Funding from the Appalachian State University Research Council (URC) grant and CAPES Print Travel fellowship that supported the visit of FAO to Universidade de S&#227;o Paulo, Ribeir&#227;o Preto - S&#227;o Paulo, Brazil, are gratefully acknowledged. This study was financed in part by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior - Brasil (CAPES) - Finance Code 001. ECPDM is grateful for the CAPES Print Travel grant that supported her visit to Appalachian State University. ECPDM is a research fellow 2 from the Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico, Brasil (CNPq).

1. Experimental wine production
<ol>
	<li>Obtain Traminnette grapes from Dynamis Estate wine in Jonesville, North Carolina vineyard, destem, and crush to release must into two separate 600 L open-top fermenters and leave on skins for about 4 days with the lids on.</li>
	<li>Punch the caps down once a day to keep skins wet and limit volatile acid (VA) production. 
	NOTE: The idea is that a lot of the aromatic precursors (monoterpenes) are situated in the skins and leave the juice in contact with the skin to increase the aromatic potential of the wine.</li>
	<li>After 4 days, pitch Saccharomyces cerevisiae var cerevisiae QA23 (Lallemand). This yeast was selected as it is a strong fermenter and is associated with enhancing the varietal character in the wine from the grapes.</li>
	<li>In one tank, Traminette R, add 20 g/hL Fermaid O when the brix is at 17.7 and 20 g/hL Fermaid O and 12.5 g/hL Fermaid K at 13.1 g/hL to achieve fermentation security, while the other tank Traminette L, add 40 g/hL Stimula Sauvignon blanc when the brix is at 17.1. Also add 20 g/hL Fermaid O when the brix is at 13.4 to enhance the varietal character of the wine.</li>
	<li>Take duplicate samples of must (day 1), yeast added (day 4), fermented must (day 15), and finished wine samples (day 30) and keep at -20 &#176;C before DNA extraction and metagenomic analysis.</li>
</ol>
2. DNA extraction for metagenomics
<ol>
	<li>Keep all duplicate samples obtained above on ice until the first centrifugation.</li>
	<li>Transfer 20 mL of the fermented sample into a sterile 50 mL screw cap tube.</li>
	<li>Add 10 mL of cold water and homogenize (vortex). Centrifuge for 1 min at 800 x g at 4 &#176;C and transfer the supernatant to a new 50 mL tube. 
	NOTE: This step will remove the solids of the sample.</li>
	<li>Repeat step 2.3 two times and pool the supernatants in the same tube.</li>
	<li>Centrifuge supernatant at 3,000 x g at 4 &#176;C for 20 min to pellet the cells. Discard the supernatant.</li>
	<li>Resuspend the pellet in 1 mL of PBS, transfer to a 2 mL screw-cap tube, and centrifuge at 14,000 x g for 2 min. Perform two more washes using PBS.</li>
	<li>At this step, freeze the pellets at -20&#176;C or follow step 2.8. It is important that all the samples are treated in the same way.</li>
	<li>Add 978 &#181;L of sodium phosphate buffer. Prepare sodium phosphate buffer, add 3.1 g of NaH2PO4&#183;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L, and adjust pH to 7.4.</li>
	<li>Add 122 &#181;L of DNA buffer (DNA spin kit) and vortex.</li>
	<li>Let it stand in the refrigerator (4 &#176;C) for 45 min-1 h. Vortex the samples every 15 min. This step can be left overnight (o/n) or until ready to continue using the DNA spin kit.</li>
