Analysis of prospective microbiology research using third-generation sequencing technology

doi:10.17520/biods.2018201

Abstract

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

Xu Yakun, Ma Yue, Hu Xiaoxi, Wang Jun. Analysis of prospective microbiology research using third-generation sequencing technology[J]. Biodiv Sci, 2019, 27(5): 534-542.

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URL: https://www.biodiversity-science.net/EN/10.17520/biods.2018201

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

Figures/Tables 3

References 43

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	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

	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