基于三代测序技术的微生物组学研究进展

doi:10.17520/biods.2018201

摘要/Abstract

摘要：

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。

关键词: 微生物, 三代测序, 16S/18S rRNA, 宏基因组

Abstract

Microbes are ubiquitous in human life. In years past, the study of microbes has only focused on single-bacteria cultures and qualitative analyses. The development of sequencing technology has greatly enhanced progress in microbiology research and more and more evidence shows that human symbiotic microbes, especially intestinal microbes, are closely related to human health. Second-generation sequencing technology is currently mainstream in microbiology research because of its high throughput, high accuracy and low cost. However, with the deepening complexity of research, the disadvantages of second-generation technology, i.e. short read length (< 450 bp), lead to subsequent challenges in data analysis and genome assembly, and limit the applicability to future research. In this context, the third-generation sequencing technology comes into being. The third-generation of sequencing (TGS) technology is also called single molecule sequencing. It directly carries out real-time sequencing of single DNA molecules without PCR amplifications. TGS technology significantly increases read length up to 2-10 kb or even 2.2 Mb. Because of its advantages of long read and no preference for GC, TGS provides a new method for full-length gene sequencing that facilitates the assembly of complete and reliable genome maps in microbes and that further reveals the diversity of microbial structures and functions. This review summarizes the technical characteristics and principles of TGS, and then mainly analyzes its applications and progress in 16S/18S rRNA gene sequencing, complete bacterial genome mapping and metagenomics research.

Key words: microbes, third-generation sequencing, 16S/18S rRNA, metagenomics

许亚昆, 马越, 胡小茜, 王军 (2019) 基于三代测序技术的微生物组学研究进展. 生物多样性, 27, 534-542. DOI: 10.17520/biods.2018201.

Xu Yakun, Ma Yue, Hu Xiaoxi, Wang Jun (2019) Analysis of prospective microbiology research using third-generation sequencing technology. Biodiversity Science, 27, 534-542. DOI: 10.17520/biods.2018201.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2018201

https://www.biodiversity-science.net/CN/Y2019/V27/I5/534

图/表 3

图1 PacBio SMRT测序原理。(a)在零模波导孔(Zero-Mode Waveguides, ZMW)中, 单个DNA分子模板与引物和聚合酶结合后, 被固定到ZMW孔底部。DNA合成开始时, 新加入的荧光标记的dNTP由于碱基配对在ZMW底部停留较长时间, 激发后发出对应的荧光信号被共聚焦显微镜实时记录; (b) 1)荧光标记胞嘧啶脱氧核苷酸; 2)胞嘧啶脱氧核苷酸进入DNA链配对, 发射荧光信号; 3)荧光基团被DNA聚合酶切除, 荧光消失; 4)标记新的脱氧核苷酸; 5)继续新一轮合成。

Fig. 1 Schematic diagram of PacBio single molecule real-time sequencing. (a) In the ZMW hole, a single DNA molecule template combined with primers and polymerase is bind to the bottom of ZMW hole. At the beginning of DNA sequencing, the newly added fluorescent labeled dNTP remained at the bottom of ZMW for a long time due to base pairing, and the corresponding fluorescent signals were recorded by confocal microscopy in real time. (b) (1) Fluorescence labeling cytosine deoxynucleotides; (2) Cytosine deoxynucleotides entering DNA chain pairing, emitting fluorescent signals; (3) Fluorescent group is removed by DNA polymerase, fluorescence disappeared; (4) Label new deoxynucleotides; (5) Continue a new round.

图2 Nanopore利用电信号检测出DNA的碱基序列。纳米孔直径很小, 仅仅允许单个核苷酸通过。当DNA单链通过的时候, 就会对离子的流动造成阻碍, 从而使流过纳米孔的电流强度发生变化。由于ATCG四种碱基的带电性质不一样, 造成电流大小的波动也不一样, 因此可根据电流的变化鉴定所通过的碱基类型。

Fig. 2 Nanopore DNA sequencing using electronic signals as detection methods. The diameter of the nanoscale is very small that only a single DNA molecule is allowed to pass through. When a single strand of DNA passes through, it blocks the flow of ions and changes the current intensity across the nanopore. Because the charge properties of the four bases of ATCG are different, the type of base passed is identified according to the change of current.

