Kyle Boyar on Cannabis Testing | The Lex Files | Ep. 2

Episode Notes:

This episode of The Lex Files happened while Kyle Boyar worked for Medicinal Genomics, the cannabis kit testing company working to democratize cannabis testing. They sell kits to identify your plants’ gender, as well as good and bad microbes. Kyle Boyar explains the science behind the tests, the intricacies of cannabis genetics and microbiota, and the daily life of a cannabis scientist.

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Medicinal Genomics & research:
www.medicinalgenomics.com

Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers
https://f1000research.com/articles/4-1422/v1

Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests
https://f1000research.com/articles/5-2471/v1

American Chemical Society’s Cannabis Chemistry Subdivision (CANN):
www.cannachem.org
www.facebook.com/canndchas
www.instagram.com/canndchas

Cannabis Science and Chemistry:
https://www.facebook.com/groups/CSAC710/

For applying to the ElSohly Award:
http://tiny.cc/ElSohlyAward

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Transcript:

Kyle Boyar Absorb all the knowledge that you get the chance to be exposed to because you never know when that knowledge might come in handy someday.

Various Quotes “This is our humble hemp patch.”
“5000 years of medical cannabis use.”
“We’re learning about other cannabinoids.”
“Marijuana is growing in every state in the Union.”

Host – Lex Pelger I’m Lex Pelger, Director of Education at CV Sciences, and this is The Lex Files.

Lex Pelger Today we speak to the scientist, Kyle Boyar, about testing cannabis. He shares about his journey from hosting electronic music events, to studying neurology, to his current role in cannabis chemistry. When this interview was recorded, Kyle worked at Medicinal Genomics, a company that sells cannabis testing kits to the public. But since then, Kyle has become the Director of Product Science at TagLeaf, a software company that has developed a Laboratory Information Management System (LIMS) for cannabis testing labs. It’s geared towards keeping labs both transparent and compliant. Congratulations, Kyle. Today we’ll be hearing about the kits sold by Medicinal Genomics that you can use to identify your plant’s gender, and to explore its microbiome. Kyle will explain how those tests work and the history and development of the techniques behind them. You don’t need a science degree to grow cannabis and as Kyle says, these test kits are designed for everyone. For any consumers of cannabis, it’s good to know how your products are being tested and what that really means. In addition to his job, Kyle also supports the cannabis science community in various ways. He volunteers at the American Chemical Society’s Cannabis Chemistry Subdivision, known as CANN, where he serves as their Vice Chair and as the Chair of their scholarship committee. With all of these angles, we’re very glad to get Kyle’s insights into the world of testing cannabis. But before we start, we should define a couple of terms that get used: Matrix, or its plural matrices, is what we call the material being tested. The matrix might be the cannabis flower, it might be an edible brownie, or it might be a concentrated extract. The matrix is the material that’s holding the cannabinoid molecules. A PCR, or polymerase chain reaction is a widely used and hugely important lab technique that amplifies small amounts of DNA. For cannabis plants, these tests directly analyze the DNA from the plant itself. But they can also be used to identify the microbes present in the plant to see if they’re good, bad, or benign. Speaking of which, when you say a bacteria is aerobic, that means it needs oxygen to live. Anaerobic bacteria do not need oxygen. In lab techniques, when you sonicate a mixture, it means that you’re hitting it with soundwaves to mix it more thoroughly, which is a very cool technique. A plate is just as it sounds. A flat surface to hold chemical reactions. Columns are the long tubes that are packed material called the stationary phase. This is where the separation takes place. The stationary phase is the material in the column that makes a sample stick to it to separate out the various molecules. And lastly, a pipett is like a turkey baster for transferring liquids. At science speed-dating events, your pipetting skills might be something that comes up. Now to share more on the science of cannabis testing, here’s Kyle Boyar.

Lex Pelger Hello everybody. I’m very pleased to have Kyle Boyar here. Thanks so much.

Kyle Boyar Thanks for having me today, Lex.

Lex Pelger I was curious about how you got into science, in general. It was neurology that you first studied, but when did you know that you wanted to be a scientist?

