Qiaquick gel elution kit

Prior to extracting genomic DNA from the stools, the samples were stored at −80°C. THSTI method⁴¹ (link) was used to extract DNA from approximately 200 mg of frozen fecal samples. The purity and content of DNA were evaluated using a Biospectrometer (Eppendorf, Germany) and 0.8% agarose gel electrophoresis. Variable regions V1-V5 of the 16S rRNA genes were amplified in 50 μL of reaction volume using 0.1 ng of template DNA with 27F (C1) and 926R (C5) primers. The 950-bp PCR products were gel purified using the QIAquick gel elution Kit (Qiagen, Germany). At THSTI in India, equimolar concentration amplicon libraries were assembled and sequenced using a Roche 454 GS FLX+ pyro-sequencer. For 16S rRNA gene sequencing through Miseq Illumina, extracted DNA was quantified with Qubit fluorometer to obtain a correct amount of the dsDNA. Genomic DNA with concentration of 5 ng/μl was selected for library preparation for 16S rRNA gene sequencing in the Illumina MiSeq NGS platform. For PCR amplification, 25μL reaction contained 2x KAPA HiFi Hot Start ReadyMix, both the primers and 2.5μL of the template DNA were used. Amplified products were cleaned using AMPure XP beads, tagged with Illumina sequencing adapters and created the sequencing library.