Metagenomic 16S rRNA Gene Sequencing

DNA extraction and PCR amplification of the variable region 4 (V4) of the 16S rRNA gene using Illumina adapted universal primers 515F/806R39 was conducted using the direct PCR protocol [Extract-N-Amp Plant PCR kit (Sigma-Aldrich, Inc.)] as previously described [24 (link),25 (link),26 (link)]. Briefly, PCRs were conducted in triplicate in 96 wells plate [denaturation for 3 min at 94 °C; 35 cycles (98 °C, 60 s; 55 °C, 60 s; 72 °C, 60 s) followed by elongation for 10 min at 72 °C]. Positive amplicons were pooled in equimolar concentrations into a composite sample that was size selected (300–500 bp) using agarose gel to reduce non-specific products from host DNA. Sequencing was performed on the Illumina MiSeq platform with the addition of 20% PhiX, and generating paired-end reads of 175b in length in each direction.