Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

Sun Chang Kim,Chul-Ho Lee,Sooin Lee,Min-Jeong Go,Sang Yup Lee,Byung-Kwan Cho,Jongoh Shin

doi:10.1038/srep29681

Abstract

Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples.

Highlights

Microbiotas are complex microbial communities containing hundreds of species-level phylotypes and are found everywhere, from humans to environments
We investigated whether the nanopore sequencing is suitable for analyzing the composition of the mouse gut microbiota at the species level and compared the results with those obtained by the short-read sequencing that has so far been widely employed in the field
Other phylogenetically similar species within different genera had more variants than species within the same genus (Supplemental Fig. S4). These findings suggested that the V3–V4 region was insufficient for analysis of the microbial community composition at the species level and that full-length 16S rRNA sequencing had the advantage of covering multiple variable regions of 16s rRNA genes

Summary

Introduction

Microbiotas are complex microbial communities containing hundreds of species-level phylotypes and are found everywhere, from humans (e.g., the microbiota within the gut) to environments. The microbiome has attracted much attention in the medical and healthcare industries, and elucidation of the microbiota composition in the human body is critical for further advancements in our understanding of related diseases and physiological states In this regard, because species in the same taxonomic units from genus up to phylum play a variety of roles, some may be crucial, others may not be correlated with the phenotype[6], it is critical to obtain the higher taxonomic resolution to species level for better understanding of the functional effects of microbiota on health and further identifying key players in a specific phenotype[7]. We investigated whether the nanopore sequencing is suitable for analyzing the composition of the mouse gut microbiota at the species level and compared the results with those obtained by the short-read sequencing that has so far been widely employed in the field

Methods

Results

Conclusion