	<li>Transfer 1 mL of sample into a Lysis solution 1 tube (DNA spin kit) and tighten the cap (write the sample number on the side of the tube and not on the cap; the kit reagents will remove anything written on the cap).</li>
	<li>Homogenize the sample in a bead-beating grinder (6.5 m/s, CY: 24 x 2) for 60 s three times. If the bead-beating grinder has the device to put ice, put 50 g of dry ice under the tubes. If not, keep the samples on wet ice for 5 min between each homogenization step.</li>
	<li>Centrifuge Lysing Matrix E tubes (DNA spin kit) for 1 min at 16800 x g(or max speed).</li>
	<li>Transfer the supernatant into a clean microtube. Thereafter, add 250 &#181;L of protein precipitation solution (PPS) reagent (DNA spin kit) and mix by shaking the tube by hand 10 times.</li>
	<li>Centrifuge for 5 min at 16800 x g to pellet the precipitate.</li>
	<li>Transfer the supernatant into a sterile 15 mL tube. Add 1 mL of binding matrix suspension (DNA spin kit) to the supernatant. 
	NOTE: Resuspend the binding matrix suspension before use until it is homogeneous.</li>
	<li>Invert the tubes by hand for 2 min, then let the tubes stand in a rack for 3 min (to allow settling of silica matrix).</li>
	<li>Carefully remove 1 mL of the supernatant. Resuspend the matrix in the remaining supernatant.</li>
	<li>Transfer 600 &#181;L of the mixture into a spin filter tube (DNA spin kit) and centrifuge for 1 min at 14500 x g.</li>
	<li>Add the remaining mixture and centrifuge.</li>
	<li>Decant flow-through and add 500 &#181;L of wash solution (DNA spin kit) into the spin filter tube and centrifuge for 1 min at 14500 x g(ensure to add EtOH to wash solution; see the product handbook). Decant the flow-through and repeat the wash two more times (3 washes in total).</li>
	<li>Decant the flow-through and centrifuge for an additional 2 min at 14,500 x g to dry the matrix of residual wash solution.</li>
	<li>Remove the spin filter and place it in a fresh catch tube (DNA spin kit). Air dry the spin filter for 5 min at room temperature (RT).</li>
	<li>Warm the DNAse-free water (DNA spin kit) at 55 &#176;C for 5 min. Add 50 &#181;L of DNAse-free water and gently stir the matrix on the filter membrane with a pipette tip for efficient elution of the DNA. Let samples stand at RT for 1 min, then centrifuge for 1 min at 14500 x g to elute DNA.</li>
	<li>Place the samples on ice. Load the sample mixture (2 &#181;L of sample + 2 &#181;L of water + 1 &#181;L of loading buffer) into a gel. Measure the DNA concentration.</li>
	<li>Store the samples at -20 &#176;C.</li>
	<li>Use a spectrophotometer to quantify the DNA extracted. 
	NOTE: A 1 &#181;L volume of DNA was dropped and quantified, and the quality of DNA was noted at an A260/A280 ratio.</li>
</ol>
3. DNA electrophoresis
<ol>
	<li>Prepare 1% agarose gel in 0.5 Tris/Borate/EDTA (TBE) buffer (20 mL for a mini gel, 70-100 mL for a medium gel). Weigh agarose+buffer, boil in microwave to melt agarose, reweigh, and add dH2O to the original weight. Cool the agarose solution to 55-60 &#176;C and pour into the gel former with comb assembly. Leave the gel to solidify.</li>
	<li>Add 1 &#181;L of 10x gel loading buffer to the sample (10 &#181;L) and load onto the gel next to a molecular weight marker. Run from negative (black) to positive (red) at 100-115 V (large gel) or 90 V (mini gel) until the blue dye is near the bottom.</li>
