title: Assessment of Bacterial Diversity of the Soil in the Western Ghats
design: Soil samples collected from all locations were pooled in equal proportion and it named as Western Ghats sample. At the same time, samples collected from Mahabaleshwar and Malshej Ghat were pooled separately and labeled as Mahabaleshwar and Malshej Ghat, respectively. These three sample were analyzed for bacterial communities and diversity present in North region of WGs. High throughput sequencing of 16S rRNA (V3-V4) gene was accomplished by Eurofins genomics India Pvt. Ltd. (India) using Illumina MiSeq platform. For sequencing, total DNA of each sample was isolated from one gram of soil using commercially available kit, Nucleospin soil, according to manufacturers instructions. Quantification of the DNA samples were carried out using NanoDrop (Thermofisher). Quality control passed samples then processed for amplicon libraries generation. The microbial diversity and community composition of the samples established by amplicon sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene using the forward primer GCCTACGGGNGGCWGCAG and reverse primer ACTACHVGGGTATCTAATCC (Eurofins Genomics Lab, India). The amplicon libraries were prepared using Nextera XT Index kit (Illumina inc.) as per the 16S metgenomic sequencing Library preparation protocol (part# 15044223 Rev. B). The QC passed amplicon with the Illumina adaptors were amplified by using i5 and i7 primers that add multiplexing index sequences as well as common adaptor required for cluster generation as per the standard Illumina protocol. The amplicon libraries were purified by AMPure XP beads and quantified using Qubit fluorometer. For quantity and quality check, amplified libraries were analyzed on 4200 Tape station system (Agilent technologies) using D1000 screen tape as per manufacturer instructions. After obtaining the mean peak size from Tape station profile, libraries were loaded on to MiSeq at appropriate concentration (10-20pM) for cluster generation and sequencing.