Kyle Boyar Well… I guess that’s an interesting question. I’ve always been fascinated with the brain, in general. That’s where the neuroscience came in. Initially, I was actually going to be an environmental studies major because, frankly, that’s what I was good at in¬… high school. I was getting 5’s on my AP tests in environmental and really when it came down to it: One, it’s sadly a little bit of a depressing subject… We’ve got things like Trump nixing the EPA (United States Environmental Protection Agency) and cutting all funding for that. Ultimately, we’re really losing that battle and yes, while I’m passionate about the environment… I didn’t at the time, see myself as pursuing a career in that space. Although, I was really good at it and was interested in it to a degree… I thought, “Well, it’s another type of science and it’s a much harder science but, why not explore the brain a bit more?” Because… How do we perceive reality? How do we take the human experience and translate it into what we have today as society builds and… just in general, all the intricacies of it? It’s a super fascinating area so I decided to go for the neuroscience degree at UC Santa Cruz. I was there for 4-years doing my degree. Meanwhile, I was actually throwing events at the time. I ended up meeting with one of the owners of a testing lab at one of my events and… [said], “look, I’m about to graduate with a neuroscience degree. I don’t have a ton of lab experience, but I hear from a friend that you run the cannabis testing labs… I think I’d be a good fit for you because I’m hungry [to participate in the cannabis field]…”He said, “Totally interested in having you.” [I] followed up with him and really didn’t get much traction after following up. They weren’t very far from the college I was at so I drafted up a resume, showed up at their door, and told the owners there, “hey I met one of your co-founders the other night and he said I’d be a good fit and I haven’t heard back from him but I want this job doing cannabis testing.” They interviewed me on the spot, and I got the job pretty much right then and there. That pretty much launched my career in cannabis testing.

Lex Pelger For all you students out there, there’s the secret. Persistence.

Kyle Boyar It’s key.

Lex Pelger And networking. What kind of events were you throwing?

Kyle Boyar These were electronic music events. This was in Santa Cruz, California. I used to have a lot of fun out in the forest. This was actually my first event, really in a formalized venue… at the Catalyst Club in downtown Santa Cruz.

Lex Pelger It’s always fascinating how many scientists have such a strong, artistic background to them. Do you think that still influences your work and thinking? Your artistic background?

Kyle Boyar Oh, absolutely… I get a lot of inspiration from music and art, in general. It’s inspiring because at the time, this was when electronic music was starting to become the next big thing. Since then I’ve watched a lot of the people that I grew up throwing events with blossom into these fantastic artists that are now headlining these massive festivals and they’re experiencing all the success in the world. And it’s very cool to see that now come around to the cannabis field. For a while it [felt] like, “Wow, I hope one day I get my time to shine like these guys,” and here we are now. The field is really blooming so it’s really cool to finally have that all come around and get to share some of that success like a lot of my friends have had in their respective industries.

Lex Pelger To get back to your science, it’s such an interesting jump to go from neurology to analytical chemistry because they sound like they might be somewhat akin to each other but when you really get close to it, they’re very different fields. What was it like for you to switch to something like that, with that kind of learning curve?

Kyle Boyar To be honest, it wasn’t a super easy transition. I was stuck in molecular biology land doing PCRs, transformations, and running gels and all that kind of stuff. When you get into chemistry more… it’s polarity and interactions with columns, and figuring out the right detector for the right job. It was definitely a very different field and realm. But you take baby steps. I started off as a laboratory technician. I definitely didn’t just jump into this and become a lab manager or director right off the bat. It was really learning and the mentorship that I got at my first job at SC [Labs] that taught me really how to think like a chemist and how to apply those principles in order to get the correct answer. It was definitely not something that happened overnight, and it took a lot of hard work. At the time, the [cannabis testing] field was so brand new. There were very few [testing] methods out there. I feel like nobody even knew what the heck a validated method was at that time. We’ve really come a long way since then. All I could say to it is just that it takes a lot of hard work and, like you said, persistence. Also, just being good with working with people. Being a sponge, really. Absorb all the knowledge that you get the chance to be exposed to because you never know when that knowledge might come in handy someday.

Lex Pelger That’s good advice. What kind of techniques were you using? What happens in a lab like SC Labs? Especially in the early days for the methods they use and the kind of work you would be doing?

