Full description of sample processing and amplicon sequencing is described in the Supplementary file. In brief, DNA was extracted from saliva and subgingival plaque using bead‐beating procedure and the Mag MiniKit (LGC Genomics, Berlin, Germany, Mag mini kit). Bacterial DNA concentration was determined by16S rRNA gene specific qPCR.[17 ] V4 hypervariable region of the 16S rRNA gene was amplified,[18 ] the amplicons were pooled equimolarly, purified and paired‐end reads were generated using the Illumina MiSeq platform (Illumina, Inc., San Diego, CA). The reads were processed into OTUs as described previously.[19 ] The most abundant sequence of each OTU was classified using the RDP classifier[20 ] and HOMD version 14.51.[21 ]
Mag minikit
Manufactured by LGC
Sourced in Germany, United Kingdom
The Mag MiniKit is a compact magnetic bead-based nucleic acid purification system designed for high-throughput sample processing. It provides a streamlined solution for the extraction and purification of DNA, RNA, or other target molecules from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Miseq platform, by Illumina (2 mentions)
Kingfisher flex dna extraction robot, by Thermo Fisher Scientific (2 mentions)
Lysing matrix beads, by MP Biomedicals (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Agarose gel, by Qiagen (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Magna lyser, by Roche (1 mentions)
Star buffer, by Roche (1 mentions)
Fastprep 96, by MP Biomedicals (1 mentions)
6 protocols using mag minikit
1
Microbiome Analysis of Saliva and Plaque Samples
2
DNA Extraction from Nasal Swabs
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DNA was previously isolated from nasal swab samples using the mag mini kit (LGC Standards, Wesel, Germany) and an adjusted protocol that included an initial bead-beating step. In short, 200 µL of nose swab medium combined with 200 µL phenol and 150 µL Lysis buffer BL (LGC Standards, Wesel, Germany) was added to a vial containing Lysing Matrix beads (MP Biomedicals, Eschwege, Germany) and subjected to bead-beating using a FastPrep-24 (MP Biomedicals, Eschwege, Germany) at 6m/s for 60 s. After centrifugation, 200 µL of the water phase (top layer) was incubated for 2 min at room temperature with 400 µL binding buffer BL (LGC Standards, Wesel, Germany), to which 10 µL mag particle suspension (LGC Standards, Wesel, Germany) had been added. The manufacturer’s protocol was then followed, with the exception that the DNA was eluted by incubating for 30 min at 55 °C instead of 10 min. Prior to 16S rRNA gene sequencing, the total number of 16S rRNA gene copy numbers within each DNA extract was measured using a 16S rRNA gene quantitative PCR as previously described [19 (link)].
3
Biofilm DNA extraction and sequencing
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Three discs were removed from the model every week and the biofilms on these discs were dispersed in 1mL sterile water by sonicating the discs for 1 min, pelleted for 15 min at 4500 rpm and stored at −80 °C for sequencing. The DNA of all samples was extracted and purified according to Cieplik et al. (Cieplik et al., 2019 (link)). In brief, the samples were added to wells of a 96-deep-well plate containing Tris-saturated phenol, 0.1 mm zirconium beads and lysis buffer and were mechanically lysed by bead-beating at 1,200 rpm for 2 min. DNA was isolated with the Mag MiniKit (LGC Genomics, Berlin, Germany).
Quantitative PCR was used to determine the bacterial DNA concentration in the biofilm samples, using universal primers specific to the bacterial 16S rRNA gene (Ciric et al., 2010 (link)). The V4 hypervariable region of the 16S rRNA gene was amplified using 1 ng DNA with 1 µM of each primer and 30 amplification cycles (Caporaso et al., 2011 (link)). Paired-end sequencing of the DNA was conducted on the MiSeq platform (Illumina, San Diego, CA, USA) with a MiSeq Reagent kit v3 and 2x251 nt at the VUmc Cancer Center Amsterdam (Amsterdam, the Netherlands). The sequence and meta data are available in the NCBI BioProject database under accession numberPRJNA614901 .
