Illustratm gfxtm pcr dna and gel band purification kit

Restriction endonucleases and Q5 High-Fidelity polymerase were purchased from New England Biolabs (Ipswich, MA, United States). Calf intestinal alkaline phosphatase (CIAP) was purchased from Invitrogen (Waltham, MA, United States). Gibson Assembly® Ultra Kit was purchased from VWR (Chester, PA, United States). All the primers employed were purchased at Sigma-Aldrich (St. Louis, MO, United States) and are described in Table 2. A QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was employed for the purification of plasmid DNA, and the Illustra^TM GFX^TM PCR DNA and Gel Band Purification Kit (GE Healthcare, Boston, MA, United States) was used for the purification of DNA amplicons from agarose gels and enzymatic reactions.

Amplicon libraries of the V4 hypervariable region of the 16S rRNA gene were generated for each of the individual samples. PCR was performed using the forward primer 515F (GTGCCAGCMGCCGCGGTAA) and the reverse primer 806R (GGACTACHVGGGTWTCTAAT) [25 (link)]. The primers included Illumina adapters and a unique 8-nt sample index sequence key [26 (link)]. The amplification mix contained 2 units of Phusion HotStart II High fidelity polymerase (Thermoscientific), 1 unit Buffer Phusion HS II [5x], including 1.5 mM MgCl₂ (Thermoscientific), 0.2 mM dNTP (Thermoscientific, Germany) and 1 μM of each primer. To each reaction 1 ng of DNA template was added. After denaturation (98°C; 30 sec), 35 cycles of denaturation (98°C; 10 sec), annealing (55°C; 30 sec), and extension (72°C; 30 sec) were performed. Individual amplicon libraries were analyzed for DNA content with the fluorescent Quant-iT™ PicoGreen^® dsDNA Assay Kit (Invitrogen). The libraries were pooled in equimolar amounts. The amplicons were purified by means of the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare, Eindhoven, the Netherlands). The quality and the size of the amplicons were analyzed on the Agilent 2100 (Santa Clara, CA, USA). The amplicon was sequenced in paired end mode on a MiSeq sequencing system (Illumina, Eindhoven, the Netherlands) with the v2 kit (Illumina) [26 (link), 27 (link)].

Genomic DNA samples were used as templates for amplification of the nifH gene of approximately 360 bp following the nested PCR protocols of Zehr et al. (1998) (link) and using a FastStart High Fidelity PCR system, dNTPack (Roche, Switzerland) with a Peltier Thermal Cycler (Bio-Rad, USA). In order to enable sample multiplexing during sequencing, barcodes were incorporated between the adapter and forward primer. Nuclease-free water was used as the negative control in each reaction. Triplicate PCRs were performed for each sample and the amplicons were pooled and subsequently purified with the illustra^TMGFX^TMPCR DNA and Gel Band Purification kit (GE Healthcare, Little Chalfont, Bucks, UK). An amplicon library was constructed with equimolar concentrations of the amplicons, and emPCR was conducted according to the Rapid Library preparation kit instructions (Roche, Switzerland). DNA beads were successfully deposited onto the PicoTiterPlate and sequenced with a GS Junior system (Roche, Switzerland).

16S rDNA amplicon sequencing of the V4 hypervariable region was performed as described previously⁵⁰ . Each sample was PCR amplified using 1 ng of template DNA with primers F515/R806, targeting the V4 hypervariable region of the 16 s ribosomal gene^{51 (link)}. In each batch of samples, a mixed pure culture isolates (mock), blanco extraction controls, pooled salivary extraction controls and pooled DNA amplification controls were included. The amount of DNA per sample was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific). The amplicon libraries were pooled in equimolar amounts and purified using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare, Eindhoven, The Netherlands). Amplicon quality and size was analyzed on the Fragment Analyzer (Advanced Analytical). Paired-end sequencing of amplicons was conducted in five separate runs on the Illumina MiSeq platform (Illumina, Eindhoven, The Netherlands). Denoising of sequence data and identification of ASVs were performed using the DADA2 (1.12.1) pipeline^{52 (link)}.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of the index case and a healthy control by Ficoll gradient centrifugation according to the manufacturer’s instructions (Ficoll-Paque PLUS, GE Healthcare).
Total RNA was isolated from PBMCs using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific). The cDNAs were synthesized from 1 μg of total RNA using random hexamers with the SuperScript IV-First Strand Synthesis System kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 20 μL.
Subsequently, cDNA of PGK1 was amplified and Sanger sequenced using specific primers surrounding the exon 9 of PGK1. The sequence of the upstream primer, located in the exon 7–8 junction of PGK1, was 5′-GTGCTCAACAACATGGAGAT-3′, and the sequence of the downstream primer located inside the exon 11 of PGK1 was 5′-TAAATATTGCTGAGAGCATCCA-3′. Amplicons were visualized on a 1% agarose gel using gel red and were purified from the gel with IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare) to be analyzed separately by direct sequencing (ABI 3500 Genetic Analyzer, Applied Biosystems, Warrington, UK) using a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK). The chromatograms were analyzed with Chromas 2.3 (Technelysium Pty Ltd.).

Restriction endonucleases, Q5 High-Fidelity polymerase and the T4 DNA Ligase (Blunt/TA Ligase Master Mix) were purchased from New England Biolabs (Ipswich, MA, USA). Calf Intestinal Alkaline Phosphatase (CIAP) was purchased from Invitrogen (Waltham, MA, USA). All the primers employed were purchased at Sigma-Aldrich (St. Louis, MO, USA). A QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was employed for the purification of plasmid DNA from cells and the Illustra^TM GFX^TM PCR DNA and Gel Band Purification Kit (GE Healthcare, Boston, MA, USA) was used for the purification of DNA amplicons from agarose gels and enzymatic reactions.