Kyle Boyar Very early on, it was… For example, potency prep was simple. Take your sample size and you have to figure out the right mass for it. You have all these different matrices with all these different concentrations, so you have to tease out the right sample mass in order to ensure that you’re within the range of your calibration of your instrument. To give the example of potency… you take your sample, you would dilute it in your solvent, and then you have to figure out a technique to actually thoroughly and completely extract all the cannabinoids from the matrix that you’re testing. That comes with trial and error, too… No one really had standardized methods or guidelines and we don’t even really have a lot of those today. AOAC (Association of Official Agricultural Chemists) has made some good progress on potency methods for things like flower and concentrates, and I believe they’ve done one for chocolate as well. But, a lot of this was just figuring this out on our own. We’d take that sample, we’d vortex, we’d sonicate, we’d do whatever we could in order to extract those cannabinoids out of the matrix and then you’d dilute it to the appropriate concentration and that just depends on what you were dealing with. You’d basically take that, put it into a 2mL autosampler vial, you’d get your injection and have your different methods set up to separate out the different cannabinoids. You have your different standards and you calibrate and make sure that everything lines up correctly. Integrating the peak the correct way… Software does that today—no problem. But this was very early on when we just had whatever was available to us. There was a lot of learning involved, figuring out what the ideal methodology was, and the best way to really approach getting the right answer.

Lex Pelger It sounds like it is so tricky. Even for testing regular cannabis flower, which is the easiest test, it’s still—you can get results all over the board. I’ve often heard that edibles in any form are the hardest thing to test because getting all the cannabinoids out using all the methods can be really tricky.

Kyle Boyar For sure. To give you a classic example of why infused products are so tricky, think about… when you’re trying to homogenize something like that, what ends up in your solution? What does that solution look like at the end of the day? Well, it’s… filled with a lot of particles that are a giant mess and who knows if you got a complete extraction or not? I’ll give some little nuggets of knowledge here. For example, with chocolates—now, of course, this method won’t work for something that’s not completely decarboxylated. And again, this was also the early days—for chocolates we found, in addition to sonicating, you’ve also got to apply some heat in order to really get a full release of those cannabinoids into the solution. Other matrices also pose tricky problems. One example of that would be: Let’s say you’re doing granola or something. Well, you have tons of little particulates in there so unless you want to completely mess up your column by injecting this stuff directly onto it, what you would do is make sure that you’ve filtered your sample properly so that you’re not gunking it up… If you are going to have a messy matrix like that, you want to ensure that you have the appropriate measures in place so that you’re not wrecking your column that costs hundreds of dollars. So, putting things in a guard column to protect that column and make sure that anything that is going in there that would cause problems is getting caught before it ends up onto the column, and then you have to spend hundreds of dollars to get a new one.

Lex Pelger >Oh, man. It must have been a learning curve.

Kyle Boyar Oh, yeah. I think with the new onslaught that we’re seeing of everyone trying to get into cannabis testing, there are so many people without this knowledge and experience that haven’t lived it yet. So, for all you big money people out there that just think that you’re going to walk in and this is going to be a cake-walk and you’re going to make millions, my advice is: Best of luck to you. Better hire someone with some experience.

Lex Pelger So, at SC Labs you got to see a lot of the nuts and bolts of testing. What was it like to switch to your current work at medicinal genomics?

Kyle Boyar I’ve actually always been really fascinated by the work that Medicinal Genomics was doing even before I was at the company. The founder, Kevin McKernan, and I actually used to share literature all the time on Facebook… I’m a moderator on this group called, “Cannabis Science and Chemistry.” I think he just saw that my ‘finger was on the pulse’, so to speak, with a lot of the research that was coming out. I had always admired his work from afar because I was turning a crank at a cannabis testing lab just making sure samples got out on time and once you learn all the analyses… what are you really doing at that point? If you’re not learning, you’re not challenging yourself, you’re not doing new things, and you’re not exploring. The transition was actually really refreshing because… I’ve always been fascinated by the work that they’ve done—and we can talk a bit more about some of the work that really got me inspired—but it really got me back on the biology train, which I had missed it for so long. It was… refreshing and I was really happy to get back into that realm. While I’m definitely no sequencing expert—I probably just know enough to be dangerous at this point—I definitely have a passion for learning new things. Every day I’m at work I’m constantly being challenged and learning new things that I didn’t know before. It’s been a really great transition, I’m actually really happy. I get to go out and fly out all over the place and interface, meet with all these people that are embarking on this new field… most of them [being] spring chickens to this. I get to impart a lot of the knowledge that I gained during my testing lab days—in the early days—and teach a new generation of scientists, which is really fulfilling.

