Biodiversity Science ›› 2019, Vol. 27 ›› Issue (5): 534-542.doi: 10.17520/biods.2018201

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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
  • Wang Jun E-mail:junwang@im.ac.cn

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

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."

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."

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.
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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