Amplicon-16S Metagenome Sequencing_Ramganga semi restored mangrove site soil microbiome
The metagenomic DNA was isolated from the received soil samples by commercially available soilKit (Nucleospin Soil). The qualities of the isolated metagenomic DNA sample quantified using NanoDrop. First amplicons were generated using the bacterial 16S V3-V4 specific primer set. Library preparation was carried out. High quality clean reads obtained, a minimum length of 100bp. Stitching the PE data into single end reads. Picking Operational Taxonomic Units (OTUs) based on sequence similarity within the reads, and picking a representative sequence for 16S rDNA sequence for a particular bacteria from each OTU against Greengenes database (version 13_8). Assigning the OTU to a taxonomic identity using reference databases. Calculated diversity metrics for the total assembled sequences and compared the types of communities, using the taxonomic assignments. OTU-picking identifies highly similar sequences across the sample provides a platform for comparisons of community structure. All the sequences from the samples were clustered into Operational Taxonomic Units (OTUs) based on their sequence similarity. Taxonomic relatedness done using UCLUST at 97% sequence similarity and each resulting cluster typically represents a species. Since each OTU may be made up of many sequences, a representative sequence for OTU is picked for downstream analysis. This representative sequence was used for taxonomic identification of the OTU.