high yielding, sugarcane rhizosphere, replicate 3
DNA was extracted using Nucleospin soil kit (MN). The quality of isolated DNA was analysed using NanoDrop by determining A260/A280 ratio. The amplicon library was prepared using Nextera XT Index Kit (Illumina Inc) as per the 16S metagenomic sequencing library preparation protocol. Primers for the amplification of bacterial specific region were designed and synthesized at Eurofins Genomics Lab. Amplification of the bacterial 16S region was carried out. 3ul of PCR product was resolved on 1.2% Agarose gel at 120 V for approximately 60 min, till the samples reached 3/4th of the gel. The primers used for amplification of 16S region are given in Table 1. The QC passed amplicons with the Illumina adaptor were amplified using i5 and i7 primers that add multiplexing index sequences as well as common adapters required for cluster generation (P5 and P7) as per the standard Illumina protocol. The amplicon libraries were purified by AMPureXP beads and quantified using Qubit Fluorometer.Table 1: Primer sequences used for amplifying 16S rRNA region.16S rRNA Forward GCCTACGGGNGGCWGCAG16S rRNA Reverse ACTACHVGGGTATCTAATCCThe amplified libraries were analyzed on 4200 Tape Station system (Agilent Technologies) using D1000 Screen tape. After obtaining the mean peak size from Tape Station profile, libraries were loaded onto MiSeq at 10pM for cluster generation and sequencing. Paired-end sequencing allows the template fragments to be sequenced in both the forward and reverse directions on MiSeq. The kit reagents were used in binding of samples to complementary adapter oligos on paired-end flow cell. The adapters were designed to allow selective cleavage of forward strands after re-synthesis of the reverse strand during sequencing. The copied reverse strand was then used to sequence from opposite end of the fragment.