Computational protocol: Dietary Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat Diet–Induced Metabolic Syndrome

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Protocol publication

[…] Mouse phenotypes were analyzed with STATISTICA, version 9.1 (StatSoft). One-way ANOVA was used to determine significance among three or more groups followed by the indicated post hoc test. Paired t tests were performed within groups (before versus after treatment), and unpaired t tests were used for independent groups. [...] 16S rRNA gene sequencing was performed on 80 paired cecal and fecal samples from 40 C57BL/6J mice consuming HFD, SPI diet, GP-SPI diet, or LFD for 13 weeks (n = 10 mice per diet group). For HFD, SPI diet, and GP-SPI diet groups, three to four paired cecal and fecal samples were randomly chosen from each of three treatment cages. The LFD group contained a total of 10 mice (in two cages), and we used all paired fecal and cecal samples from the LFD group for 16S sequencing. DNA was extracted using the PowerSoil bacterial DNA extraction kit (MoBio, Carlsbad, CA) and PCR-amplified using barcoded universal bacterial primers targeting variable region 4 of the 16S rRNA gene: 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Integrated DNA Technologies, Coralville, IA). The following thermocycler protocol was used: denature at 94°C for 3 min and 35 cycles of 94°C for 45 s, 50°C for 30 s, and 72°C for 90 s, with a final extension at 72°C for 10 min (). Triplicate reactions for each sample were pooled and amplification was confirmed by 1.5% gel electrophoresis. Amplicons were cleaned with the Ampure XP kit (Agencourt, Danvers, MA) and quantified using the Quant-iT Picogreen dsDNA Assay kit (Invitrogen, Carlsbad, CA). Barcoded amplicons from all 80 samples were pooled and sequenced on one lane of an Illumina HiSeq, resulting in >2 × 107 150-bp single-end reads. We obtained 253,135 ± 9,112 sequences per sample (range 138,728–460,521). To avoid bias owing to differences in sampling depth, we conducted analyses at a subsampled depth of 100,000 sequences. Sequences were analyzed on the Harvard Odyssey computational cluster using the Quantitative Insights Into Microbial Ecology (QIIME) software package (). Operational taxonomic units were picked at 97% similarity against the Greengenes database () (constructed by the “” script), which we trimmed to span only the 16S rRNA region flanked by our sequencing primers (positions 521–773). Bray-Curtis principal coordinate analysis was performed using the QIIME script “” Statistical analyses of microbial community structure (permutational multivariate ANOVA [PERMANOVA] and analysis of similarities [ANOSIM]) were performed using the QIIME script “” Microbial biomarker discovery was performed using the LEfSe algorithm (), with an LDA score of three or above set as the threshold for significance. […]

Pipeline specifications

Software tools Statistica, QIIME, LEfSe
Applications Miscellaneous, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Mus musculus, Akkermansia muciniphila
Diseases Metabolic Diseases, Neoplasms, Glucose Intolerance
Chemicals Glucose, Nitric Oxide, Polyphenols