	<li>Stain gel for 30 min in 1 &#181;L/mL ethidium bromide or SYBR green (nitrile gloves and goggles needed), rinse in water, and view under an ultraviolet (UV) light source. From the duplicate samples, select the highest yield for further processing.</li>
</ol>
4. High throughput sequencing
<ol>
	<li>Amplify the V4 hypervariable region of the 16S rRNA gene using specific primers 515F (5&#39; -GTGYCAGCMGCCGCGGTAA-3&#39;) and 806R (5&#39;-GGACTACNVGGGTWTCTAAT-3&#39;)22. Set the polymerase chain reaction (PCR) conditions at 94 &#176;C for 2 min, 30 cycles of 94 &#176;C for 20 s, 58 &#176;C for 20 s, 65 &#176;C for 1 min, and 65 &#176;C for 10 min (final extension).</li>
	<li>Carry out PCR reactions with a high-fidelity PCR master mix (Table of Materials).</li>
	<li>Generate libraries with DNA library preparation kit and then sequence using paired-end sequencing on a sequencing platform. 
	NOTE: Here, the libraries generated with NEBNext Ultra II DNA Library Prep Kit were sequenced using paired-end Illumina sequencing (2 &#215; 250 bp) on the novaseq6000 platform.</li>
	<li>Follow the sequencing steps:
	<ol>
		<li>Step 1: Shear the DNA with an enzyme, usually by a method that selects for a general size.
		<ol>
			<li>Add 3.5 &#181;L of sequencing buffer and 1.5 &#181;L of enzyme mix to 25 &#181;L of DNA (ca. 1 &#181;g,) mix 10 times with a pipette and spin quickly.</li>
			<li>Place in a thermocycler with the following conditions: preheat lid at 75&#176;C, (1) 30 min for 20 &#176;C (2) 30 min for 65 &#176;C (3) hold at 4&#176;C.</li>
		</ol>
		</li>
		<li>Step 2: Add adaptors via ligation
		<ol>
			<li>Add 15 &#181;L of ligation master mix, 1.25 &#181;L of adapter, 0.5 &#181;L of ligation enhancer, and 30 &#181;L of end prep DNA mix with a pipette.</li>
			<li>Incubate at 20 &#176;C for 15 min in a thermocycler and then add 1.5 &#181;L uracil-specific excision reagent (USER) enzyme to the ligation mix to obtain a total of 48.26 &#181;L. Incubate 37 &#176;C for 15 min.</li>
			<li>Add 43.5 &#181;L of magnetic beads to the adapter ligation DNA, mix 20 times with a pipette, and incubate for 5 min at RT. Separate the beads with a magnetic rack, then incubate for 5-10 min, and discard the supernatant.</li>
			<li>Wash the pellet with 100 &#181;L of 80% ethanol, dry for 30 s, and wash again. Elute beads and pellet in 8.5 &#181;L of 10 mM Tris HCl/0.1x Tris-EDTA(TE)/HPLC H20, mix with a pipette and incubate at RT for 2 min.</li>
			<li>Incubate in the magnetic rack and remove 7.5 &#181;L of eluted DNA as supernatant. Place the elute in a new tube and conduct a PCR step.</li>
			<li>First, add 12.5 &#181;L of master mix, 2.5 &#181;L of index primer i7 primer, and 2.5 &#181;L of universal PCR primer/i5 primer to 7.5 &#181;L adapter-ligated DNA to obtain 25 &#181;L of PCR reaction mix. Use the following PCR condition: preheat the lid to 103 &#176;C. 98 &#176;C for 30 s, 98 &#176;C for 10 s, 65 &#176;C for 75 s, 65 &#176;C for 5 min and hold at 4 &#176;C after 10 cycles.</li>
			<li>Clean the 25 &#181;L amplified DNA. Add 22.5 &#181;L of magnetic beads and mix 20 times. Incubate at RT for 5 min and remove 50 &#181;L of the supernatant. Wash the beads with 100 &#181;L of 80% ethanol, and remove all ethanol by drying the beads gently for 30 s.</li>
			<li>Resuspend the dark brown beads in 16 &#181;L of water, mix 20 times with a pipette, and place in the magnetic rack for 30-60 s. Transfer 15 &#181;L to a new tube.</li>
