Amplification and Sequencing of Soil 16S rRNA

Soil DNA was used for 16S rRNA gene amplification using V3 and V4 primer sets (V3F-5’ CCTACGGGNBGCASCAG3’; V4R-5’ GACTACNVGGGTATCTAATCC3’) (Takahashi et al., 2014 (link)). The amplified products were checked by running on 2% (w/v) agarose gel. The PCR products were purified for paired-end library preparation. Approximately 5 ng/μL of the PCR amplicon was used for library preparation using the NEB Next Ultra DNA library preparation kit (New England Biolabs, United Kingdom), as per the manufacturer’s protocol. Library size and quality were determined using Agilent 2200, TapeStation (Agilent Technologies, United States). High-throughput sequencing was performed using Illumina HiSeq 2500 platform with 2*250 cycle chemistry to obtain the metagenomic data.