Quantitative PCR was used to determine the bacterial DNA concentration in the biofilm samples, using universal primers specific to the bacterial 16S rRNA gene (Ciric et al., 2010 (link)). The V4 hypervariable region of the 16S rRNA gene was amplified using 1 ng DNA with 1 µM of each primer and 30 amplification cycles (Caporaso et al., 2011 (link)). Paired-end sequencing of the DNA was conducted on the MiSeq platform (Illumina, San Diego, CA, USA) with a MiSeq Reagent kit v3 and 2x251 nt at the VUmc Cancer Center Amsterdam (Amsterdam, the Netherlands). The sequence and meta data are available in the NCBI BioProject database under accession number
4
Microbiome Analysis of Biofilm Samples
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Two of the four replica biofilms of each condition were used for microbiome analysis. DNA was isolated as described previously, using phenol bead beating followed by Agowa nucleic acid isolation according to the manufacturer's instructions (LGC Genomics, Mag mini kit) (9 (link)). Briefly, cells were mixed with TRIS-saturated phenol, pH 8, 0.1 mm zirconium beads, and Mag lysis buffer and were mechanically disrupted four times at 1,200 rpm for 2 min. The DNA containing phase was added to binding buffer and magnetic titanium beads. After washing the beads, according to the Agowa Mag mini DNA extraction protocol, the DNA was eluted from the beads with 63 µl elution buffer (Agowa). The amount of bacterial DNA after purification was determined by QPCR, using a universal primers-probe set targeting the 16S rRNA gene (15 (link)).
For amplification of the V4 hypervariable region of the 16S rRNA gene 100 pg DNA was used as described previously (15 (link)) except that 5 µM of each primer was used and 30 cycles were performed. Using Fragment analyzer, the generated amplicons were checked for quality, pooled equimolarly, and purified from agarose gel (Qiagen, Roermond, The Netherlands). Paired-end sequencing of the amplicons was conducted on the Illumina MiSeq platform (Illumina Inc., San Diego, CA) using the Illumina MiSeq reagent kit V2 to generate 200-bp paired-end reads.
For amplification of the V4 hypervariable region of the 16S rRNA gene 100 pg DNA was used as described previously (15 (link)) except that 5 µM of each primer was used and 30 cycles were performed. Using Fragment analyzer, the generated amplicons were checked for quality, pooled equimolarly, and purified from agarose gel (Qiagen, Roermond, The Netherlands). Paired-end sequencing of the amplicons was conducted on the Illumina MiSeq platform (Illumina Inc., San Diego, CA) using the Illumina MiSeq reagent kit V2 to generate 200-bp paired-end reads.
5
Fecal DNA Isolation and Purification
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Fecal samples were thawed on ice and ~ 200 mg from each sample was added to sterile water at a ratio of 1:3. Homogenization involved bead beating using a MagNaLyser (Roche, Basel, Switzerland) twice at 6500 rpm for 20 s with 1 minute cooling at 4°C between runs as described previously [30 (link)]. DNA was extracted using the Mag Mini LGC kit (LGC Genomics, Hoddesdon, UK) according to the manufacturer's recommendations using a KingFisher Flex DNA extraction robot (Thermo Fisher Scientific, Waltham, MA, USA). Adequate DNA quality and quantity in samples were ensured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). DNA samples were stored at -20°C until processing.
6
Fecal Sample Preparation for Microbiome Analysis
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Right after dissection, fecal samples were collected from the colon and placed in 400 μL S.T.A.R buffer (Roche, USA) with acid-washed glass lysing beads (approximately 0.2g <106 μm, 0.2g of 425–600 μm and 2–4 beads of 2mm Sigma-Aldrich) and stored at -80°C for further processing. All samples were homogenized twice on FastPrep 96 (1,800 rpm, 40 sec, 5 min cooling step in-between, MP BioMedicals). Processed samples were then centrifuged (13,000 rpm, 10 min) and 50 μL supernatant was transferred to 96-well plates for protease treatment and DNA extraction using Mag Mini LGC kit (LGC Genomics, UK) on KingFisher Flex DNA extraction robot (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.
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