Lex Pelger So, you’re their West Coast Field Applications Scientist… What would your work look like day-to-day?

Kyle Boyar That’s funny. I actually just had a conversation about this right before jumped on this podcast… My day-to-day is… it’s really support for the products that we provide. Medicinal Genomics provides three different product SKUs (stock-keeping units) primarily. First would be our PathoSEEK® Microbial Testing and that’s coupled with our SenSATIVAx® DNA extraction. We’ve designed two of those DNA extractions for different matrices. One of them would be for plant and flower matrices, and the other one would be for infused products and extracts. What that looks like, basically, is troubleshooting for all these different SKUs… Besides the SenSATIVAx® and the PathoSEEK®, we’ve got our youPCR® line which is… a do-it-yourself PCR [test] at home. What’s really cool about this is they’ve got these mini-PCRs now that are… portable. Some of them can even operate directly from your phone. So, people who are out here in the field that want to get rapid answers for… Does this plant have powdery mildew or not? Do I want to actually take this clone back into my grow room? Am I going to give my room ‘plant AIDS’, essentially? It’s supporting all those products… When it comes to sequencing, we offer something called, StrainSEEK®, and that comes in two different varieties. One is a smaller panel that covers 3.2 megabases (Mb)—3.2 million bases—and that’s looking at, primarily, your cannabinoid and terpene synthase genes, and that whole family. Then we’ve got a whole-genome sequence as well, which is… exactly what it sounds like. It’s a whole-genome shotgun, it’s the entire thing. That’s useful in the context of things like intellectual property where you’re trying to show that your cultivar that you’ve bred is truly unique. Really my day-to-day is answering questions about that service. For the testing labs and manufacturers or producers that are running any of these assays, [when they say], “So, I’m getting weird data. What do I do to fix the problem and how do I get more accurate data out of—where in my process am I going wrong?” A lot of it is teaching these people different tips and tricks in order to ensure they get the best result possible… A lot of the time it’s a lot of chemists that I work with… Testing labs, the majority of the analyses that they do are chemist[ry], so they hire chemists. Many times, they’re brand new, out of college, don’t have a lot of experience. But they know a bit about chemistry. Many of them don’t know anything about molecular biology. Some of them have never even done a PCR before. In the worst cases, have never really used a pipette. That has come across a couple times. So, we provide protocols and everything in order to help guide these testing labs. But, of course, everyone wants to get into testing and not everyone has VC (venture capital) funding or all the backing in the world. A lot of people are trying to do it out of their own pocket, so we get a lot of folks that want to take our protocols and ‘trim the fat’, so to speak. When you’re trimming fat, you’re really cutting corners and that’s really going to compromise your data. It’s really looking into what [the labs] are doing in their processes and where they could improve on those processes in order to actually arrive at a better answer; or if they’re not getting an answer at all, figuring out why that is.

Lex Pelger That’s fascinating. So, you get to work with growers on the ground all the way up to chemists, with these various products?

Kyle Boyar Exactly. What I like to say about youPCR® is we’ve essentially made molecular biology ‘stoner-proof’ with it. It’s a cool way to get people who otherwise wouldn’t be even holding a pipette, to embark on this cool scientific journey. At the same time, [they] also help out with their operations and learn more about the plant as they go.

Lex Pelger Can you define PCR for us?

Kyle Boyar So, PCR, is polymerase chain reaction. This was invented by a guy named Kary Mullis. He was actually taking hallucinogens on the beach, as the story goes, and he had this idea. I think he was working at Life Technologies, at the time. He was thinking, “How could we get DNA amplification to happen? We know that if you heat DNA, the double-strand DNA, to a certain heat—what we call a ‘hot start’ at 95 [degrees Celsius]—you’ll get those two strands to come apart.” His next idea was, “We have complementary base-pairing that happens with DNA. If we have these short little pieces of DNA—what we now know as primers—that are complementary to our upstream of our target sequence; if I can get them to anneal—that’s the part where you cool it down from the hot start—… to their complementary base-pairs, then I also throw in a polymerase into that reaction mixture, then won’t the polymerase just recognize this as something where it just has to run with it? If I just do this heating and cooling over and over again, will I get an amplification of my target sequence that I’m hoping for?” He’s thinking outside the box here. He’s in an altered state of mind. Then sure enough, he gave it a go and it worked. So, that’s the story of PCR and the general mechanics of how it works.

