高通量测序技术在线虫多样性研究中的应用

doi:10.17520/biods.2022266

摘要/Abstract

摘要：

土壤线虫多样性是土壤生态学研究的热点之一, 然而对土壤线虫群落组成及多样性的研究通常受到分类学和方法学的限制。当前, 分子生物学技术的快速发展丰富了我们对土壤线虫多样性的认识, 但也存在一定的局限性。本文综述了常用分子生物学技术如变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)、末端限制性片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)、实时荧光定量PCR (quantitative real-time PCR, qPCR)和高通量测序(high-throughput sequencing, HTS)技术近年来在线虫多样性研究中的应用, 重点从土壤线虫DNA提取方法、引物和数据库的选择、高通量测序技术和形态学鉴定结果的比较等方面阐述了高通量测序技术在线虫多样性研究中的优势与不足, 并提出选择合适的线虫DNA提取方法结合特定引物和数据库进行注释分析, 仍是今后使用高通量测序技术开展线虫多样性研究的重点。当研究目标是土壤线虫多样性时, 优先推荐富集线虫悬液提取DNA的方法, 因此, 研究人员应根据具体目标选择最优组合开展实验研究。

关键词: 分子生物学技术, 高通量测序, 土壤线虫多样性, 线虫DNA提取, 形态学鉴定

Abstract

Background & Aims: Soil nematode diversity is one of the hot topic in soil ecology research. However, in-depth studies on soil nematode community composition and diversity are limited by the established taxonomy and methodology. At present, the development of molecular biological techniques has elevated our understanding of the diversity of soil nematodes, but it also included set-backs. This paper summarizes the research progress in molecular biology technology, including denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), quantitative real-time PCR (qPCR) and high-throughput sequencing (HTS) technology, and the advantages and disadvantages of high-throughput sequencing in nematode research. We elaborate these techniques based on the extraction of soil nematode DNA, the selection of primers and reference databases, as well as the comparison between high-throughput sequencing and morphological identification analysis.
Prospects: The selections of soil nematode DNA extraction methods, in combination with proper primers and reference databases remain the key point to study nematode diversity. When focusing on nematode classification, we recommend DNA extraction after enrichment of nematodes suspension. Conclusively, researchers can then select the optimal combination to conduct experiments according to their research objectives.

Key words: molecular biological techniques, high-throughput sequencing, soil nematode diversity, nematode DNA extraction, morphological identification

孙翌昕, 李英滨, 李玉辉, 李冰, 杜晓芳, 李琪 (2022) 高通量测序技术在线虫多样性研究中的应用. 生物多样性, 30, 22266. DOI: 10.17520/biods.2022266.

Yixin Sun, Yingbin Li, Yuhui Li, Bing Li, Xiaofang Du, Qi Li (2022) Application of high-throughput sequencing technique in the study of nematode diversity. Biodiversity Science, 30, 22266. DOI: 10.17520/biods.2022266.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2022266

https://www.biodiversity-science.net/CN/Y2022/V30/I12/22266

图/表 5

参考文献 51

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分子生物学技术 Molecular biology techniques	优点 Advantages	缺点 Disadvantages	主要文献 References
变性梯度凝胶电泳 Denaturing gradient gel electrophoresis (DGGE)	相比形态学鉴定简单快速, 提高物种鉴定速度和时间成本效益 Compared with morphological identification, it is simple and rapid, and can improve the speed and time cost effectiveness of species identification	灵敏度不高, 不适合测量群落多样性; 在线虫稀有物种的代表性上鉴定不充分; 不能够直接评估物种丰富度 Low sensitivity, not suitable for measuring community diversity; the representation of rare nematode species was not adequately identified; species richness cannot be directly assessed	1993; 2005; 2008; 2013
末端限制性片段长度多态性分析 Terminal restriction fragment length polymorphism (T-RFLP)	操作简单; 有利于处理大量样本; 成本相对较低, 可以跨电泳运行比较数据 The manipulation is simple and good for processing a large number of samples; the cost is relatively low and data can be compared across electrophoresis runs	不能准确反映线虫群落结构组成, 结果有一定偏差, 可能高估线虫群落的多样性 The nematode community composition could not be accurately reflected, and the results may overestimate the diversity of nematode communities	2012; 2014; 2015
实时荧光定量PCR Quantitative real-time PCR (qPCR)	灵敏度高、特异性强; 检测结果可定量; 适用于大量样本; 可以确定特定线虫类群丰度 High sensitivity and specificity; the detection results can be quantified; suitable for large samples; the abundance of specific nematode trophic groups can be determined	不能提供样本中所有线虫群落的多样性信息, 无法计算线虫的富集指数和通道指数; 对引物要求较高, 容易出现非特异性条带; 成本也较高 The diversity information of all nematode communities in the samples could not be provided, and the enrichment index (EI) and channel index (CI) of nematode communities could not be calculated; high requirements for primers, prone to non-specific bands; the cost is higher	2006; 2010; 2010; 2012
高通量测序 High-throughput sequencing (HTS)	成本低; 测序量大、灵敏度高、用时短; 提供更高的分类学分辨率 Lower in cost; large amount in sequencing, high sensitivity and short time; provides higher taxonomic resolution	只能以相对丰度表征物种数量; 现有引物不是线虫特异性引物; 线虫参考数据库有待充实 Species numbers can only be represented by relative abundance; existing primers are not nematode-specific primers; the nematode reference database needs to be enriched	2017; 2018; 2020; 2021

