高通量测序技术在线虫多样性研究中的应用
收稿日期: 2022-05-13
录用日期: 2022-07-14
网络出版日期: 2022-09-09
基金资助
中国科学院战略性先导科技专项子课题(XDA28020202);国家自然科学基金(41877047);中国科学院国际合作重点项目(151221KYSB20200014)
Application of high-throughput sequencing technique in the study of nematode diversity
Received date: 2022-05-13
Accepted date: 2022-07-14
Online published: 2022-09-09
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。
孙翌昕, 李英滨, 李玉辉, 李冰, 杜晓芳, 李琪 . 高通量测序技术在线虫多样性研究中的应用[J]. 生物多样性, 2022 , 30(12) : 22266 . DOI: 10.17520/biods.2022266
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.
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