Microbiome sample collection tube

Fecal samples were collected from mice while they were individually housed in metabolic cages and then flash-frozen for downstream analysis. Frozen samples (2 per mouse at each time point) were immersed in the Transnetyx microbiome sample collection tube (1.5 mL, prefilled with DNA stabilization liquid and glass beads) and sent to Transnetyx (Cordova, TN) for automated DNA extraction (Qiagen DNeasy 96 PowerSoil Pro QIAcube HT extraction kit), KAPA HyperPlus library prep, and shallow shotgun whole genome sequencing (WGS; minimum read depth of 2 million paired-end reads) on an Illumina NovaSeq instrument. Shallow WGS provides species/sub-strain level taxonomic resolution, as well as identification of non-bacterial organisms that may be present in the sample. Data analysis and visualization of taxonomy, alpha-diversity, beta-diversity, and PCA clustering were performed using One Codex (http://www.onecodex.com). The One Codex Database contains >115,000 completed microbial reference genomes, including 62,000 distinct bacterial genomes; human and mouse genomes were included to exclude host reads. Individual sequences were compared by exact alignment using k-mers, where k=31, as previously described (8 (link)–10 (link)). Relative species abundance was estimated from the sequencing depth and coverage across the reference genomes.