			<li>Determine the DNA concentration using a fluorometer. Mix 90 &#181;L of buffer + 1 &#181;L dye + 2 &#181;L of DNA library sample, and read the concentration. Dilute the DNA to 2 nM, load 27-30 &#181;L to the flow cell, and place it in the sequencer.</li>
		</ol>
		</li>
		<li>Step 3: Sequence the adaptors, hybridize the libraries generated above to the matrix of the patterned flow cell, and capture DNA per the manufacturer&#39;s instructions. Use it as the template for the second synthesis.
		<ol>
			<li>Then, amplify the second strand into a clonal cluster. Linearize the cluster and block the active sites. Add sequencing primer to provide a site for sequencing by synthesis in the sequence per the manuacturer&#39;s protocol. 
			NOTE: Chemically, the modified nucleotides bind to the DNA template strand using natural complementarity. Each nucleotide has a fluorescent tag and a reversible terminator that blocks the inclusion of the next base. Therefore, the presence of a fluorescent signal indicates which nucleotide is inserted, and the terminator is cleaved for the next base to bind.</li>
		</ol>
		</li>
		<li>Step 4: Amplify the single-bound strand to give clusters of sequences that are sequenced in tandem by synthesis. 
		NOTE: The clusters give a brighter signal than a single strand through bidirectional scanning and two-channel sequencing chemistry. The sequencing software automatically transfers base call (*.cbcl) files to the specified output folder location for data analysis.</li>
	</ol>
	</li>
</ol>
5. Bioinformatics
<ol>
	<li>Generate the sequence data as FASTQ files. Analyze to include the following parts.
	<ol>
		<li>Part I:
		<ol>
			<li>Save the FASTQ files obtained from the sequencer, click to open a spreadsheet file, and create mapping/metadata files.</li>
			<li>Open the Nephele website (https://nephele.niaid.nih.gov/),&#160;upload the FASTQ files, read QC, filtering, and trimming in QIIME222. Deposit the raw sequences as sequence read archive (SRA) and GenBank in NCBI BioProject (https://submit.ncbi.nlm.nih.gov/subs/bioproject/)</li>
		</ol>
		</li>
		<li>Part II: Go to the Nephele website (https://nephele.niaid.nih.gov/),&#160;do the demultiplexed paired-end reads, filter substitution, and chimera errors, and merge using DADA223. Use the Naive Bayes classifier trained on the Silva version 132 99% OTU database to perform bacterial taxonomic assignment at 97% similarity24.</li>
		<li>Part III: Open the MicrobiomeDB website, click to upload files, and generate OTU alpha-diversity, beta-diversity, relative abundance, and rarefaction curves (https://microbiomedb.org/mbio/app).</li>
		<li>Part IV: Open a spreadsheet and transfer data to make heatmaps, Venn diagrams, and linear discriminant analysis effect size (https://microbiomedb.org/mbio/app).</li>
	</ol>
	</li>
</ol>
6. Statistical analysis
<ol>
	<li>Open QIIME222, and determine the significant differences in alpha diversity between groups using the alpha-group-significance script. This will also perform the Kruskal-Wallis test.</li>
	<li>Determine the differences in beta diversity between groups by using PERMANOVA, including a pairwise test25.</li>
	<li>Obtain the significant differences in the bacterial community structure amongst the groups using MicrobiomeDB. A p-value &#8804;0.05 was considered statistically significant.</li>
</ol>

