CTRL_MS_33_9
From the extracted DNA, the hypervariable V1-V3 region of the 16S rRNA gene (506 bp) was amplified using the corresponding primers (27F: 5-AGA GTT TGA TCM TGG CTC AG-3; 533R: 5-TTA CCG CGG CKG CTG GCA CG-3. PCR was prepared in a final volume of 25 L using 300 ng of template DNA and a primer concentration of 0.4 M. The reaction was performed using the LongAmp Hot Start Taq 2 Master Mix (New England Biolabs) setting up the same conditions than in Domingo-Bretn et al., 2024. Amplicons were purified using AMPure XP magnetic beads (Backman Coulter, Brea, CA, USA) with a beads/sample ratio of 0.6, and eluted in a final volume of 30 L of water. All samples were then quantified by fluorescence using the PicoGreen kit (Thermo Fisher, Waltham, MA, USA), and the presence of the specific ~500 bp band was verified by agarose gel electrophoresis (1.5% w/v in Tris-EDTA buffer). Library preparation for sequencing was performed using the Native Barcoding Kit 96 V14 (Oxford Nanopore Technologies, Oxford, UK). All PCR products were diluted equimolarly to a final concentration of 200 femtomoles in a total volume of 12.5 L. Next, end repair of the sequences was carried out using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA), following the manufacturer's instructions. Then, in a first ligation reaction, unique barcoding sequences were added. Once this step was completed, the sequences from each sample were molecularly identified, allowing all samples to be pooled into a single tube. Subsequently, a purification step using magnetic beads was performed, followed by a second ligation reaction to attach the sequencing adapters. Another purification step was carried out after the second ligation, and the final DNA concentration in the library was quantified by fluorescence using PicoGreen. Sequencing of the libraries was performed using the MinIONTM sequencing device from the Oxford Nanopore platform. A total of 35 femtomoles of library were loaded onto an R10.4.1/FLO-MIN114 flow cell. The acquisition of raw sequencing data and the demultiplexing process (identification of the barcodes for each recorded sequence) were carried out using the MinKNOW software v23.07.8. The basecalling process (assignment of the corresponding base to each position) was performed using the Dorado v0.7 (https://github.com/nanoporetech/dorado; last accessed: 25 August 2024) algorithm with the most accurate model developed to date (SUP model).