title: Elucidating bacterial community interaction on metal surface and inhibiting the biofilm formation for control of biocorrosion in Cooling Water System
design: Protocol: DNA Extraction: DNA extraction is done using the suitable method for the sample type from commercially available kits such as QIAGEN, ZYMO RESEARCH, Thermo Fisher DNA extraction was done as per the manufacturer's recommendation. PCR Amplification of V3-V4 Region of 16s Gene: Composition of TAQ Master MIX:1) High-Fidelity DNA Polymerase 2) 0.5mM dNTPs3) 3.2mM MgCl24) PCR Enzyme Buffer Primer Details:16sF:-5 AGAGTTTGATGMTGGCTCAG316sR:-5 TTACCGCGGCMGCSGGCAC3Conditions Used:40ng of Extracted DNA is used for amplification along with 10pM of each primer Initial Denaturation:95 degree C25 Cycles of the following condition: Denaturation @ 95 deg C for 15 sec, Annealing @ 60 deg C for 15 sec, Elongation @ 72 deg C for 2 mins Final Extension at 72 deg C for 10 mins and Hold at 4 deg C. Overview of Sequencing Protocol: The amplicons from each sample were purified with Ampure beads to remove unused primers and an additional 8 cycles of PCR was performed using Illumin a bar coded adapters to prepare the sequencing libraries. Libraries were purified using Ampure beads and quantitated using Qubitds DNA High Sensitivity assay kit. Sequencing was performed using Illumina Miseq with 2x300PE v3 sequencing kit. Bioinformatics protocol: Raw data QC is done using FASTQC and MULTIQC, followed by trimming of adapters and low quality reads by TRIMGALORE. The trimmed reads are further taken for processing which includes merging of paired end reads, chimeri a removal and OUT abundance calculation and estimation correction this is achieved by QIIME / MOTHUR / KRAKEN / BRACKEN workflows. This workflow enables highly accurate investigations at genus level. The databases used are SILVA / GREENGENES / NCBI Each read is classified based on % coverage and identity. The 16S workflow will be useful in identifying pathogens in a mixed sample or understanding the composition of a microbial community. DNA QC :Extracted DNA from the samples were subjected to Nano Drop and GEL Check before being taken for PCR amplification: The Nano Drop readings of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs quality PCR Ampliqon QC: The amplified 16s PCR Product is purified and subjected to GEL Check and Nano drop QC. The Nano Drop readings of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs quality.
library_name: EMMR-SKR-NGS1