Gut Microbiome 16S rRNA Gene Sequencing Analysis

Illumina MiSeq paired-end rRNA gene sequencing (V3-V4 region) was used to determine the bacterial composition of fecal genomic DNA extracted from mouse and donor specimens (N = 403 samples). Processing of frozen fecal specimens, DNA extraction, library preparation, sequencing, quality control, and primary bioinformatics analysis were performed by the Genomics Core, CWRU, and the Beijing Genomics Institute in Shenzen, China. The selection of operational taxonomic units (OTUs) was performed using the QIIME1 open reference picking and USEARCH (v7.0.1090) to perform clustering at 97% similarity. Operational taxonomic unit representative sequences were taxonomically classified using Ribosomal Database Project (RDP) Classifier v.2.2 trained on the Greengenes database, using 1 confidence value as cutoff. Based on OTU abundance information, the relative abundance of each OTU in each sample was calculated, and the principal component analysis (PCA) of OTU was done with the relative abundance value. The software used in this step was package “ade4” of software R(v3.1.1). Downstream analyses of OTU tables used principles based on QIIME1.²⁶ See details in online supplementary materials.