Profiling the Bacterial Community in Grapes Using Amplicon Metagenomics

<ol>
	<li>Skinkis, P. A., Bordelon, B. P., Wood, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+monoterpene+constituents+in+traminette,+gew&#252;rztraminer,+and+riesling+winegrapes.">Comparison of monoterpene constituents in traminette, gew&#252;rztraminer, and riesling winegrapes.</a> American Journal of Enology and Viticulture. 59, 440-445 (2008).</li><li>Bordelon, B. P., Skinkis, P. A., Howard, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+training+system+on+vine+performance+and+fruit+composition+of+Traminette.">Impact of training system on vine performance and fruit composition of Traminette.</a> American Journal of Enology and Viticulture. 59, 39-46 (2008).</li><li>Reisch, B. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traminette' Grape. New York Food and Life Science Bulletin. , (1996).</li><li>Clemente-Jimenez, J. M., Mingorance-Cazorla, L., Mart&#305;&#769;nez-Rodr&#305;&#769;guez, S., Las Heras-V&#225;zquez, F. J., Rodr&#305;&#769;guez-Vico, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+and+oenological+properties+of+wine+yeasts+isolated+during+spontaneous+fermentation+of+six+varieties+of+grape+must.">Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must.</a> Food Microbiology. 21, 149-155 (2004).</li><li>Attila, K. &. #. 1. 9. 5. ;., Kluz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+microflora+of+wine+grape+berries.">Natural microflora of wine grape berries.</a> Journal of Microbiology, Biotechnology and Food Sciences. 4 (special issue 1), 32-36 (2015).</li><li>Swiegers, J. H., Bartowsky, E. J., Henschke, P. A., Pretorius, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+and+bacterial+modulation+of+wine+aroma+and+flavor+in+Microbial+modulation+of+wine+aroma+and+flavour.">Yeast and bacterial modulation of wine aroma and flavor in Microbial modulation of wine aroma and flavour.</a> Australian Journal of Grape and Wine Research. 11 (2), 139-173 (2008).</li><li>Alonso-del-Real, J., P&#233;rez-Torrado, R., Querol, A., Barrio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominance+of+wine+Saccharomyces+cerevisiae+strains+over+S.+kudriavzevii+in+industrial+fermentation+competitions+is+related+to+an+acceleration+of+nutrient+uptake+and+utilization.">Dominance of wine Saccharomyces cerevisiae strains over S. kudriavzevii in industrial fermentation competitions is related to an acceleration of nutrient uptake and utilization.</a> Environmental Microbiology. 21 (5), 1627-1644 (2019).</li><li>Virdis, C., Sumby, K., Bartowsky, E., Jiranek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactic+acid+bacteria+in+wine:+Technological+advances+and+evaluation+of+their+functional+role.">Lactic acid bacteria in wine: Technological advances and evaluation of their functional role.</a> Frontiers in Microbiology. 11, 3192 (2021).</li><li>Burns, T. R., Osborne, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+malolactic+fermentation+on+the+color+and+color+stability+of+Pinot+noir+and+Merlot+Wine.">Impact of malolactic fermentation on the color and color stability of Pinot noir and Merlot Wine.</a> American Journal of Enology and Viticulture. 64, 370-377 (2013).</li><li>Piao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+bacterial+community+and+its+temporal+succession+during+the+fermentation+of+wine+grapes.">Insights into the bacterial community and its temporal succession during the fermentation of wine grapes.</a> Frontier of Microbiology. 6, 809 (2015).</li><li>Figdor, D., Gukabivale, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+against+the+odds:+Microbiology+of+root+canals+associated+with+post-treatment+disease+Endodontic+Topics.">Survival against the odds: Microbiology of root canals associated with post-treatment disease Endodontic Topics.</a> 18, 62-77 (2011).</li><li>Bokulich, N. A., Joseph, C. M. L., Greg Allen, G., Benson, A. K., Mills, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+reveals+significant+bacterial+diversity+of+botrytized+wine.">Next-generation sequencing reveals significant bacterial diversity of botrytized wine.</a> Plos ONE. 7 (5), e36357 (2012).</li><li>Berbegal, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metagenomic-based+approach+for+the+characterization+of+bacterial+diversity+associated+with+spontaneous+malolactic+fermentations+in+wine.">A metagenomic-based approach for the characterization of bacterial diversity associated with spontaneous malolactic fermentations in wine.</a> International Journal of Molecular Science. 20, 3980 (2019).</li><li>Leveau, J. H., Tech, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grapevine+microbiomics:+bacterial+diversity+on+grape+leaves+and+berries+revealed+by+high-throughput+sequence+analysis+of+16S+rRNA+amplicons.">Grapevine microbiomics: bacterial diversity on grape leaves and berries revealed by high-throughput sequence analysis of 16S rRNA amplicons.</a> International Symposium on Biological Control of Postharvest Diseases: Challenges and Opportunities. 905, 31-42 (2010).