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: RNA-Seq, Low quantity, SSU rRNA, OTU, Microbial community
Background
Ribosomal RNA (rRNA) accounts for more than 90% of the total microbial RNA, and is suitable for the analysis of microbial communities as an indicator of microbial physiological activity to synthesize proteins (Blazewicz et al., 2013). The study of microbial community transcripts, including rRNA and mRNA, in a particular environment (Double RNA metatranscriptomics) has advantages in providing both functional and taxonomic information on microbes (Urich et al., 2008), but has failed to perform OTU-based community comparisons. Although diversity indices could be calculated and compared using the V3 region of 16S rRNA sequences when gel-extracted SSU rRNA is analyzed, only a third of the resulting 16S rRNA sequences were found to be suitable for such analyses (Li et al., 2014). Besides, such method usually requires high quantities of RNA (Li et al., 2014). We recently developed a modified RNA-Seq method that uses an immediate adaptor ligation step at the 5’ end of the RNA prior to reverse transcription. Consequently, we can obtain more 16S RNA reads which can be used for OTU-based community and diversity analysis especially regarding low-RNA-yield samples such as tap water, shower curtain and human skin (Yan et al., 2017), and also mudflat sediment samples (Yan et al., 2018).
Materials and Reagents
Equipment
Software
Procedure
Note: Details of sample collection, storage and transport for low-biomass samples (tap water, shower curtain, mudflat water, leaf surface and human skin) please see the method described by Yan et al. (2017). Regarding mudflat sediment samples, details of sample collection and nucleic acid extraction are described in our recent work (Yan et al., 2018).
Data analysis
Notes
Acknowledgments
This protocol was adopted from our previous study (Yan et al., 2017). This work was supported by the National Natural Science Foundation of China (NSFC) [grant number 31170114]. We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
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