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Progress+in+environmental+transcriptomics+based+on+next-generation+high-throughput+sequencing

生物多样性 2013, 21 (4): 401–410 Doi: 10.3724/SP.J.1003.2013.10031 Biodiversity Science http: //http://www.wendangku.net/doc/c16b4ba31eb91a37f1115cfd.html

基于新一代高通量测序的环境微生物

转录组学研究进展

蔡元锋贾仲君*

(中国科学院南京土壤研究所土壤与农业可持续发展国家重点实验室, 南京 210008)

摘要:环境微生物转录组学是一门新兴学科, 它以复杂环境样品中的微生物mRNA为研究对象, 利用近年兴起的RNA-Seq高通量测序技术, 在整体水平上对环境微生物的基因表达水平和调控规律进行研究。本文概述了环境微生物转录组研究从样品的采集保存、RNA提取、mRNA的富集、cDNA合成直到高通量测序及数据分析的基本流程。总结了该技术面临的主要瓶颈: 环境样品mRNA含量低、腐植酸等干扰杂质多、rRNA去除程度有限。针对 RNA 的提取、纯化以及mRNA的富集这些重点步骤, 详细阐述了近年来在提高mRNA的得率与纯度上的方法学进展。

重点介绍了高通量测序数据的处理及分析方法, 从测序数据的质量控制、序列组装、rRNA的鉴定及去除、功能基因注释及分类到差异表达基因的鉴定。最后总结了近年来环境微生物转录组学在新基因的发现、不同环境条件下微生物的基因表达及调控规律研究、有机物的代谢路径分析等3个主要研究领域的广泛应用。随着测序技术及生物信息学分析工具的发展进步, 环境微生物转录组学将具有更广阔的应用前景。

关键词:转录组学, RNA-seq, 高通量测序, 环境微生物学

Progress in environmental transcriptomics based on next-generation high-throughput sequencing

Yuanfeng Cai, Zhongjun Jia*

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008

Abstract: Environmental transcriptomics, which focuses on microbial mRNA derived from complex envi-ronmental samples using the RNA-Seq method, allows investigation of expression and patterns of regulation of functional genes in natural microbial communities. This review outlines the basic protocol of environ-mental transcriptomics, from sample collection and preservation, total RNA isolation, mRNA enrichment, cDNA synthesis to high-throughput sequencing and data analysis. Main technological problems are pointed out, such as low yield of mRNA in environmental samples, contamination of mRNA by various impurities like humic substances and limited degree of rRNA removal. Recent progresses in specific methodologies to improve the quantity and quality of mRNA, especially in RNA extraction, purification and the enrichment of mRNA, are outlined. Bioinformatics methods that deal with the large volume of RNA-Seq data are ad-dressed, such as quality control of the sequence data, sequence assembly, detection and removal of rRNA, gene annotation and functional classification, and detection of differently expressed genes. The widely appli-cation of environmental transcriptomics, including detection of new genes, study of gene expression and regulation of microorganisms in different environments, and the analysis of metabolic pathways of special organic substances, are also highlighted. Environmental transcriptomics, combined with the further develop-ment of sequencing technology and bioinformatics tools in the future, are likely to be comprehensively used in the study of environmental microbiology.

Key words: transcriptomics, RNA-Seq, high-throughput sequencing, environmental microbiology

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收稿日期: 2013-01-31; 接受日期: 2013-03-09

基金项目: 国家自然科学基金重大项目(41090281)

∗通讯作者Author for correspondence. E-mail: jia@http://www.wendangku.net/doc/c16b4ba31eb91a37f1115cfd.html

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