DNA extraction, amplicon sequencing, and metagenomics analysis
Microbial genomic DNA was isolated from fecal samples with the MoBio PowerFecal DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA) following the manufacturer’s protocols. Fungal DNA was extracted using the E.Z.N.A.TM Fungal DNA Mini Kit (OMEGA Bio-tek, Inc., Norcross, GA) according to manufacturer’s instructions. Successful DNA isolation was confirmed by 1% agar gel electrophoresis. Bacterial amplicon libraries were prepared by amplifying the V4 hypervariable region of 16S rRNA genes, while fungal amplicon libraries were prepared by amplifying the ITS1 region, as described previously (Huang et al 2015). Amplicon sequencing was performed on the Illumina Hiseq 2500 (diet conversion experiment) and Illumina Miseq 2500 (lifestyle- shift experiment) platforms to generate 150 bp paired-end reads.
Metagenomic binning (n=60, fecal samples from OBD) was conducted using metabat2 to bin samples from single-sample assemblies and the co-assembly, as described previously (Stewart et al 2018) and in further detail below.