A Modified Low-quantity RNA-Seq Method for Microbial Community and Diversity Analysis Using Small Subunit Ribosomal RNA

Yong-Wei Yan; Ting Zhu; Bin Zou; Zhe-Xue Quan

doi:10.21769/BioProtoc.2828

A Modified Low-quantity RNA-Seq Method for Microbial Community and Diversity Analysis Using Small Subunit Ribosomal RNA

Bio Protoc. 2018 May 5;8(9):e2828. doi: 10.21769/BioProtoc.2828.

Authors

Yong-Wei Yan¹, Ting Zhu¹, Bin Zou¹, Zhe-Xue Quan¹

Affiliation

¹ Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai, China.

Abstract

We propose a modified RNA-Seq method for small subunit ribosomal RNA (SSU rRNA)-based microbial community analysis that depends on the direct ligation of a 5' adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10-100 ng) and does not require a DNA removal step. Using this method, we could obtain more 16S rRNA sequences of the same regions (variable regions V1-V2) without the interference of DNA in order to analyze OTU (operational taxonomic unit)-based microbial communities and diversity. The generated SSU rRNA sequences are also suitable for the coverage evaluation for bacterial universal primer 8F (Escherichia coli position 8 to 27), which is commonly used for bacterial 16S rRNA gene amplification. The modified RNA-Seq method will be useful to determine potentially active microbial community structures and diversity for various environmental samples, and will also be useful for identifying novel microbial taxa.

Keywords: Low quantity; Microbial community; OTU; RNA-Seq; SSU rRNA.