Illumina MiSeq paired end sequencing
PCR amplification of 16s rRNA
Two sets of PCRs targeting the V4 hypervariable region of the 16S ribosomal ribonucleic acid (rRNA) gene using the primers 515F and 806R were performed. Amplicons were cleaned using the Agencourt® AMPure® XP PCR Purification kit (Beckman Coulter, California, USA) . Each of the purified PCR products was quantified using the QuantiFluor® dsDNA System quantification kit on Promega Glomax®-Multi Detection system (Promega, Wisconsin, USA). Product sizes were confirmed using agarose gel electrophoresis. PCR products were pooled to a normalized concentration of 120 ng. Gel excision and purification was performed on the final pool using a 1.6% agarose gel (25). The purified library size was determined using the Agilent 2100 bioanalyzer using hsDNA kit; and quantified using the KAPA qPCR quantification kit (KAPA Biosystems, Massachusetts, USA)