生物多样性 ›› 2019, Vol. 27 ›› Issue (5): 534-542.doi: 10.17520/biods.2018201

• 综述 • 上一篇    下一篇

基于三代测序技术的微生物组学研究进展

许亚昆1, 2, 马越1, 2, 胡小茜1, 王军1, *()   

  1. 1 中国科学院微生物研究所, 北京 100101
    2 中国科学院大学, 北京 100049
  • 收稿日期:2018-07-30 接受日期:2018-12-25 出版日期:2019-05-20
  • 通讯作者: 王军 E-mail:junwang@im.ac.cn
  • 基金项目:
    科技部重点研发子课题(2018YFC2000504);国家自然科学基金(31771481)

Analysis of prospective microbiology research using third-generation sequencing technology

Xu Yakun1, 2, Ma Yue1, 2, Hu Xiaoxi1, Wang Jun1, *()   

  1. 1 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101
    2 University of Chinese Academy of Sciences, Beijing 100049
  • Received:2018-07-30 Accepted:2018-12-25 Online:2019-05-20
  • Contact: Wang Jun E-mail:junwang@im.ac.cn

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

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

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。"

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。"

表1

三代测序技术的比较"

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.
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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