表1 三代测序技术的比较

Table 1 Comparison of three generation sequencing technologies

	技术平台 Technical platform	测序原理 Principle of sequencing	测序读长 Read length	优点 Advantages	缺点 Limitations
第一代 The first generation	Sanger	可中断测序 Chain-terminating sequencing	600-1,000 bp	读长长; 准确率高; 能很好地处理一些重复序列和多聚序列 Long reads; high accuracy; good ability to deal with repetitive and homopolymer regions.	通量低; 样品制备成本高, 难以做大量的平行测序 Low throughput; high cost of Sanger sample preparation; making massively parallel sequencing prohibitive.
第二代 The second generation	Roche/454	焦磷酸测序 Pyrosequencing	200-400 bp	在二代测序中读长最长; 高通量 Longest read lengths among the second-generation; high throughput.	样品制备较难; 难于处理重复和同种碱基多聚区域 Challenging sample preparation; hard to deal with repetitive/homopo- lymer regions.
	Illumina	边合成边测序 Sequencing by synthesis	2 × 150 bp	高通量 Very high throughput	读长短 Short reads
	ABI/Solid	连接测序 Sequencing by ligation	25-35 bp	高通量; 成本低 High throughput; low cost.	测序运行时间长; 读长短, 造成后续的数据分析困难和基因组拼接困难 Long sequencing runs (days); short reads, resulting in difficulties in subsequence data analysis and genome assembly.
第三代 The third generation	PacBio SMRT	边合成边测序/ DNA聚合酶 Sequencing by synthesis/DNA polymerase	~1,000 bp	高平均读长; 不需要扩增; 最长单个读长接近100 kb Long average read length; no amplification of sequencing fragments; longest individual reads approach 100 kb.	错误率高; 依赖DNA聚合酶的活性 Low accuracy; dependence on DNA polymerase activity.
第三代 The third generation	Nanopore	电信号测序/ 核酸外切酶 Electronic signals sequencing/exonuclease	最大记载2.2 M Maximum record 2.2 M	读长超长; 电学测序; 方便携带 Over-long read; electronic sequencing; portable.	错误率高 High sequencing error

表1 三代测序技术的比较

Table 1 Comparison of three generation sequencing technologies

	技术平台 Technical platform	测序原理 Principle of sequencing	测序读长 Read length	优点 Advantages	缺点 Limitations
第一代 The first generation	Sanger	可中断测序 Chain-terminating sequencing	600-1,000 bp	读长长; 准确率高; 能很好地处理一些重复序列和多聚序列 Long reads; high accuracy; good ability to deal with repetitive and homopolymer regions.	通量低; 样品制备成本高, 难以做大量的平行测序 Low throughput; high cost of Sanger sample preparation; making massively parallel sequencing prohibitive.
第二代 The second generation	Roche/454	焦磷酸测序 Pyrosequencing	200-400 bp	在二代测序中读长最长; 高通量 Longest read lengths among the second-generation; high throughput.	样品制备较难; 难于处理重复和同种碱基多聚区域 Challenging sample preparation; hard to deal with repetitive/homopo- lymer regions.
	Illumina	边合成边测序 Sequencing by synthesis	2 × 150 bp	高通量 Very high throughput	读长短 Short reads
	ABI/Solid	连接测序 Sequencing by ligation	25-35 bp	高通量; 成本低 High throughput; low cost.	测序运行时间长; 读长短, 造成后续的数据分析困难和基因组拼接困难 Long sequencing runs (days); short reads, resulting in difficulties in subsequence data analysis and genome assembly.
第三代 The third generation	PacBio SMRT	边合成边测序/ DNA聚合酶 Sequencing by synthesis/DNA polymerase	~1,000 bp	高平均读长; 不需要扩增; 最长单个读长接近100 kb Long average read length; no amplification of sequencing fragments; longest individual reads approach 100 kb.	错误率高; 依赖DNA聚合酶的活性 Low accuracy; dependence on DNA polymerase activity.
第三代 The third generation	Nanopore	电信号测序/ 核酸外切酶 Electronic signals sequencing/exonuclease	最大记载2.2 M Maximum record 2.2 M	读长超长; 电学测序; 方便携带 Over-long read; electronic sequencing; portable.	错误率高 High sequencing error

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