Analysis of the gut microbiota by 16S rRNA sequencing and taxonomic assignment of reads

Genomic DNA was extracted from fecal pellets using enzymatic and mechanical lysis as previously described (Yuan et al. 2012). Fecal pellets from individual mice were collected in sterile containers and stored at −80°C. All pellets were then thawed on ice and one pellet (~ 38mg) per mouse transferred into a sterile 2.0 mL glass tube with cell lysis buffer composed of 50 μL lysozyme (10 mg/ml, Sigma-Aldrich, St. Louis, MO, USA), 6 μL mutanolysin (25 KU/ml; Sigma-Aldrich, St. Louis, MO, USA), and 3 μL lysostaphin (4000 U/ml, Sigma-Aldrich, St. Louis, MO, USA) and 41 μL of TE50 buffer (10 mM Tris·HCl and 50 mM EDTA, pH 8.0). After incubating at 37°C for 1 hour, pellets were mechanically disrupted by vortexing at high speed. 600 mg of 0.1-mm-diameter zirconia/silica beads (BioSpec, Bartlesville, OK, USA) were added to the mixture to lyse bacterial cells by using Mini-BeadBeater-96 (BioSpec) at 2100 rpm for 1 minute. Further isolation and purification of the total genomic DNA from crude lysates were processed using QIAamp DNA Mini Kit (Qiagen, Hilden, GER) according to the manufacturer’s handbook except that DNA was eluted into two separate tubes using two 100 μL aliquots. A Nanodrop 2000 was used to quantify genomic DNA in each sample according to manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, DNA was purified on the Qiagen fecal DNA kit (Qiagen, Venlo, Netherlands). Bacterial 16S rRNA genes were amplified by PCR using barcoded primers flanking hypervariable regions V1 to V3 (Escherichia coli positions 27F to 534R). The variable V1–V3 regions of 16S rRNA genes in each sample were amplified in two rounds of PCR with dual barcode indexing prior to analysis on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). The first PCR round amplified the target specific regions in 16S rRNA genes (E. coli positions 27F-534R), while the second attached sample specific barcodes and Illumina sequencing adapters. The V1–V3 regions of 16S rRNA genes were amplified in 96-well microtiter plates using the HotStar HiFidelity PCR kit (Qiagen) and 100 ng of template DNA in a total reaction volume of 50μl with the universal 16S rRNA primers 27F and 534R. The first round of PCR was run in a PTC-100 thermal controller (MJ Research, St. Bruno, Quebec, CAN) using the following cycling parameters: 2 min of denaturation at 95°C, followed by 20 cycles of 1 min at 95°C (denaturing), 1 min at 51 °C (annealing), and 1 min at 72 °C (elongation), with a final extension at 72°C for 10 min. The presence of amplicons was confirmed by agarose gel electrophoresis and staining with Gel Red. The second PCR was run in a total reaction volume of 20μl using the following cycling parameters: 10 min of denaturation at 95°C, followed by 10 cycles of 15 s at 95°C (denaturing), 30 s at 51 °C (annealing), and 1 min at 72 °C (elongation), with a final extension at 72 °C for 3 min. Negative controls without a template were included for each primer pair. The following procedure was performed by the Genomics Resources Core of the Institute for Bioinformatics and Evolutionary Studies (IBEST) at the University of Idaho: Amplicon concentrations were quantified by fluorometry (GeminiXPS, Molecular Devices, Sunnyvale, CA, USA) using PicoGreen, then equimolar amounts of the PCR amplicons were pooled in a single tube. Short DNA fragments and amplification primers were removed from the pool amplicons using AMPure beads (Beckman-Coulter, Indianapolis, IN, USA), and then the purified amplicons were qualified on an Advanced Analytical Fragment Analyzer (Ankeny, IA). This cleaned amplicon pool was then quantified using the KAPA Illumina library quantification kit (KAPA Biosciences, Wilmington, MA, USA) on the Applied Biosystems StepOnePlus real-time PCR system. This concentration was used as a starting point to normalize the pool to 10nM. Amplicons were sequenced using an Illumina MiSeq platform and a 300bp paired-end protocol (Illumina, Inc., San Diego, CA) with custom sequencing primers. dbcAmplicons were used to demultiplex all amplicons from other reads in the run after identifying the dual-barcoded pair, and further by using the locus-specific primers as an additional “barcode.” The final amplicons were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at the University of Idaho.

Sequence reads were cleaned, filtered, and taxonomically assigned using the Ribosomal Database Project (RDP) (http://rdp.cme.msu.edu) Naive Bayesian Classifier to the first RDP level with a bootstrap score of ≥50 for phylum to genus classification. Data were then processed using R scripts to calculate taxon relative abundance based on the total number of sequences within each sample. To simplify community composition analysis, we only included taxa constituting: a) at least 1% of the community in two or more samples or b) 5% of the community in at least one sample. Taxonomically assigned reads that did not meet this threshold were combined into an “Other Bacteria” category, along with reads that could not be taxonomically assigned beyond the level of bacteria. Qualitative assessments of changes in genus-level community richness and diversity were performed by calculating Shannon’s and Simpson’s indices(Payne et al. 2011). Hierarchical clustering and principal components analysis (PCoA) (Ravel et al. 2011) were used to assess similarities and differences in bacterial community composition across HFD male and female mice.