1. Poster Print Size: This poster template is 24” high by 48” wide. It can be used to print any poster with a 1:2 aspect ratio including 30x60, 36x72, 42x84, and 48x96. Placeholders: The various elements included in this poster are ones we often see in medical, research, and scientific posters. Feel free to edit, move, add, and delete items, or change the layout to suit your needs. Always check with your conference organizer for specific requirements. Image Quality: You can place digital photos or logo art in your poster file by selecting the Insert, Picture command, or by using standard copy & paste. For best results, all graphic elements should be at least 150-200 pixels per inch in their final printed size. For instance, a 1600 x 1200 pixel photo will usually look fine up to 8“-10” wide on your printed poster. To preview the print quality of images, select a magnification of 100% when previewing your poster. This will give you a good idea of what it will look like in print. If you are laying out a large poster and using half-scale dimensions, be sure to preview your graphics at 200% to see them at their final printed size. Please note that graphics from websites (such as the logo on your hospital's or university's home page) will only be 72dpi and not suitable for printing. [This sidebar area does not print.] Change Color Theme: This template is designed to use the built-in color themes in the newer versions of PowerPoint. To change the color theme, select the Design tab, then select the Colors drop-down list. The default color theme for this template is “Office”, so you can always return to that after trying some of the alternatives. Printing Your Poster: Once your poster file is ready, visit www.genigraphics.com to order a high-quality, affordable poster print. Every order receives a free design review and we can deliver as fast as next business day within the US and Canada. Genigraphics® has been producing output from PowerPoint® longer than anyone in the industry; dating back to when we helped Microsoft® design the PowerPoint® software. US and Canada: 1-800-790-4001 Email: info@genigraphics.com [This sidebar area does not print.] Multiplex qPCR and Cannabis Microbiome Sequencing Reveals Several Bacteria and Fungi Native to Cannabis Flowers Kevin McKernan, Jessica Spangler, Lei Zhang, Vasisht Tadigotla, Yvonne Helbert, Kellie Dodd, Douglas Smith Medicinal Genomics Corporation Jessica Spangler Medicinal Genomics Corporation Email: Jessica.Spangler@medicinalgenomics.com Research and Development Contact 1. McKernan, K.J., Spangler, J., Helbert, Y., Zhang, L. & Tadigotla, V. DREAMing of a patent-free human genome for clinical sequencing. Nat Biotechnology 31, 884-887 (2013). 2.McKernan, K.J. et al. Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment. PLoS One 9, e96492 (2014). 3.Stewart, E.J. Growing Unculturable Bacteria. Journal of Bacteriology 194, 4151-4160 (August 2012). 4.Kusari, P., Kusari, S., Spiteller, M., & Kayser, O. Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens. Fungal Diversity 60, 137–151 (2013). 5. Langouet, S. t al. Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer research 55, 5574-5579 (1995). 6. Langouet, S. et al. Metabolism of aflatoxin B1 by human hepatocytes in primary culture. Advances in experimental medicine and biology 387, 439-442 (1996). 7. Yamaori, S., Ebisawa, J., Okushima, Y., Yamamoto, I. & Watanabe, K. Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: role of phenolic hydroxyl groups in the resorcinol moiety. Life sciences 88, 730-736 (2011). References Introduction Our current assay offerings for use with SenSATIVAx TM and PathogINDICAtor TM are listed below. Presence/Absence Tests E.coli E.coli STEC only Salmonella Aspergillus (A.niger, A.fumigatus, & A.flavus species specific assays coming soon) MGC Offerings As many regulations are beginning to mandate “heat killing” of microbial content, we must remain aware of how these drying techniques often confound culture- based methods used to monitor colony forming units (CFU). Even though this “heat kill” process may be effective at sterilizing some of the culturable microbial content it does not eliminate various pathogenic toxins like Aflatoxin or the DNA that encodes the Aflatoxin gene. This is of particular importance as Aflatoxin is a carcinogen. The clearance of Aflatoxin requires the liver enzyme CYP3A4 and this liver enzyme is potently inhibited by Cannabinoids 5-7. With the publication of the Cannabis genome and many pathogenic microbial genomes, we designed several multiplexed quantitative PCR (qPCR) assays to detect pathogenic DNA in a background of host Cannabis DNA. One of these qPCR assays is designed to detect total yeast and mold and thus targets the 18S rDNA ITS (Internal Transcribed Spacer) regions. ITS regions are routinely used to sequence the microbiomes of samples and identify and itemize the collection of microbial communities present in a given sample. The addition of next-generation sequencing primer tails to this yeast and mold qPCR assay enables reflexive sequencing of samples testing positive for yeast and mold. Next- generation sequencing thus reveals a digital record of the microbiological community on a given Cannabis sample. Cannabis samples that tested positive for yeast and mold with qPCR and culture-based techniques were sequenced to precisely identify the collection of microbes present in the failing qPCR samples. Microbes harmful to both the plant and humans were identified (Botrytis, Magneporthe, Fusarium and Aspergillus) while over 180 different microbes including endophytes were also present. This qPCR assay presents a real-time tool for both quality testing and geospatial monitoring of microbial communities in cannabis production