Metagenomic 16S rRNA gene sequencing protocol

The hypervariable V3–V4 region of the prokaryotic 16SrRNA gene from the total bacterial DNA (10 ng) was amplified using the primers, viz. Pro341F (5′-CCTACGGGNBGCASCAG-3′) and Pro805R (5′-GACTACNVGGGTATCTAATCC-3′) (Takahashi et al., 2014 (link)). The amplified product was gel-purified to remove non-specific amplification, if there is any. Metagenomic library preparation was done using the NEBNext Ultra DNA library preparation kit (New England Biolabs) using equimolar quantities of PCR amplicon (5 ng). The library quantity and quality were estimated in Agilent 2200 TapeStation. The sequencing was then performed on an Illumina HiSeq 2500 platform (2 × 300 paired-end sequencings) (AgriGenome Labs Private Limited, Kochi, India). The high-quality samples were refined and used for metagenomics analysis.