Computational protocol: Bamboo shoot fiber prevents obesity in mice by modulating the gut microbiota

Similar protocols

Protocol publication

[…] Total genome DNA was extracted from the cecal content using a QIAamp DNA Stool minikit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Genomic DNA concentration was normalized to 1 ng/μl. The V3-V4 region (~450 bp) of the 16S rRNA gene was amplified using forward primer 341F (5′-CCTACGGGNGGCWGCAG-3′) attached to the Roche B adapter for Illumina library construction and reverse primer 802R (5′-TACNVGGGTATCTAATCC-3′) attached to the Roche A adapter and a 10-nt barcode [5′-A-adapter-N (10) + 16S primer-3′]. PCR reactions were performed as follows: 10 ng of the purified DNA, 15 μl of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 200 nmol/L of forward and reverse primers, and nuclease-free water in a final volume to 30 μl. PCR cycling condition consisted of an initial denaturation of 1 min at 98 °C, 30 cycles of 10 s at 98 °C, 30 s at 50 °C, and 5 min at 72 °C. PCR products were quantified (400–450 bp), pooled in equimolar ratios, purified (GeneJET Gel Extraction Kit, Thermo Scientific), and used for the Illumina HiSeq/MiSeq platform sequencing to a depth of at least 20,000 reads per sample (mean reads per sample 48,410 ± 984).Reads were merged by using FLASH processed using the QIIME (Quantitative Insights Into Microbial Ecology) analysis pipeline. Paired-end joined sequences were grouped into operational taxonomic units (OTUs) using the GreenGenes database and the UPARSE algorithm with a 97% threshold of pairwise identity. Samples were grouped according to the diet groups. A representative sequence for each OTU was aligned and the RDP classifier was used to annotate taxonomic information for each representative sequence. Analysis was performed at each taxonomical level (Phylum, Class, Order, Family, Genus, Species), separately. In-house Perl scripts were used to analyze alpha- (within samples) and beta- (among samples) diversity. Unweighted unifrac for Principal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with Arithmetic mean (UPGMA) Clustering tree were used to assess the variation between experimental groups (beta diversity). Alpha diversity was calculated for all the samples. […]

Pipeline specifications

Software tools QIIME, UPARSE, RDP Classifier
Databases Greengenes
Application 16S rRNA-seq analysis
Organisms Mus musculus, Bacteroidetes