facilities. This highly sensitive assay allows for the detection of very low levels of contamination. This can be used to proactively monitor for outbreaks before they can spread. The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food-borne illnesses. As a result, the detection of mold and bacteria on agricultural products has become an important safety consideration. This risk extends itself to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products. As a result, third-party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets. Medicinal Genomics has developed a novel PCR-based assay for the detection of pathogenic microbes in Cannabis materials. This process consists of a proprietary system for DNA extraction called SenSATIVAx TM and a novel PCR assay called PathogINDICAtor TM. PathogINDICAtor TM utilizes a novel qPCR-based assay that is contamination-free and provides an internal plant DNA control for every reaction. DNA detection is based on a 5’ nuclease assay that directly measures the amount of plant and microbe DNA in a given sample. This technique provides robust sensitivity, specificity, and multiplexing capability. Positive results can then be confirmed through microbiome sequencing. PathogINDICAtor TM is able to provide a contamination-free technique through a proprietary process known as DREAM PCR 1, 2, which uses methylation-specific restriction enzymes to prevent the carryover of amplified products from one reaction to the next. We present favorable comparisons to conventional techniques. 5mC5hmC Enzyme digests - + Restriction Enzyme In PCR DREAM PCR 5hmCTP Cannabis DNA (lightly methylated) DREAM PCR DNA (heavily methylated) Figure 2 (below): Process of a typical 5' nuclease fluorescent assay. PathogINDICAtor TM includes 5’ universal tails and 5hmCTP in PCR. Figure 3: Sample containing plant and microbe DNA. (Plant DNA = Green, Microbial DNA = blue). X-axis is cycles (every 1.5 minutes). Y-axis is relative fluorescence units in a log 10 scale. Figure 4: Sample with no microbe DNA present. (Plant DNA = Green, Microbial DNA detection = Blue) Figure 7: After the positive result using PathogINDICAtor TM, the product was next-generation sequenced on and Illumina MiSeq with our Yeast & Mold Detection Assay to reveal the microbes contained in the sample. Using OneCodex to analyze the data, this sample is primarily Byssochlamys, which produces mycotoxin and should be detected for safety. (Sample 3 in Figure 8 is also a representation of this sample) In general, this data can be used for many purposes, most notably to determine the possible contaminates in a greenhouse or grow room prior to wider infestation. Yeast/Mold (10,000 CFU/g) SimPlate®(CFU/g) 3M TM Petriflim TM (CFU/g)Biolumix PathogINDICAtor TM (Cq) 00pass21.71 (fail) Total Aerobic (100,000 CFU/g) SimPlate®(CFU/g) 3M TM Petriflim TM (CFU/g)Biolumix PathogINDICAtor TM (Cq) 24000 (pass) pass24.03 (pass) Figure 6: To aid in validation of SenSATIVAx TM and PathogINDICAtor TM, we utilized three alternative culture-based assays (3M TM Petriflim TM, SimPlate®, and Biolumix). When a sample does not correlate we reflex to next-generation sequencing of that sample to confirm our assay is correct. Total Coliform (1,000 CFU/g) SimPlate®(CFU/g) 3M TM Petriflim TM (CFU/g)Biolumix PathogINDICAtor TM (Cq) 00passCq>40 Total Entero (1,000 CFU/g) SimPlate®(CFU/g) 3M TM Petriflim TM (CFU/g)Biolumix PathogINDICAtor TM (Cq) 00passCq>40 SimPlate®: Technique 1 Total ColiformTotal EnteroTotal Aerobic Total Yeast & Mold 3M TM Petriflim TM : Technique 2 Total Coliform Total Entero Total Aerobic Total Yeast & Mold Total Aerobic Total Yeast & Mold Internal Control PathogINDICAtor TM Biolumix : Technique 3 Total Coliform Total Entero Total Aerobic Total Yeast & Mold Discussion 12345678910 Figure 8: A look at multiple sequenced PathogINDICAtor TM positive cannabis samples and the microbiomes found on each. This data can help in determining trends within multiple cannabis samples. Notice that 9 of 10 have Penicillium as a part of the microbiome, which is a known endophyte 4. Sample 1 is the Australian Bastard sample referenced in the presentation titled “Genomic, Terpene, and Cannabinoid Profiles of a Putatively Novel Cannabis Species”. Threshold Tests Total Yeast & Mold (18S) Total Aerobic Bacteria (16S) Total Enterobacteriaceae (16S) Total Coliform Figure 1 (above): DREAM PCR generates heavily methylated DNA that cannot survive digestion. Step 1: Primers and probe bind to target DNA. Step 2: PCR occurs, primers are extended on forward and reverse DNA strands. Step 3: Probe is degraded as a result of polymerization and fluorescent signal is generated. Step 4: Target DNA is amplified and fluorescent signal can be measured and quantified. + Examples of PathogINDICAtor TM Data Mold PathogINDICAtor TM Internal Control Yeast & Mold Signal Biolumix *Incorrect Passing Result Blue line represents the 1,000 CFU threshold while the green line represents the 10,000 CFU threshold. *Failing Result Figure 5: With only 1% of microbes able to grow into colonies 3, there is the potential for false negative results with conventional culture-based methods. The cannabis sample has visible mold growing, however, the culture-based method does not detect the microbes. PathogINDICAtor TM detects and fails the sample. Example 1: Comparison of a Visually-Contaminated Sample Internal Control Microbial DNA The Microbiome Profile of the Sequenced Positive Result Samples Relative Read Count Example 2: Comparison of Multiple Methods Kellie Dodd Medicinal Genomics Corporation Email: Kellie.Dodd@medicinalgenomics.com Business Development Contact John Geanacopoulos Medicinal Genomics Corporation Email: jg@medicinalgenomics.com Sales Contact www.medicinalgenomics.com