Pathogen lysis tube

Mechanical lysis of cell walls was accomplished with bead beating. One aliquot of enriched urine samples (200 μl) was transferred into Pathogen Lysis Tubes (Qiagen) with glass beads, and 50 μl of Buffer ATL (containing Reagent DX, Qiagen) was added according to the manufacturer’s instructions. The Pathogen Lysis Tubes were then attached to a horizontal platform on a vortex mixer and vortexed for 10 min at maximum speed. After that, the Pathogen Lysis Tubes were removed and briefly spined to collect any drops from the inside of the lid. DNA was extracted from the supernatant using the IndiSpin Pathogen Kit as described in Method 1.

Milk samples (5–10 mL) were thawed and centrifuged at 4000 × g for 20 min to separate fat and cells from whey. Thereafter, total DNA was isolated from the pellets by using the MasterPure Complete DNA and RNA Purification Kit (Epicenter) according to the manufacturer's instructions with some modifications (Simón-Soro et al., 2015 (link)). Two hundred and fifty microliters of saline solution and 250 μl of lysis buffer were added to the pellets, together with Pathogen Lysis Tubes (QIAGEN) glass beads. Both chemical and physical cells disruption was performed after mixing vigorously the samples in a TissueLyser II (QIAGEN) during 5 min at 30 Hz, incubating in dry ice 3 and 5 min at 65°C in a thermoblock, repeating the process 2 times. Fifty microliters of lysozyme (20 mg/ml) and 5 μl of lysostaphin (20 μg/ ml) were added to the tubes, and the samples were incubated for 1 h at 37°C. Two microliters of proteinase K were added and samples were incubated for 15 m at 65°C. The reaction was ended putting tubes on ice, and proteins were precipitated using 350 μl of the protein precipitation agent, discarding the pellets. DNA was precipitated using isopropanol, washed with 70% Ethanol and resuspended with 30 μl TE buffer. The total DNA isolated was quantified with a NanoDrop ND-1000 (ThermoScientific) spectrophotometer.

Every morning, two milliliters of saliva were collected from each participant into a drool collection kit before brushing teeth. The saliva samples were transferred into storage tubes and stored in a freezer. For DNA extraction, 100 μL of whole saliva was diluted with 400 μL of phosphate buffered saline to reduce viscosity, and the diluted saliva was transferred to a Pathogen Lysis Tube S (QIAGEN N.V.) and mechanically disrupted for 10 min using a Disruptor Genie (Scientific Industries). The resultant solution was subsequently processed using the QIAamp UCP Pathogen Mini Kit (QIAGEN N.V.) according to the manufacturer’s instruction. Double stranded DNA concentrations were measured using the Qubit HS Assay Kit (ThermoFisher SCIENTIFIC). Additionally, the collected saliva was thawed and centrifuged at 1500 g for 15 min, and the supernatants were utilized in an ELISA testing below.

Media samples were centrifuged at 6000xg to pellet bacteria and resuspended in 500µL buffer ATL (Qiagen). Pellets were subjected to mechanical lysis at maximum speed for 10 minutes in Pathogen lysis tubes (Qiagen), before DNA extraction using QIAamp UCP Pathogen Mini Kit (Qiagen), according to the manufacturer's protocol.

Frozen tumor tissues were aseptically sliced into appropriate sizes and placed in 1.5 ml sterile tubes. We utilized β-mercaptoethanol and a rotor-stator homogenizer to disrupt the tissue and homogenize the lysate. Metagenomic DNA and total RNA were simultaneously extracted using QIAGEN Pathogen Lysis Tubes and QIAGEN AllPrep DNA/RNA Mini Kit. DNA/RNA was quantified for concentration and purity using a NanoDrop spectrophotometer and stored at −20 °C. DNA samples were applied to library construction and whole-genome sequencing on the Illumina platform and yielded 2 × 150 bp paired-end reads. After discarding the adaptor contamination and low-quality reads, an average of 80 Gb of clean data per sample was obtained. Meanwhile, samples of RNA were sequenced for the whole transcriptome on the Illumina platform (paired-end, 2 × 150 bp reads) and yielded an average of 15 Gb of clean data per sample.