Platinum hot start master mix

Bacterial isolates were identified by 16S rRNA sequencing. DNA was extracted from pure cultures of each isolate using either a crude extraction (boil in water for 10 min) or the DNeasy Blood and Tissue Kit (Qiagen, Carlsbad, CA). PCR was performed in 25 μL reactions using Platinum Hot-Start Master Mix (Thermo Fisher, Waltham, MA) and 0.2 μM the 27F/1492R universal primers (Frank et al., 2008 (link)). PCR conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 51°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 10 min. PCR quality and fragment size were verified by gel electrophoresis (1% agarose gel, 10 V/cm). PCR products were cleaned with the Gel/PCR DNA fragment extraction kit (IBI Scientific, Dubuque, IA) and the DNA concentration was quantified using the Qubit 4 (Invitrogen, Carslbad, CA). Cleaned PCR products were sequenced using both 27F and 1492R primers on an ABI 3730 capillary sequencer (Thermo Fisher) by the Oregon State University Center for Genome Research and Biocomputing (CGRB, Corvallis, OR). Consensus sequences were generated from the forward and reverse sequences for each isolate using SeqTrace (Stucky, 2012 (link)). Taxonomy was assigned using the EZBioCloud Database (Yoon et al., 2017 (link)).

DNA was extracted using the Quick-DNA Fecal/Soil Microbe Microprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. 16S-rRNA V4 amplicons were generated using 515F and 806R primers^{50 (link),51 (link)}. PCRs were conducted using Platinum HotStart MasterMix (ThermoFisher Scientific, Waltham, MA, USA) with the following conditions: 94 °C for 3 min, 30 cycles of 94 °C for 45 s, 50 °C for 60 s, 72 °C for 90 s, and a final annealing hold of 72 °C for 10 min. Amplicon barcodes and Illumina-specific sequences were added with an additional round of PCR and sequenced with MiSeq V3 chemistry at the Pennsylvania State University Hershey Medical Campus Genomics Core Facility (Hershey, PA, USA).
Amplicon sequences were processed and binned into operational taxonomic units (OTUs) using mothur v.1.43.0^{52 (link)}. Briefly, contigs were formed, trimmed, and aligned to the Silva SEED database (v132). Chimeric sequences were detected and removed from samples using VSEARCH as implemented in mothur. Sequences were classified using the RDP reference taxonomy training set (v16) and sequences that were classified as eukaryote, plastids, or “unknowns” were removed. We opted to use 97% similarity to bin OTUs. Samples were then randomly subset to 25,000 sequences per sample and the resulting OTU tables were used for the following downstream analyses.