</li><li>Rubiola, S., Macori, G., Civera, T., Fanning, S., Mitchell, M., Chiesa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+full-length+16s+RNA+metabarcoding+and+whole+metagenome+sequencing+suggests+the+use+of+either+is+suitable+for+large-scale+microbiome+studies.">Comparison between full-length 16s RNA metabarcoding and whole metagenome sequencing suggests the use of either is suitable for large-scale microbiome studies.</a> Foodborne Pathogens and Disease. 19, 495-504 (2022).</li><li>Bokulich, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+among+wine+grape+microbiome,+metabolome,+and+fermentation+behavior+suggest+microbial+contribution+to+regional+wine+characteristics.">Associations among wine grape microbiome, metabolome, and fermentation behavior suggest microbial contribution to regional wine characteristics.</a> Mbio. 7 (3), e00631-e00716 (2016).</li><li>Siren, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-omics+and+potential+applications+in+wine+production.">Multi-omics and potential applications in wine production.</a> Current Opinion in Biotechnology. 56, 172-178 (2019).</li><li>Kidd, S. P., Bastian, S. E. P., Eisenhofer, R., Welsh, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+viable+grapevine+microbiome+to+enhance+the+quality+of+wild+wines.">Monitoring the viable grapevine microbiome to enhance the quality of wild wines.</a> Microbiology Australia. 44 (1), 13-17 (2023).</li><li>Lorenz, P., Eck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomics+and+industrial+applications.">Metagenomics and industrial applications.</a> Nature Reviews Microbiology. 3, 510-516 (2005).</li><li>Tosi, E., Azzolini, M., Guzzo, F., Zapparoli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+different+fermentation+behaviours+of+two+indigenous+strains+of+Saccharomyces+cerevisiae+and+Saccharomyces+uvarum+isolated+from+Amarone+wine.">Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine.</a> Journal of Applied Microbiology. 107 (1), 210-218 (2009).</li><li>Stefanini, I., Cavalier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+approaches+to+investigate+the+contribution+of+the+vineyard+environment+to+the+quality+of+wine+fermentation:+potentials+and+difficulties.">Metagenomic approaches to investigate the contribution of the vineyard environment to the quality of wine fermentation: potentials and difficulties.</a> Frontiers in Microbiology. 9, 991 (2018).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QIIME+allows+analysis+of+high-throughput+community+sequencing+data.">QIIME allows analysis of high-throughput community sequencing data.</a> Nature Methods. 5, 335-336 (2010).</li><li>Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A., Holmes, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DADA2:+High-resolution+sample+inference+from+Illumina+amplicon+data.">DADA2: High-resolution sample inference from Illumina amplicon data.</a> Nature Methods. 13, 581-583 (2016).</li><li>Quast, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SILVA+ribosomal+RNA+gene+database+project:+improved+data+processing+and+web-based+tools.">The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.</a> Nucleic Acids Research. 41 (Database issue), D590-D596 (2013).</li><li>Anderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+non-parametric+multivariate+analysis+of+variance.">A new method for non-parametric multivariate analysis of variance.</a> Austral Ecology. 26 (1), 32-46 (2001).</li><li>Bolyen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=interactive,+scalable+and+extensible+microbiome+data+science+using+QIIME+2.">interactive, scalable and extensible microbiome data science using QIIME 2.</a> Nature Biotechnology. 37, 852-857 (2019).</li><li>Fadiji, A. E., Babalola, O. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomics+methods+for+the+study+of+plant-associated+microbial+communities:+A+review.">Metagenomics methods for the study of plant-associated microbial communities: A review.</a> Journal of Microbiological Methods. 170, 105860 (2020).</li><li>van Hijum, S. A., Vaughan, E. E., Vogel, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+state-of-art+sequencing+technologies+to+indigenous+food+fermentations.">Application of state-of-art sequencing technologies to indigenous food fermentations.</a> Current Opinion in Biotechnology. 24, 178-186 (2013).</li><li>V&#237;quez-R, L., Fleischer, R., Wilhelm, K., Tschapka, M., Sommer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jumping+the+green+wall:+The+use+of+PNA-DNA+clamps+to+enhance+microbiome+sampling+depth+in+wildlife+microbiome+research.">Jumping the green wall: The use of PNA-DNA clamps to enhance microbiome sampling depth in wildlife microbiome research.</a> Ecology and Evolution. 10 (20), 11779-11786 (2020).</li><li>Bukin, Y. S., Galachyants, P., Yu, I. V., Morozov, S. V., Bukin, A. S., Zakharenko, T. I., Zemskaya, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+16S+rRNA+region+choice+on+bacterial+community+metabarcoding+results.">The effect of 16S rRNA region choice on bacterial community metabarcoding results.</a> Scientific Data. 6, 190007 (2019).</li></ol>