分子生物学技术 Molecular biology techniques	优点 Advantages	缺点 Disadvantages	主要文献 References
变性梯度凝胶电泳 Denaturing gradient gel electrophoresis (DGGE)	相比形态学鉴定简单快速, 提高物种鉴定速度和时间成本效益 Compared with morphological identification, it is simple and rapid, and can improve the speed and time cost effectiveness of species identification	灵敏度不高, 不适合测量群落多样性; 在线虫稀有物种的代表性上鉴定不充分; 不能够直接评估物种丰富度 Low sensitivity, not suitable for measuring community diversity; the representation of rare nematode species was not adequately identified; species richness cannot be directly assessed	1993; 2005; 2008; 2013
末端限制性片段长度多态性分析 Terminal restriction fragment length polymorphism (T-RFLP)	操作简单; 有利于处理大量样本; 成本相对较低, 可以跨电泳运行比较数据 The manipulation is simple and good for processing a large number of samples; the cost is relatively low and data can be compared across electrophoresis runs	不能准确反映线虫群落结构组成, 结果有一定偏差, 可能高估线虫群落的多样性 The nematode community composition could not be accurately reflected, and the results may overestimate the diversity of nematode communities	2012; 2014; 2015
实时荧光定量PCR Quantitative real-time PCR (qPCR)	灵敏度高、特异性强; 检测结果可定量; 适用于大量样本; 可以确定特定线虫类群丰度 High sensitivity and specificity; the detection results can be quantified; suitable for large samples; the abundance of specific nematode trophic groups can be determined	不能提供样本中所有线虫群落的多样性信息, 无法计算线虫的富集指数和通道指数; 对引物要求较高, 容易出现非特异性条带; 成本也较高 The diversity information of all nematode communities in the samples could not be provided, and the enrichment index (EI) and channel index (CI) of nematode communities could not be calculated; high requirements for primers, prone to non-specific bands; the cost is higher	2006; 2010; 2010; 2012
高通量测序 High-throughput sequencing (HTS)	成本低; 测序量大、灵敏度高、用时短; 提供更高的分类学分辨率 Lower in cost; large amount in sequencing, high sensitivity and short time; provides higher taxonomic resolution	只能以相对丰度表征物种数量; 现有引物不是线虫特异性引物; 线虫参考数据库有待充实 Species numbers can only be represented by relative abundance; existing primers are not nematode-specific primers; the nematode reference database needs to be enriched	2017; 2018; 2020; 2021

提取方法 Extraction method	优点 Advantages	缺点 Disadvantages	土壤样品量 Soil sample weight (g)	线虫提取试剂盒 Nematode DNA extraction kit	文献 References
土壤中提取线虫DNA Nematode DNA was extracted from soil	无需富集步骤, 样本量较大时减小工作量 No enrichment step, suitable for work with large sample size	适合土壤线虫的土壤试剂盒数量少, 且结果不稳定, 在不同的实验条件下影响试剂盒的提取成功率 The kinds of soil kit suitable for soil nematodes were few; the extraction efficiency is instable and affected by experimental conditions	0.25	PowerLyzer^TM PowerSoil^? DNA Isolation Kit	2015
			0.25	PowerLyzer Soil DNA extraction kit	2020
			10	PowerMax Soil DNA isolation kit	2018
			10	DNeasy PowerSoil Kit	2021
			25	NucleoSpin^? Soil	2017
				Precellys^? Soil DNA Kit
				PowerLyzer^? Soil DNA Isolation Kit
				PowerSoil^? Soil DNA Isolation Kit
				PowerMax^? Soil DNA Isolation Kit
				E.Z.N.A. Mag-Bind^? Soil
富集线虫悬液提取DNA Nematode DNA was extracted from the enriched suspension	避免了放大其他物种(如真菌和植物)的DNA Avoiding amplification of other species DNA, such as fungi and plants	费时费力, 可能会偏向于线虫的特定属或发育阶段 Prefer to some particular genus or developmental stage of nematodes 在处理大量样品时, 富集工作易成为瓶颈 Enrichment process is a restriction when handling large number of samples	100	Clear Detections Nematode DNA extraction and purification kit?	2018
			40	MO BIO UltraClean^? Tissue & Cells DNA Isolation kit	2018
			40	MO BIO UltraClean^? Tissue & Cells DNA Isolation kit	2018	100	DNeasy Blood & Tissue Kit	2020; 2022

