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.