Illumina MiSeq paired end sequencing
paired end
Amplicon library construction involved two PCR steps. First, the V4-V5 hypervariable region of 16S rRNA gene (rDNA) was PCR amplified from the environmental DNA, using 515F and 926R primer pairs. The first PCR primer pair included handle sequences and V4-V5 region specific primer sequences. The handle sequence in the first PCR primer acted as an annealing site for the second PCR primer pair. The second PCR primer consisted of an adapter sequence and handle sequence specific bases.  The reverse primer of second PCR also contained a 8 nucleotide barcode to facilitate multiplexing of samples. Different barcodes were used for different samples. Products of the second PCR were purified and sequenced using Illumina MiSeq platform.