Metagenome Sequencing and Analysis of Wastewater Samples Collected from River Mahananda, Siliguri, West Bengal, India
The project focuses on the metagenomic analysis of wastewater from the Mahananda River influenced by urban discharge, aiming to explore microbial diversity and potential functional pathways related to wastewater contamination in River Mahananda, Siliguri, West Bengal, India.Briefly, river water samples were collected from the middle stream of the Mahananda River in Siliguri, West Bengal, India near Suryasen Park, a site influenced by urban wastewater discharge. One liter of water, collected from the designated sampling point at two distinct time points, on the same sampling day, were combined in equal volume to ensure a representative sample for the subsequent analyses. The combined water sample was filtered through a sterile coarse filter paper and eventually through a 0.22 micrometer membrane filter using a filtration unit attached to a vacuum pump. Post-filtration, the membrane filter was carefully cut into small pieces using a sterile scissor, and metagenomic DNA was extracted from the cut membrane filter papers using the QIAGEN DNeasy Power Water Kit following the manufacturer protocol.The Kapa HyperPlus kit was used to perform library preparation. Briefly, 100 ng of DNA was subjected to enzymatic fragmentation to produce dsDNA fragments followed by end repair, polyA-tailing and adapter ligation. Generated library was then amplified with minimal PCR cycles to avoid any bias. The amplified library was subjected to clean-up using magnetic beads. The expected library size was checked by TapeStation analysis. The libraries were normalized, pooled, and sequenced using the Illumina NovaSeq X Plus system. Using a 10B flow cell, 300 cycles kit, 5 Gb data per sample with 150 bp read length were generated. Demultiplexed Fastq files were checked for quality using FastQC, and later MultiQC was used to analyze the overall quality of the sequences.