Computational protocol: Modulation of Microbiota-Gut-Brain Axis by Berberine Resulting in Improved Metabolic Status in High-Fat Diet-Fed Rats

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Protocol publication

[…] Fresh stool sample were collected in month 8 and immediately stored at −80 °C for subsequent analysis. The sample sizes were 5 in control group,5 in HFD group, and 4 in HFD + BBR group. There is no difference in sampling between the groups studied. Genomic DNA of microbiota was extracted from fecal sample by TIANamp stool DNA kits (TIANGEN). DNA was quantified by the Nanodrop 2000. The extracted DNA from each sample was used as the template to amplify the V3 and V4 hypervariable regions of ribosomal 16S rRNA genes. Briefly, the purified 1 μg of genomic DNA were fragmented to an average size of 300-400 bp and ligated with adapters. The PCR was performed using a primer cocktail that anneals to the ends of the adapters to enrich DNA fragments that have adapter molecules on both ends and followed by clean up and quantification. Sequencing was performed using a 300-bp paired-end sequencing protocol on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at Oebiotech Company, Shanghai, China. Raw paired-end reads were subjected to quality filtering using Trimmomatic software before paired-end read assembling with FLASH software. All chimeras of assembling sequences were eliminated to reach high-quality sequences. [...] All microbiota sequences were assigned to Operational Taxonomic Units (OTUs) using the UCLUST algorithm in CD-HIT with 97% threshold of pairwise identity, and the most abundant sequence of each OTU was selected as the representative sequence and subjected to RDP classifier for taxonomical assignment with a bootstrap cutoff of 50%. The rarefaction estimates and Shannon-Wiener index were calculated using QIIME. The representative sequences of OTUs were used to generate a phylogenetic tree using FasTree. The phylogenetic tree was then used for unweighted UniFrac principal coordinates analysis (PCoA). The relative abundances of gut microbiota in each sample and other measurement data were expressed as mean ± SEM and evaluated with one-way analysis of variance (ANOVA) with Fisher's least significant difference (LSD) post hoc test. Statistical significance was accepted as p ℋ 0.05 (two-sided significance testing). All statistical analyses were performed using SPSS 22.0 statistical software (Chicago, IL, USA). […]

Pipeline specifications

Software tools Trimmomatic, UCLUST, CD-HIT, RDP Classifier, QIIME
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Rattus norvegicus
Chemicals Berberine, Glucose