The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline

Abstract

BackgroundSubstantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples.ResultsWe have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart).ConclusionThe high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

Adipose tissue microbiota in humans: an open issue

“…The discrepancy between our results in humans and those in mice 14 , despite using the same experimental procedures, may be related to intrinsic differences in bacterial load in mice and humans. However technical limitations cannot be completely excluded.…”

Early-Life Sugar Consumption Affects the Rat Microbiome Independently of Obesity

“…The detection of sequencing fragments was performed by using MiSeq Illumina technology (Illumina Inc.). The 16S targeted sequences were then clustered into operational taxonomic units (OTUs) before taxonomic assignment and analyzed by using the bioinformatics pipeline as described previously (38,39). The OTUs and taxonomy classifications were tabulated, and to account for differences in raw counts across the samples the tables were log normalized by using Equation 1:…”

Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis

“…Implantation of endotoxin derived from highly virulent bacteria causes delayed demyelination and innate immune activation22. Recent studies demonstrate the presence of bacterium-encoded RNA and DNA sequences, particularly those derived from alpha-Proteobacteria, as well as bacterial proteins, in brains from humans (in the presence or absence of neurological disease), nonhuman primates23, rodents24 and other species25.…”