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

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.

Polygala tenuifolia extract inhibits lipid accumulation in 3T3-L1 adipocytes and high-fat diet–induced obese mouse model and affects hepatic transcriptome and gut microbiota profiles

“…Mouse gut microbiota profiles were determined through 16S rRNA gene sequencing of genomic DNA from stool [43]. After 5-week PTE treatment, DNA was extracted from mouse stool samples using a QIAamp DNA Stool Mini Kit (Qiagen, Germany).…”

Heterogeneity Spacers in 16S rDNA Primers Improve Analysis of Mouse Gut Microbiomes via Greater Nucleotide Diversity

“…The 16S rDNA primers recognize highly conserved regions that flank hypervariable regions, subsequently yielding amplicons with highly homogenous sequences. Moreover, the clustering technique and fluorescent identification inherent to Illumina technology can make it difficult to discern sequences with long repeats, inverted repeats or GGC [10,13]. Bases A and C are more prone to substitution errors [13].…”

“…Several studies have compared the microbial composition and diversity across different 16S rDNA primers, demonstrating that the level of taxonomic resolution detected varies depending on the hypervariable region targeted [6–9]. The V3–V4 16S rDNA primers have been commonly used across the microbiome field for soil, various animal models and humans [6,7,10,11]. As compared with sequencing other hypervariable regions, the V3–V4 hypervariable region provides a broad taxonomic range of bacteria, thus capturing more microbial diversity with decreased taxonomic bias [6,7,12].…”

“…Given the importance for taxonomic identification of each nucleotide within the 16S rDNA amplicon, miscalling of one base increases the probability of misidentifying the bacterium [10]. To address these shortcomings in the standard Illumina V3–V4 primers, different techniques have been employed to increase the quality of the data, including quality trimming, read overlapping, targeting multiple hypervariable regions and dual-indexing of the samples to reduce error rates [4,10,13–16]. The aforementioned techniques, however, do not address the inherent issue of low nucleotide diversity.…”

Rapid sequencing‐based diagnosis of infectious bacterial species from meningitis patients in Zambia

“…Advances in DNA sequencing technology have now enabled obtaining real‐time DNA sequencing data using the nanopore‐based sequencer MinION (Oxford Nanopore Technologies, Oxford, UK) . Recently, remarkable performances of the MinION system have been reported for rapid bacterial identification based on sequencing of full‐length 16S rRNA gene amplicons . Using this sequencer in tandem with two laptop computers, we have developed a portable and rapid bacterial composition analysis system for the on‐site diagnosis of infectious diseases .…”