The authors have no conflicts of interest to declare.

The protocol of metagenomics starts from the sampling of the grape must, and when yeast was added to the must, the fermenting wine and final wine samples. This was followed by duplicate DNA extraction that was successfully extracted from these samples. The quantities obtained varied in concentration from 15 ng/ &#181;L to 87 ng/ &#181;L. This shows that the DNA extraction protocol is effective for metagenomic studies of wine. Although the quality of the DNA at A260/A280 varies, this may be attributed to different paramet...

Traminette grape is characterized by production of superior wine quality, in addition to appreciable yield and partial resistance to several fungal infections1,2,3. The natural fermentation of grapes relies on associated microorganisms, wine production environment, and fermentation vessels4,5. Oftentimes, many wineries rely on wild yeast and bacteria for fermentation, production of alcohol, esters, aroma, and flavor development6.
The goal of this study is to examine the bacterial composition of grapes and monitor their dynamics during fermentation. Although, the modern use of starter cultures such as Saccharomyces cerevisiae for primary fermentation, where alcohol is produced, is common to different wine styles7. In addition, secondary fermentation, where malic acid is decarboxylated by Oenococcus oeni to lactic acid, improves the organoleptic and taste profile of the wine and reduces the acidity of wine8,9. With the recent advances in the use of culture-independent methods, it is now possible to determine different microbes associated with the wine grape and the species that are transferred to must and participate in the fermentation at different times up to the final product10.
The roles and dynamics of wild bacteria from different grapes transferred to the must during wine fermentation are poorly understood. The taxonomy of many of these bacteria is not even known, or their phenotypic properties are uncharacterized. This makes their application in coculture fermentation still poorly underutilized. However, microbiological culture-based analysis has been used to determine the bacterial population associated with grapes and wine10. It is widely known that selective culture plating is tedious, prone to contamination, has a low reproducibility, and output can be doubtful; it also misses bacterial species whose growth requirements are unknown. Previous studies indicate that culture-independent, 16S rRNA gene-based methodologies offer a more dependable and cost-effective approach to characterizing complex microbial communities11. For example, sequencing the hypervariable regions of the 16S rRNA gene has been successfully employed to study bacteria in grapes leaves, berries, and wine12,13,14. Studies have shown that the use of either 16S rRNA metabarcoding or whole metagenomic sequencing is suitable for microbiome studies15. There is emerging information about the possible linkage of bacterial diversity to their metabolic attributes during wine production, which could help in the determination of oenological properties and terroir16.
The need to maximize the advantages of the metagenomic tools using next-generation sequencing (NGS) to study the grape and wine microbial ecology has been emphasized16,17. Also, the use of culture-independent methods based on high throughput sequencing to profile microbial diversity of the food and fermentation ecosystem has become very relevant and valuable to many laboratories and is recommended for industrial use18,19. It provides an advantage of detection and taxonomic profiling of the present microbial populations and the contribution of environmental microbes, their relative abundance, and alpha and beta diversity20. The sequencing of the variable region of the 16S region has become an important gene of choice and has been used during different microbial ecological studies.
While many studies focus on fungi, especially yeast, during wine fermentation21, this study reported the 16S amplicon sequencing and bioinformatic tools used to study the bacteria during Traminette grapes fermentation for wine production.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose gel&#160;</td>
			<td>Promega, Madison, WI USA</td>
			<td>V3121</td>
			<td>Electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>FastPrep DNA spinKit for soil&#160;</td>
			<td>MP Biomedicals, Solon, OH USA</td>
			<td>116560-200</td>
			<td>DNA extraction&#160;</td>
		</tr>
		<tr>
			<td>FastQC software</td>
			<td>Babraham Institute, United Kingdom</td>
			<td></td>
			<td>Bioinformatics&#160;</td>
		</tr>
		<tr>
			<td>Fermaid O</td>
			<td>Scott Laboratory, Petaluma, CA USA&#160;</td>
			<td></td>
			<td>Fermentation&#160;</td>
		</tr>
		<tr>
			<td>High-Fidelity PCR Master Mix&#160;</td>
			<td>New England Biolabs, USA</td>
			<td>F630S</td>
			<td>Polymerase chain reaction for sequencing&#160;</td>
		</tr>
		<tr>
			<td>NEBNext Ultra</td>
			<td>New England Biolabs, USA</td>
			<td>NEB #E7103</td>
			<td>DNA Library Prep</td>
		</tr>
		<tr>
			<td>NEBNext Ultra II DNA Library Prep Kit&#160;</td>
			<td>Illumina, San Diego, CA&#160; USA</td>
			<td></td>
			<td>DNA sequencing&#160;</td>
		</tr>
		<tr>
			<td>NovaSeq Control Software (NVCS)</td>
			<td>Illumina, San Diego, CA&#160; USA</td>
			<td></td>
			<td>DNA sequencing&#160;</td>
		</tr>
		<tr>
			<td>Novaseq6000 platform&#160;</td>
			<td>Illumina, San Diego, CA&#160; USA</td>
			<td></td>
			<td>DNA sequencing&#160;</td>
		</tr>
		<tr>
			<td>QuiBit</td>
			<td>Thermoscientific, Waltham, MA, USA</td>
			<td></td>
			<td>DNA quantification&#160;</td>
		</tr>
		<tr>
			<td>Quickdrop spectrophotometer&#160;</td>
			<td>Molecular device, San Jose, CA, USA</td>
			<td></td>
			<td>DNA quantification&#160;</td>
		</tr>
		<tr>
			<td>Sodium Phosphate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>342483</td>
			<td>DNA extraction buffer</td>
		</tr>
		<tr>
			<td>Stimula Sauvignon Blanc</td>
			<td>Scott Laboratory, Petaluma, CA USA&#160;</td>
			<td></td>
			<td>Fermentation&#160;</td>
		</tr>
	</tbody>
</table>

profiling the bacterial community of fermenting traminette grapes during wine production using metagenomic amplicon sequencing

