Nanodrop

A commercial kit (E.Z.N.A. Stool DNA Kit, Omega Bio-Tek Inc., Doraville, Georgia, USA) was used for DNA extraction and DNA quantity and quality were assessed by spectrophotometry (Nano Drop, Roche, Mississauga, Canada). The V4 region of the 16S rRNA gene was then amplified using primers 564F and 785R [15 (link)] and amplicons were sequenced by Illumina MiSeq using 2X250 chemistry, providing fully overlapping paired end reads. Negative (PCR water instead of template) controls were included in all runs.

DNA from stool samples was extracted using QIAamp® Fast DNA Stool Mini (Cat No./ID: 51604, QIAGEN) following the manufacturer’s “Fast DNA Stool Mini Handbook” for fast purification of genomic DNA. Eluted DNA was collected in 1.5 mL DNA Lo-Bind microcentrifuge tubes, and the quantity and quality of DNA were assessed by Qubit 2.0 DNA HS Assay (Thermo Fisher, Massachusetts, USA) and NanoDrop® (Roche, USA) to meet the sequencing requirements.

DNA extraction was performed using a commercial kit (E.Z.N.A. Stool DNA Kit, Omega Bio-Tek Inc., Doraville, Georgia, USA) following the manufacturer’s “stool DNA protocol for pathogen detection”. DNA quantity and quality were accessed by spectrophotometry (NanoDrop, Roche, Mississauga, Canada).

The mucosal samples were rinsed with sterile saline only once or twice to remove visible ingesta. This step was performed with care, in order to prevent any disruption of the mucus layer. DNA was extracted from mucosal tissues and intestinal content samples using the QIAamp DNA stool mini kit for pathogen detection (Qiagen, Montreal, QC, Canada) as per manufacturer’s instructions.
The DNA was diluted to a final concentration of 20 ng/μL for PCR. The 16S rRNA genes were amplified targeting the V3-V4 region [22 (link)]. The V3-V4 region of the 16S rRNA gene was amplified in a PCR reaction mixture containing 25 μL of Kapa 2G Fast Hot Start Ready Mix 2×, 1.3 μL of MgCl₂ (50 mM) (Invitrogen, Burlington, ON, Canada), 1.0 μL of BSA (2 mg/mL) (Bio-Rad, Mississauga, ON, Canada), 16.7 μL of nuclease-free water, 2 μL of DNA and 2 μL of forward (S-D-Bact-00564-a-S-15 5′-AYTGGGYDTAAAGNG-3′) and reverse (S-D-Bact-0785-b-A-18 5′-TACNVGGGTATCTAATCC-3′) primers (10 pMol/μL).
PCR products were then purified with magnetic beads and DNA quantification was measured by spectrophotometry using the NanoDrop^® (Roche, Mississauga, ON, Canada). The library was pooled and sequencing was at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform using a V3 kit (2 × 300 cycles).

DNA extraction was performed using the E.N.Z.A. Stool DNA Kit (Omega Bio-Tek Inc., Doraville, Georgia, USA) following the manufacturer’s protocol for pathogen detection. Quantity and quality of extracted nucleic acids were assessed by spectrophotometry (NanoDrop, Roche, Mississauga, ON, Canada).