This review provides new insights into the application of sequencing technology in the clinical diagnosis of microorganisms.     

单分子实时测序技术的原理与应用

单分子DNA测序技术是近10年发展起来的新一代测序技术,也称为第三代测序技术,包括单分子实时测序、真正单分子测序、单分子纳米孔测序等技术。文章介绍了单分子实时(Single-molecule real-time,SMRT)测序技术的基本原理、性能以及应用。与Sanger测序法和下一代测序技术相比,SMRT测序具有超长读长、测序周期短、无需模板扩增和直接检测表观修饰位点等特点,为研究人员提供了新选择。同时,SMRT测序的低准确率备受争议(约85%),其中约93%的错误是插入缺失,因此,其数据应用于基因组组装前需先对数据进行纠错处理。目前,SMRT测序在小型基因组从头测序和完整组装中已有良好应用,并且已经或将在表观遗传学、转录组学、大型基因组组装等领域发挥其优势,促进基因组学的研究。     

转录组研究新技术:RNA-Seq及其应用

转录组是特定组织或细胞在某一发育阶段或功能状态下转录出来的所有RNA的集合。转录组研究能够从整体水平研究基因功能以及基因结构,揭示特定生物学过程以及疾病发生过程中的分子机理。RNA-Seq作为一种新的高效、快捷的转录组研究手段正在改变着人们对转录组的认识。RNA-Seq利用高通量测序技术对组织或细胞中所有RNA反转录而成的cDNA文库进行测序,通过统计相关读段(reads)数计算出不同RNA的表达量,发现新的转录本;如果有基因组参考序列,可以把转录本映射回基因组,确定转录本位置、剪切情况等更为全面的遗传信息,已广泛应用于生物学研究、医学研究、临床研究和药物研发等。文章主要介绍了RNA-Seq原理、技术特点及其应用,并就RNA-Seq技术面临的挑战和未来发展前景进行了讨论,为今后该技术的研究与应用提供参考。     

现代微生物识别技术在水产养殖环境研究中的应用

养殖环境的恶化已成为制约水产业健康发展的瓶颈。鉴于微生物在养殖水体中的重要作用,本文从技术角度对近年来发展起来的现代微生物分子识别技术进行了概括介绍,并综述了以核酸为靶分子和以微生物表面抗原为靶分子的两类微生物识别技术在水产养殖中的应用进展,为水产养殖环境的改善及其病害防治提供参考。     

Recent development and future directions of enzyme technology in Japan

Fundamental research in enzymology have remarkably developed during the past thirty years. However, the actual application of enzymes has been rather limited for several technical and economical reasons. Such limiting factors are now being overcome year after year. Immobilized enzyme and cell technology might be a kind of break-through in this field. From the industrial view-point, enzyme technology is now coming into its real developmental stage. Enzyme technology has recently been finding several directions for its development. These directions may be classified as follows : (1) new industrial catalysts, (2) Tools for food production and processing, (3) pharmaceutical uses, (4) analytical and measurement tools, (5) aids for screening new physiologically active substances, and (6) aids for creation of new sources of energy and raw materials. The authors wish to demonstrate such directions of enzyme technology using actual examples studied and/or operated mainly in Japan.