16S rRNA-seq gut microbiome: Mexico
The taxonomic identification of bacteria relied on the sequences derived from the V3 and V4 variable regions of the 16S rDNA gene. To construct the libraries, the Illumina protocol (16S Metagenomic Sequencing Library Preparation) (Cod. 15044223 Rev. B) was followed. Initially, microbial genomic DNA was stored at a concentration of 5 ng/L in 10 mM Tris pH 8.5. Subsequently, fluorometric quantification using the Qubit fluorometer (ThermoFisher Scientific) ensured accurate adjustments to the DNA concentration. The DNA was then subjected to amplification using specific primers targeting the V3-V4 regions of the 16S rRNA gene [18], followed by ligation to an adapter, which facilitated the multiplexing step through the incorporation of indices to distinguish individual samples via the Nextera XT Index Kit (FC-131-1096). Prior to sequencing, the library was quantified once again using the Qubit fluorometer. Sequencing was carried out with 2 x 300 bp paired-end reads (MiSeq v3 reagent kit (MS-102-3001)) on a MiSeq sequencer, following the manufacturer's guidelines (Illumina).