2.7. Library preparation and sequencing
Libraries were constructed using 50 ng of genomic DNA by a 2-step PCR-based workflow. In round 1 PCR, the 16S rRNA gene V3 to V4 regions were first amplified for 26 cycles using region-specific proprietary primers developed at Genotypic Technology Pvt. Ltd., Bangalore, India, using KAPA HiFi Hot Start PCR Kit (KAPA Biosystems Inc., Boston, MA). The PCR amplicons were further amplified for 10 cycles to add Illumina sequencing barcoded adaptors (Nextera XT v2 Index Kit, Illumina, CA). The libraries were normalized and pooled for multiplex sequencing. The normalized sample was denatured for 5 minutes using 0.2 N NaOH and neutralized by HT1 Buffer (Illumina). Denatured libraries were further diluted to the concentration of 13 pM and loaded into an Illumina MiSeq v3 600 cycles cartridge (Illumina) to read 275 bp length for forward (Read 1) and reverse (Read 2) reads in paired-end mode.After the completion of the sequencing, the data were demultiplexed using bcl2fastq software v 2.20 (Illumina, USA),