Dneasy powersoil extraction kit

Microbial DNA was extracted from different body site samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen, Germantown, MD, USA) according to manufacturer’s protocol. Samples that did not have enough biomass were not able to be sequenced. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515f/806r [25 (link)] in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscientific, Waltham, MA, USA). PCR was performed in 20 μL vol and included 2 μL (7.5 μM concn) of forward and reverse primers, 12.5 μL of Hot Start Taq 2X Master Mix (New England BioLabs Inc., Ipswich, MA., USA), 3.5 μL of deionized water, and 2 μL of sample DNA. Thermal cycle conditions were 95 °C for 3 min for the initial denaturing step, followed by 30 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx. 350 bp). The Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing, following the manufacturer’s guidelines [25 (link)]. Reagent controls using certified DNA free water were run through library preparation and PCR and did not generate libraries. For quality control, samples submitted for sequencing included a random blank sample of technical replicates.

Microbial DNA was extracted from fecal samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen) according to manufacturer's protocol. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515/806 in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscieniftic). PCR was performed in 20 μl vol and included: 2 μl (7.5 μM concn) of forward and reverse primers, 12.5 μl of Hot Start Taq 2X Master Mix (New England BioLabs Inc.), 3.5 μl of deionized water, and 2 μl of sample DNA. Thermal cycle conditions were 95°C for 3 min for initial denaturing step, followed by 30 cycles of 95°C for 30 s, 50°C for 1 min, and 72°C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx 350 bp). Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing following the manufacturer's guidelines. Reagent controls using certified DNAfree water were run through library preparation and PCR and did not generate libraries. For quality control, samples submitted for sequencing included a random blank sample of technical replicates.

Microbial DNA was extracted from fecal samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen, USA) according to manufacturer’s protocol, with the initial step of the protocol beginning with vertexing the sample with beads to break up the material. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515f/806r [25 (link)] in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscieniftic, Waltham, MA). PCR was performed in 20 μl vol and included: 2 μl (7.5 μM concn) of forward and reverse primers, 12.5 μl of Hot Start Taq 2X Master Mix (New England BioLabs Inc., Ipswich, MA., USA), 3.5 μl of deionized water, and 2 μl of sample DNA. Thermal cycle conditions were 95˚C for 3 min for initial denaturing step, followed by 30 cycles of 95˚C for 30 s, 50˚C for 1 min, and 72˚C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx 350 bp). Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing following the manufacturer’s guidelines [25 (link),26 (link)]. Reagent controls using certified DNA free water were run through library preparation and PCR and did not generate libraries. For quality control, samples submitted for sequencing included a random blank sample of technical replicates.