Metagenomic Profiling of Gut Microbiome

DNA was extracted from the Pakistani and Finnish participants’ fecal samples using the same procedure, Repeated Bead Beating (RBB) method,^{73 (link)} with the following modifications for automated DNA purification: Approximately, 0.25 g of fecal samples and 340 µL and 145 µL of lysis buffer were used on the first and second rounds of bead beating, respectively. Then, 200 µL of the clarified supernatant collected from the two bead beating rounds was used for DNA extraction with the Ambion Magmax™ −96 DNA Multi-Sample Kit (4413022, Thermo Fisher Scientific, USA) using the KingFisherTM Flex automated purification system (ThermoFisher Scientific, USA). DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA). Library preparation and Illumina MiSeq sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene using primers 341 F/785 R were performed as previously described.^{74 (link)} To characterize the gut fungal community, we performed ITS-1 sequencing for the Pakistani samples using a two-step PCR protocol described in detail elsewhere.^{75 (link)} PCR-amplicons of the ITS-1 region were generated using ITS1F and ITS2 primers.^{75 (link),76 (link)}