提取方法 Extraction method	优点 Advantages	缺点 Disadvantages	土壤样品量 Soil sample weight (g)	线虫提取试剂盒 Nematode DNA extraction kit	文献 References
土壤中提取线虫DNA Nematode DNA was extracted from soil	无需富集步骤, 样本量较大时减小工作量 No enrichment step, suitable for work with large sample size	适合土壤线虫的土壤试剂盒数量少, 且结果不稳定, 在不同的实验条件下影响试剂盒的提取成功率 The kinds of soil kit suitable for soil nematodes were few; the extraction efficiency is instable and affected by experimental conditions	0.25	PowerLyzer^TM PowerSoil^? DNA Isolation Kit	2015
			0.25	PowerLyzer Soil DNA extraction kit	2020
			10	PowerMax Soil DNA isolation kit	2018
			10	DNeasy PowerSoil Kit	2021
			25	NucleoSpin^? Soil	2017
				Precellys^? Soil DNA Kit
				PowerLyzer^? Soil DNA Isolation Kit
				PowerSoil^? Soil DNA Isolation Kit
				PowerMax^? Soil DNA Isolation Kit
				E.Z.N.A. Mag-Bind^? Soil
富集线虫悬液提取DNA Nematode DNA was extracted from the enriched suspension	避免了放大其他物种(如真菌和植物)的DNA Avoiding amplification of other species DNA, such as fungi and plants	费时费力, 可能会偏向于线虫的特定属或发育阶段 Prefer to some particular genus or developmental stage of nematodes 在处理大量样品时, 富集工作易成为瓶颈 Enrichment process is a restriction when handling large number of samples	100	Clear Detections Nematode DNA extraction and purification kit?	2018
			40	MO BIO UltraClean^? Tissue & Cells DNA Isolation kit	2018
			40	MO BIO UltraClean^? Tissue & Cells DNA Isolation kit	2018	100	DNeasy Blood & Tissue Kit	2020; 2022

线虫提取方式 Nematode extraction method	试剂盒 Nematode DNA extraction kit	引物 Primers	数据库 Databases	与形态学结果比较 Comparison with morphological results				文献 References
线虫提取方式 Nematode extraction method	试剂盒 Nematode DNA extraction kit	引物 Primers	数据库 Databases	形态学 Morphology	高通量测序 HTS	共有 Common	营养类群 Trophic groups	文献 References
土壤中提取线虫DNA Nematode DNA was extracted from soil	PowerSoil DNA Isolation Kit (MoBioLaboratories, Carlsbad, CA, USA)	3NDf/ 1132-rmodR	NCBI SRA database	27属 27 genera	42属 42 genera	15属 15 genera	HTS更偏向植物寄生线虫 HTS prefers to plant parasitic nematodes	2021
富集线虫悬液提取DNA Nematode DNA was extracted from the enriched suspension	MO BIO UltraClean^? Tissue & Cells DNA Isolation kit	NF1F/18Sr2bR	The SILVA 111 database	21科 21 families	30科 30 families	-	HTS偏向食细菌线虫, 形态学偏向食真菌线虫 HTS prefers to bacterivores nematodes; morphology prefers to fungivores nematodes	2018
	The Clear Detections Nematode DNA extraction and purification kit? (Clear Detections, Wageningen, the Netherlands)	3NDf/1132r	PR2 database	71属 71 genera	101属 101 genera	-	HTS与形态学更偏向植物寄生线虫 HTS and morphology prefer to plant parasitic nematodes	2018
		3NDf/1132r	PR2 database	71属 71 genera	101属 101 genera	-		2018	DNeasy Blood & Tissue Kit	NF1F/18Sr2bR	NCBI NT database	81属 81 genera	77属 77 genera	36属 36 genera	HTS与形态学更偏向植物寄生线虫 HTS and morphology prefer to plant parasitic nematodes	2020
	Ion Torrent PGM with the Ion PGM Hi-Q Sequencing Kit and the Ion 318 Chip Kit (Life Technologies)	NF1F/18Sr2bR	SILVA 132 eukaryotic database	46属 46 genera	51属 51 genera	27属 27 genera	HTS中食细菌线虫和杂食性线虫的相对丰度高于形态学鉴定 The relative abundance of bacterivores nematodes and omnivorous nematodes in HTS was higher than morphological identification	2022