16S rDNA Sequencing of Gut Microbiome

Total DNA from the stool samples was extracted using the ZymoBiomics DNA/RNA mini kit (ZymoResearch Scientific, Irvine, CA, USA) in accordance with the manufacturer’s protocol. DNA from each sample was amplified using specific primers targeting the V1–V3 region of 16S rDNA [37 (link)]. PCR reaction contained 1 ng of DNA, 5xFIREPol MasterMix (Solis BioDyne, Tartu, Estonia) and 2  µM of each primer (10 µM). The reaction conditions for PCR amplification were 95 °C for 15 min; 25 cycles of 95 °C for 20 s, 56 °C for 30 s and 72 °C for 1 min; and final elongation at 72 °C for 5 min. The products of amplification were verified by agar electrophoresis. DNA libraries for Illumina sequencing were prepared using the index PCR reaction with input of 1 ng of DNA. The reaction conditions for the PCR were 95 °C for 15 min; 12 cycles of 95 °C for 10 s, 55 °C for 30 s and 72 °C for 90 s; and final elongation at 72 °C for 5 min. Index PCR amplification products were purified using 1.8x Agencourt AMPure XP magnetic beads (BeckmanCoulter, Brea, CA, USA). DNA libraries were validated by Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Mixed amplicons were pooled and sequenced using an Illumina MiSeq platform via a 300 bp paired-end reads Illumina sequencing system (Illumina, San Diego, CA, USA).