Microbial Diversity Analysis of Industrial Cooling Tower Biofilms by Metagenomic Sequencing
Extraction protocol: DNA extraction is done using Xploregen kit.  Add 60 ul of solution C1 and invert several times or vortex briefly [Note: Solution C1 maybe added to the power bead tube before adding sample]  Vortex at max speed for 10 mins. [Note: If using the 24-place vortex adapter for more than 12 preps, increase the vortex time by 5-10 mins]  Centrifuge tube at 10,000 x g for 30s  Transfer the supernatant to a clean 2ml collection tube. [Note: expect between 400 to 500 ul of supernatant. Supernatant may still contain some particles.]  Add 250 ul of soln. C2 and vortex for 5 sec. Incubate at 4 degree C for 5 mins.  Centrifuge the tube for 1 min at 10,000 X g  Avoiding the palette, transfer up to 600 ul of supernatant to a clean 2ml collection tube.  Add 200 ul of soln. C3 and vortex briefly. Incubate at 4 degree C for 5 mins. [Note: You can skip 5 min incubation. However, if you have already validated the extraction with this incubation, we recommend you to retain the step.]  Centrifuge the tube for 1 min at 10,000 X g.  Avoiding the palette, transfer up to 600 ul of supernatant to a clean 2ml collection tube.  Shake to mix soln. C4 and add 1200ul to the supernatant. Vortex for 5 s.  Load 675ul onto an MB Spin Column and centrifuge at 10,000 X g for 1 min. Discard flow through.  Repeat the above step twice, until all of the samples have been processed.  Add 500 ul of the solution C5. Centrifuge for 30s at 10,000 X g.  Discard the flow through. Centrifuge again for 1 min at 10,000 X g.  Carefully place the MB spin column into a clean 2ml collection tube. Avoid splashing any solution C5 onto the column.  Add 100ul of the soln. C6 to the center of the white filter membrane. Alternatively, you can use sterile DNA-free PCR grade water for this step.  Centrifuge at room temperature for 30s at 10,000 X g. Discard the MB spin column. The DNA is now ready for downstream application. [Note: Solution C6 is 10 mM Tris-HCl, pH 8.5. We recommend storing DNA frozen (-20 degree C- -80 degree C) as soln. C6 does not contain EDTA.] Role of buffer:  C1 buffer- Lysis of cell  C2 buffer- removal of Proteins and RNA  C3 buffer- to remove other residues  C4 buffer- binding buffer (binds with DNA)  C5 buffer- wash buffer (to wash DNA)  C6 buffer- Elution of DNA PCR Amplification of 16s Region: Composition of TAQ Master MIX:  High-Fidelity DNA Polymerase  0.5mM dNTPs  3.2mM MgCl2  PCR Enzyme Buffer. Primer Details: 16sF: - 5 AGAGTTTGATGMTGGCTCAG 3 16sR: - 5 TTACCGCGGCMGCSGGCAC 3 Conditions Used:  40ng of Extracted DNA is used for amplification along with 10pM of each primer  Initial Denaturation - 95 degree C  25 Cycles of the following condition,  Denaturation @ 95 degree Celsius for 15 sec, Annealing @ 60 degree Celsius for 15 sec, Elongation @ 72 degree Celsius for 2 mins  Final Extension at 72 degrees Celsius for 10 mins and hold at 4 degrees Celsius. 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 Nanodrop QC  The Nano Drop readings of 260/280 at an ~ value of 1.8 to 2 is used to determine the DNAs quality. 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 Illumina barcoded adapters to prepare the sequencing libraries. Libraries were purified using Ampure beads and quantitated using Qubit dsDNA High Sensitivity assay kit. Sequencing was performed using Illumina Miseq with 2x300PE v3-v4 sequencing kit. Bioinformatics Protocol | Metagenomics: The database used for 16s V3-V4 region is NCBI Step1:  The bcl data received from the sequencer is de-multiplexed into .fastq raw data. Step2:  The de-multiplexed data quality will be checked using Fastqc (Version 0.11.9) and  Multiqc (Version 1.10.1) tools. Step3:  The QC passed samples are qualified for further analysis and we use our own pipeline for metagenomics (Biokart Pipeline) for 16s metagenomic. The workflow of the pipeline is as follows. Step4:  Once the run is over we get the final raw OTU table from which we start visualization of the analysis. Step5:  The abundance feature tables and the top ten organisms in each samples are constructed using Microsoft excel (2024). Step6:  The other analyses like Heatmap, core microbiome, Dendrogram, Alpha diversity, Beta diversity, PCOA plot, Rarefaction curve, KEGG pathway etc. were built using  Microbiome analyst (online tool: https://www.microbiomeanalyst.ca/).