Bacterial diversity profiling of mangrove sediment employing Metagenomic approach

The study aimed to identify the bacterial metagenomic profiling of mangrove rhizosphere soil sample by targeting the V3-V4 regions. The metagenomic bacterial DNA of Rhizophora apiculata rhizosphere soil was isolated by DNA Power Soil Kit. Isolated DNA was quantified using Nanodrop 2000. DNA quality and integrity was checked by 1% agarose gel electrophoresis. PCR amplification of targeted regions was performed by using specific primers connecting with barcodes. The PCR products with proper size were selected by 2% agarose gel electrophoresis. Same amount of PCR products from each sample was pooled, end-repaired, A-tailed and further ligated with Illumina adapters. Libraries were sequenced on a paired-end Illumina platform to generate 250bp paired-end raw reads. The experimental procedures of DNA library preparation are PCR Amplification i) PCR Amplification, ii) Size selection, iii) End repair & A-tailing, iv) Adapter ligation and v) Purification. Sequenced reads (raw reads) containing low quality reads and primes/adapters, affecting the analysis quality were filtered to get clean reads by removing i) reads whose length less than 60bp after trimming the primers and adapters from end of reads ii) reads containing N > 10% (N represents the base cannot be determined) and iii) reads containing low quality (Q score <= 5) base which is over 50% of the total base. Among the metagenomes, Vibrio genus was the most abundant of about 32% of total bacteria.