Fecal microbiota 16s rRNA gene sequencing and sequences analysis

16S rRNA gene amplification and sequencing were done using the Illumina MiSeq technology following the protocol of Earth Microbiome Project with their modifications to the MOBIO PowerSoil DNA Isolation Kit procedure for extracting DNA (www.earthmicrobiome.org/emp-standard-protocols)^44,45. Bulk DNA was extracted from feces collected on P21 and P105 using a PowerSoil-htp kit from MoBio Laboratories (Carlsbad, CA, USA) with mechanical disruption (bead-beating). The 16S rRNA genes, region V4, were PCR amplified from each sample using a composite forward primer and a reverse primer containing a unique 12-base barcode, designed using the Golay error-correcting scheme, which was used to tag PCR products from respective samples⁴⁵. We used the forward primer 515 F 5′-AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGG TAA-3′: the italicized sequence is the 5′ Illumina adapter B, the bold sequence is the primer pad, the italicized and bold sequence is the primer linker and the underlined sequence is the conserved bacterial primer 515 F. The reverse primer 806 R used was 5′-CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT-3′: the italicized sequence is the 3′ reverse complement sequence of Illumina adapter, the 12X sequence is the golay barcode, the bold sequence is the primer pad, the italicized and bold sequence is the primer linker and the underlined sequence is the conserved bacterial primer 806 R. PCR reactions consisted of Hot Master PCR mix (Quantabio, Beverly, MA, USA), 0.2 μM of each primer, 10–100 ng template, and reaction conditions were 3 min at 95 °C, followed by 30 cycles of 45 s at 95 °C, 60 s at 50 °C and 90 s at 72 °C on a Biorad thermocycler. PCRs products were purified with Ampure magnetic purification beads (Agencourt, Brea, CA, USA), and visualized by gel electrophoresis. Products were then quantified (BIOTEK Fluorescence Spectrophotometer) using Quant-iT PicoGreen dsDNA assay. A master DNA pool was generated from the purified products in equimolar ratios. The pooled products were quantified using Quant-iT PicoGreen dsDNA assay and then sequenced using an Illumina MiSeq sequencer (paired-end reads, 2 × 250 bp) at Cornell University, Ithaca.

Forward and reverse Illumina reads were joined using the fastq-join method^46,47, sequences were demultiplexed, quality filtered using Quantitative Insights Into Microbial Ecology (QIIME, version 1.8.0) software package⁴⁸. QIIME default parameters were used for quality filtering (reads truncated at first low-quality base and excluded if: (1) there were more than three consecutive low quality base calls (2), less than 75% of read length was consecutive high-quality base calls (3), at least one uncalled base was present (4), more than 1.5 errors were present in the bar code (5), any Phred qualities were below 20, or (6) the length was less than 75 bases). Sequences were assigned to operational taxonomic units (OTUs) using UCLUST algorithm⁴⁹ with a 97% threshold of pairwise identity, and classified taxonomically using the Greengenes reference database 13_8⁵⁰. A single representative sequence for each OTU was aligned and a phylogenetic tree was built using FastTree⁵¹. The phylogenetic tree was used for computing the unweighted UniFrac distances between samples^52,53. rarefaction were performed and used to compare abundances of OTUs across samples. Principal coordinates analysis (PCoA) plots were used to assess the variation between experimental group (beta diversity). Alpha diversity curves were determined for all samples using the determination of the number of observed species. LEfSE (LDA Effect Size) was used to investigate bacterial members that drive differences between groups⁵⁴. The threshold on the logarithmic LDA score for discriminative features was set to 2.0, and the alpha values for the factorial Kruskal-Wallis test and pairwise Wilcoxon test between subclasses were set to 0.05.

In addition to fecal samples collected from the animals used in this current study, the 16s sequences previously generated from²⁶ were reanalyzed by combining gene sequences from both male and female mice treated with water (male: 12; female: 12), CMC (male: 11; female: 12), and P80 (male: 10; female: 9).