Zirconium glass beads

Microbial DNA was extracted by sampling 300 mg of feces from each sample. Genomic DNA was isolated by adding bead-beating using a QIAamp Power Fecal DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Zirconium glass beads (400 mg; 0.1 mm diameter, BioSpec Products, Bartlesville, OK, USA) were added to the extraction system followed by vigorous vortexing (twice) using a FastPrep-24 Instrument (MP Biomedicals, Solon, OH, USA) at a speed of 6.0 m/s for 90 s. Extracted DNA was confirmed using agarose gel electrophoresis.
The 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) forward primer and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) reverse primer were used to amplify the V3-V4 hyper-variable region of the 16S rRNA gene. PCR conditions were: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and elongation at 72 °C for 30 s for 25 cycles. Finally, extension was done at 72 °C for 5 min. The PCR product was purified and used to construct a library, and then, paired-end sequencing (2 × 250) was performed on a MiSeq platform (Illumina, San Diego, CA, USA) from Novogene Co. Ltd. (Beijing, China).

After tissue homogenates in sealed containers were thawed on ice, 400 μl (equivalent to 160 mg of tissue) was added to tubes containing 1.2 ml of ASL lysis buffer (QIAamp DNA stool kit; Qiagen) and 400 mg of 0.1-mm-diameter zirconium glass beads (BioSpec Products). Then, 800 μl of the PBS control and 800 μl of the skin swab control were also added to tubes containing ASL buffer and beads. Mechanical and chemical lyses were performed on all samples by bead beating at 4,800 rpm for 60 s at room temperature and then 60 s on ice (repeated twice) (Mini-beadbeater-1; BioSpec Products), after which the suspension was incubated at 95°C for 5 min. Subsequent procedures were performed using the Qiagen QIAamp DNA stool kit according to the manufacturer's protocol, with the exception of the last step, in which the column was eluted with 120 μl of elution buffer. DNA was stored at −20°C until further use.

After thawing on ice, 2 ml of milk were spun down at 20,000×g for 10 min and the supernatant was discarded. The pellet was then homogenized in 1.4 ml of ASL buffer (QIAamp^® DNA Stool Kit, QIAGEN: Valencia, CA, USA) and 400 mg of 0.1-mm diameter zirconium-glass beads (BioSpec Products, Bartlesville, OK, USA). Mechanical and chemical lyses were performed by bead beading at 4800 rpm for 60 s, then 60 s on ice (repeated twice) using a mini-beadbeater-1 (BioSpec Products) and then incubated at 95 °C for 5 min. Subsequent procedures were performed using the QIAGEN QIAamp^® DNA Stool Kit according to the manufacturer’s protocol, with the exception of the last step in which the column was eluted with 120 μl of elution buffer. DNA was stored at −20 °C until further use.
A no template PCR control and a DNA extraction kit reagent control were sequenced alongside the samples. We observed that the taxon abundances in the controls were uncorrelated with the abundances in the experimental samples, and the distance between the controls and samples was large. Thus, we conclude that the controls had different profiles than that of the milk samples (Additional file 7: Figure S4).