Magna pure lc dna isolation kit 3

Fecal samples stored in RNAlater^® were diluted with PBS solution (dilution1:2). To remove fecal debris, the samples were centrifuged at 2000 rpm at 4 °C for 2 min. Total DNA was extracted from pelleted bacterial cells in the robotic workstation The MagNA Pure LC Instrument (Roche) using the MagNA Pure LC DNA isolation kit III (Bacteria, Fungi) (Roche) according to the manufacturer’s instructions. The region V3-V4 of the 16S rRNA gene was amplified by PCR and used to amplicon library construction following Illumina instructions. Metagenomic libraries were constructed using NEXTERA XT kit according to the manufacturer’s instructions (Illumina). Sequencing was performed with the Kit V3 (2 × 300 cycles) in MiSeq platform (Illumina, Eindhoven, Netherlands) in the Centre for Public Health Research (FISABIO-Salud Pública, Valencia, Spain). All sequences were deposited in the public European Nucleotide. Archive server under accession number PRJEB32411.

Total genomic DNA was isolated from previously frozen samples using a combination of mechanical and enzymatic lysis followed by bacterial DNA extraction with the MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi; Roche, Mannheim, Germany) according to previously described methods (Klymiuk et al., 2016 (link); Kump et al., 2018 (link)) on a MagNA Pure LC 2.0 instrument (Roche Diagnostics, Mannheim, Germany). Hypervariable region V4-specific PCR amplification was performed as described by Klymiuk et al. (2016) (link) using the primer set 515F-5′-GTGCCAGCMGCCGCGGTAA and 806R-5′-GGACTACHVGGGTWTCTAAT synthesized at Eurofins (MWG, Ebersberg, Germany). PCR reaction for each sample was performed in triplicates. Amplicons were purified as previously described (Klymiuk et al., 2016 (link)), quantified, pooled, and finally sequenced on a MiSeqII desktop sequencer (Illumina, Eindhoven, Netherlands) using V3 chemistry (MiSeq Reagent Kits v6, Illumina, Eindhoven, Netherlands) and a paired-end protocol according to manufacturer’s instructions.

Isolation of total genomic DNA, PCR, and FLX sequencing were performed as recently described in Kump et al. [28 (link)]. Briefly, a MagnaLyser Instrument (Roche Diagnostics, Mannheim, Germany) and a MagNA Pure LC 2.0 Instrument (Roche Diagnostics) with the MagNA Pure LC DNA Isolation Kit III (bacteria, fungi) were used for automated DNA isolation according to the manufacturer’s instructions. For library preparation, 454 one-way read strategy (Lib-L kit, Primer A, Primer B; Roche 454 Life Science, Branford, CT, USA) amplifying the 16S rRNA hyper variable regions V1–2 with the target specific primers F27-AGAGTTTGATCCTGGCTCAG and R357-CTGCTGCCTYCCGTA was used. FLX fusion primers with FLX adaptor, key, and MID sequences were used for amplification (supplementary table S1). For each sample, a PCR was carried out with 50 ng total genomic DNA in a 25-µL reaction volume, performed in triplicate. Amplicons were purified according to Kump et al. [28 (link)] and pooled and sequenced using the GS FLX Titanium Sequencing Kit XLR70 (Roche 454 Life Science, Branford, CT, USA) according to manufacturer’s instructions.

Total nucleic acids (TNA) were extracted from 100 μL of EDTA-anticoagulated blood using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostic) as previously described [2 (link)]. The saliva and rectal swabs were incubated in PBS at 40 °C for 10 min prior to TNA extraction as previously described [25 (link)]. TNA was extracted from 100 μL of urine using the MagNA Pure LC DNA Isolation Kit III (Bacteria, Fungi) (Roche Diagnostic). As pre-isolation steps, Bacterial lysis Buffer and Proteinase K were added, and the mixture was incubated at 65 °C for 10 min. The subsequent procedure was performed according to the manufacturer’s instructions.
TNA was eluted into 100 μL of elution buffer and stored at −20 °C until further use. During all nucleic acid extractions, negative controls of 100 μL of PBS were concurrently prepared with each batch of samples to monitor cross-contamination.
All TNA samples were tested using TaqMan® real-time qPCR for the presence and quantity of M. haemofelis and “Cand. M. turicensis” on an ABI PRISM 7700/7500 Sequence Detection System (Applied Biosystems, Rotkreuz, Switzerland) and a 10-fold serial dilution of plasmid standards as previously described [2 (link),14 (link)]. For all PCR reactions, positive (plasmid standard) and negative controls (nuclease free water) were included in each assay.

Tongue biofilm was taken from the middle third of the tongue dorsum with a sterile spatula [7 (link)]. The spatula was transferred into 2.0 ml of phosphate-buffered saline (PBS). After shaking vigorously for 30 s, the spatula was removed. Microbial suspensions in PBS were kept at −80°C until further processing. After supragingival plaque was removed with a cotton roll, subgingival plaque was collected from the mesiobuccal pocket of the most distally located, clinically examined, upper tooth in the periodontally examined quadrants. Paper points (ISO 35; Roeka, Langenau, Germany) were inserted until the pocket base for 10 seconds. To avoid cross contamination, the sampling site was confined with cotton rolls. Paper points were stored at −80°C. Before DNA extraction, 230 µl of lysis buffer and 20 µl of proteinase K (both MagNA Pure LC DNA Isolation Kit III, Roche, Mannheim, Germany) were added and samples were incubated at 65°C for 10 min and then at 95°C for 10 min. DNA was extracted as described elsewhere [7 (link)] and stored at −20°C.

Stool samples of patients and donors were immediately frozen and stored at −20°C. DNA extraction from stool samples was performed by mechanical lysis with a MagnaLyser Instrument (Roche Diagnostics, Mannheim, Germany) and subsequent total bacterial genomic DNA isolation with the MagNA Pure LC DNA Isolation Kit III (bacteria, fungi) in a MagNA Pure LC 2.0 Instrument (Roche Diagnostics) according to the manufacturer's instructions.12 For amplification of bacterial 16S rRNA gene, the template‐specific sequence 515F‐5′‐GTGCCAGCMGCCGCGGTAA‐3′ and 806R‐5′‐GGACTACHVGGGTWTCTAAT‐3′, targeting the hypervariable region V4 of the 16S rRNA gene were used.26 PCR reactions for each sample were performed in triplicates. Subsequently, the amplicons were purified according to standard procedures, quantified, pooled and sequenced with the MiSeq Reagent Kits v3 (600 cycles, Illumina, Eindhoven, Netherlands) according to manufacturer's instructions with 20% OhiX (Illumina). The generated FASTQ files were used for microbiota analysis.