PCR amplification of the bacterial 16S rRNA gene V3–V4 region using high throughput sequencing and a bioinformatics analysis

The V3–V4 region of the 16S ribosomal RNA (rRNA) genes was amplified for each sample. A set of 8-nucleotide barcodes was added to the universal forward primer 338 F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer 806 R (5′-GGACTACHVGGGTWTCTAAT-3′), which were targeted at the domain Bacteria. PCR amplification was then performed as described previously²¹. The PCR products were sequenced using the Illumina Miseq PE300 platform. High-quality sequences were extracted from the raw reads. After the removal of the barcodes and primer, the extracted sequences were processed mainly using the QIIME (v1.7.0) suite of software tools²².