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have facilitated a better understanding of both culturable and non-culturable microbes in grapes and wine. During industrial fermentation, microbes, known and unknown, are often responsible for product development and off-flavor. Therefore, profiling the bacteria from grape to wine can enable an easy understanding of in situ microbial dynamics. In this study, the bacteria of Traminette grapes must undergoing fermentation, and the final wine were subjected to DNA extraction that yielded 15 ng/&#181;L to 87 ng/&#181;L. The 16S amplicon of the hypervariable region of the V4 region was sequenced, relatively abundant bacteria consisting of phyla Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Fusobacteriota and followed by the Verrucomicrobiota, Halobacterota, Desulfobacterota, Myxococcota, and Acidobacteriota. A Venn diagram analysis of the shared unique operational taxonomic units (OTU) revealed that 15 bacteria phyla were common to both grape must, fermenting stage, and final wine. Phyla that were not previously reported were detected using the 16S amplicon sequencing, as well as genera such as Enterobacteriaceae and Lactobacillaceae. Variation in the organic nutrient use in wine and its impact on bacteria was tested; Traminette R tank containing Fermaid O and Traminette L stimulated with Stimula Sauvignon blanc + Fermaid O. Alpha diversity using the Kruskal-Wallis test determined the degree of evenness. The beta diversity indicated a shift in the bacteria at the fermentation stage for the two treatments, and the final wine bacteria looked similar. The study confirmed that 16S amplicon sequencing can be used to monitor bacteria changes during wine production to support quality and better utilization of grape bacteria during wine production.

Author Spotlight: Exploring the Fermentation Microbiome Through Next-Generation Sequencing

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 sequence stands out as a pivotal technology that is currently driving advancements in our research field

The quantity and quality of DNA extracted from grapes must, fermenting wine, and final wine were first determined; the quantity value ranges from 15-87 ng/&#181;L (Table 1).
Sequencing and bioinformatics 
The Illumina high throughput sequencer generated a FASTQ file that was imported to the Nephele and viewed on QIIME 2 platform26. Firstly, FastQC software was used to check for the sequence quality. Then, it was tr...

Watch this Scientific Journal Video about Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing at JoVE.com

Here, we present a protocol to describe amplicon metagenomic for determining the bacterial community of Traminette grapes, fermenting grapes, and final wine.

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have ...

profiling-bacterial-community-fermenting-traminette-grapes-during

Research

JoVE Journal

Biology

501 Views.  Appalachian State University. 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 sequence stands out as a pivotal technology that is curr...

Video: Author Spotlight: Exploring the Fermentation Microbiome Through Next-Generation Sequencing

DNA Extraction from Fermented Grape for Metagenomic Analysis

To begin, transfer 20 milliliters of the fermented grape sample into a sterile 50 milliliter screwed cap tube. Then add 10 milliliters of cold water into the tube and vortex for homogenization. Centrifuge the sample at 800 G for one minute at four degrees Celsius.Transfer the supernatant to a new 50 milliliter tube and centrifuge at 3000 G for 20 minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in one milliliter of PBS. Transfer the cell suspension to a two milliliter screw cap tube and centrifuge at 14, 000 G for two minutes.Now resuspend 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, 800 G.Transfer the supernannt into a clean microtube 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, 800 G 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 rack for three minutes. After removing one milliliter of the supernatant, resuspend 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 DNAse-free water on the filter membrane and gently stir the matrix with a pipette tip. Centrifuge at 14, 500 G for one minute to elute the DNA. Load the sample onto an agarose gel.Finally, measure the DNA concentration.

High-Throughput Sequencing and Bioinformatic Analysis in Grape Must and Wine for Microbial Diversity

To begin, use specific primers 515f and 806r to amplify the V4 hypervariable region of the 16S rNA 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 in 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. Resuspend he 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 fluorimeter. 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 End QIIME2, Perform Filtering and Trimming.Next, using DADA2, execute demultiplexed 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 winemaking displayed the dominance of phyla such as Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, and Fusobacteriota.At the genus level, important genera 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.