Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing

Research scope focuses on the understanding of the fermentation microbiome. This is to answer questions such as how diverse microbes in natural fermentation are, and what gene coding for metabolite is necessary for flavor and safety? One of the latest advancement in our field involves understanding how the dynamics of the microbiome correlate with the metabolic profile observed in fermented foods and beverages.

The next generation sequences stands out as a pivotal technology that is currently driving advancements in our research field. To begin, transfer 20 milliliters of the fermented grape sample into a sterile 50-milliliter screw-cap tube. Then, add 10 milliliters of cold water into the tube and vortex for homogenization.

Centrifuge the sample at 800g for one minute at four degrees Celsius. Transfer the supernatant to a new 50-milliliter tube and centrifuge at 3, 000g for 20 minutes at four degrees Celsius. After discarding the supernatant, re-suspend the pellet in one milliliter of PBS.

Transfer the cell suspension to a two-milliliter screw-cap tube and centrifuge at 14, 000g for two minutes. Now, re-suspend the pellet in 978 microliters of sodium phosphate buffer of pH 7.4 and 122 microliters of DNA buffer. Vortex the tube and place it at four degrees Celsius for 45 minutes to one hour.

Transfer one milliliter of the sample into a Lysis solution one-tube and tighten the cap. Homogenize the sample three times in a bead-beating grinder for 60 seconds each. Then, centrifuge the Lysing Matrix E-tube for one minute at 16, 800g.

Transfer the sate into a clean micro tube and add 250 microliters of protein precipitation solution to the tube. Shake the tube 10 times to mix the contents. Centrifuge the sample for five minutes at 16, 800g and transfer the supernatant into a sterile 15-milliliter tube.

Then, add one milliliter of binding-matrix suspension to the supernatant and mix by inverting the tubes for two minutes before placing them in a rack for three minutes. After removing one milliliter of the supernatant, re-suspend the matrix in the remaining supernatant. Transfer 600 microliters of the mixture into a spin-filter tube and centrifuge.

Then, decant the flow through and add 500 microliters of wash solution into the spin-filter tube. Remove the spin filter and place it in a fresh catch tube for five minutes. After drying, add 50 microliters of warm DNA's free water on the filter membrane and gently stir the matrix with a pipette tip.

Centrifuge at 14, 500g for one minute to elute the DNA. Load the sample onto agarose gel. Finally, measure the DNA concentration.

To begin, use specific primers 515F and 806R to amplify the V4 hypervariable region of the 16S rRNA gene. Perform PCR using the given conditions. Next, add 3.5 microliters of sequencing buffer and 1.5 microliters of enzyme mix to the tube.

After mixing and spinning the sample, set up a thermocycler reaction. Now, add the adapter ligation mixture into the tube and incubate at 20 degrees Celsius for 15 minutes in a thermocycler. Add 1.5 microliters of uracil-specific extension reagent enzyme to the ligation mix, and incubate at 37 degrees Celsius for 15 minutes.

Now, add 43.5 microliters of magnetic beads to the adapter ligation DNA and mix 20 times. After five minutes, place the tube in a magnetic rack for five to 10 minutes for bead separation and discard the supernatant. Wash the pellet with 100 microliters of 80%ethanol and dry the beads for 30 seconds.

To elute the beads and pellet, add 8.5 microliters of 10 millimolar Tris-Hcl into the tube and incubate for two minutes. Place the tube in a magnetic rack and remove 7.5 microliters of eluded DNA as supernatant into a new tube. Add PCR reagents to the adapter ligated DNA-containing tube to set up a 25 microliter reaction.

To clean amplified DNA, add 22.5 microliters of magnetic beads into the tube and mix 20 times. After five minutes, remove 50 microliters of supernatant and wash the beads with 100 microliters of 80%ethanol. Re-suspend the dark brown beads in 16 microliters of water and mix 20 times.

Place the tube in the magnetic rack for 30 to 60 seconds and transfer 15 microliters to a new tube. Mix 90 microliters of buffer, one microliter of dye and two microliters of DNA library sample and read the DNA concentration on a fluorometer. After diluting the DNA to two nanomolar, load 27 to 30 microliters of the flow cell and place it in the sequencer.

Save the FASTQ files obtained from the sequencer. Click to open a spreadsheet file and create a mapping or metadata file. Open the Nephele website, upload the FASTQ files and read QC.In QIIME 2, perform filtering and trimming.

Next, using DADA2, execute de-multiplexed paired-end reads, filter substitution, chimera errors and merging. Use the Naive Bayes classifier trained on the SILVA version 132, 99%Operational Taxonomic Unit, or OTU database, to perform bacterial taxonomic assignment at 97%similarity. Open the MicrobiomeDB website, click to upload the files and generate OTU alpha diversity, beta diversity, relative abundance and rarefaction curves.

Finally, open a spreadsheet and transfer data to make heat maps, Venn diagrams and linear discriminant analysis effect size. A heat map based on the relative abundance of bacteria at different stages of wine-making displayed the dominance of phyla such as Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, and Fusobacteriota. At the genus level, important genara like Enterobacteriaceae and Lactobacillaceae were identified.

A Venn diagram analysis revealed 15 shared unique OTUs from the grape must to the final wine. The results of the Amplicon sequencing based on the V4 variable region also indicated the alpha diversity in the two Traminette R and L.Beta diversity analysis showed a shift in bacteria during fermentation, but the bacterial composition in the final wine was similar to that in the must and yeast-added stages.