Lex Pelger Just a quick note here. In a PCR mix, you also need magnesium present as well as dinucleotide triphosphates (dNTPs), the building blocks of DNA.

Lex Pelger What’s really fascinating about what your company does is—I think, especially—is the microbial testing. You’re really working with the pathogenic bacteria, the toxigenic fungi, and the beneficial microbes that can grow on cannabis. Can you talk about how a grower who wants to be a doing much better job would be using this to test what’s on their plants?

Kyle Boyar Absolutely. Firstly, this is slightly different from PCR, in the sense that this is quantitative PCR. It’s quantitative because this amplification event… instead of just a primer, now you have a primer and a probe. That probe has a quencher attached to it. Whenever it’s just sitting in solution, the fluorescence is not allowed to happen. But, when you get this amplification event, what ends up happening is that quencher gets removed and then fluorescence is emitted. This fluorescence is what the instrument is measuring and that’s how you get quantitative data out of the PCR reaction. That’s why we think it’s also a really powerful tool is because if you can get quantitative data out of this DNA amplification, you have targeted primers that are very specific to your target sequence, then you can get highly specific. What’s really great about this is, because it’s targeted, you get a much better answer and you can get this answer much more rapidly than commonly used methods. Things like plating… it takes sometimes up to a week for some of these different fungi to grow on these medias so it’s really helpful to be able to… in a business case… you can get a same-day answer rather than waiting. When we have people that are waiting on results to release product in the market it’s really helpful. But, to jump back to your original question, which is, how can this give people a better insight into cultivation and produce, ultimately, better product? It’s a good way of being able to rapidly screen for safety. We know that there [are] a lot of pathogens out there that can be found in cannabis and some of them are actually found commonly—they’re endophytes. That means [that] they actually reside within the cannabis plant, they’re not just on the surface. Environmental factors, things like: if you’re cultivating outdoors or, in general, if you were growing in an area that’s not well insulated or there’s not filtration happening of the air that’s incoming into your grow room, then fungal spores can get in there… In the case of something like salmonella, are you fertilizing with something like chicken sh*t? If you are, then you run the risk of potentially having salmonella on your product; or coliforms or other things that could, potentially, not be so great to the end user. So, having these rapid screens and the availability of these tests to get quick answers is extremely valuable. Also, like I was saying about the specificity, often times when we look at the culture plating methods that are available currently that are commonly used in food when we sequence the stuff that’s actually growing on those plates, it’s often not the target organism that they’re actually trying to measure. To give you an example, Medicinal Genomics did a study looking at some of the different methods that are currently available. In this case, we looked at 3M™ Petrifilm Plates and we also looked at Biomérieux and their culture-based system… the Tempo®. In both of these cases, oftentimes what we found when we sequenced for total yeast and mold, we ended up finding up to 60% bacteria was growing on these plates. So, ultimately, people were getting these inflated counts. A lot of cultivators who spend tons of money on this testing to get their stuff to market are, ultimately, having their products failed because people are using, one: an antiquated technology that probably really shouldn’t be in use anymore. At least, not as widely as it is currently. And two: you’re basically getting the wrong answer. If you’re not being selective for the organism of interest, then how can you really trust the data that’s coming out of these things? Ultimately, it’s going to lead to more failures and then people are going to look to things like fungicides. One that might ring a bell to your listeners here is Eagle® 20[EW], or myclobutanil. That one is commonly used on things like grapes in wine country. But that’s a different route of administration when you’re consuming grapes. You’re not smoking grapes. What’s really tricky about myclobutanil is, there is a cyano group on there. So, a cyanide group, essentially. C with a triple-bond to N. What happens when you heat this stuff is that cyano group will pop off. What happens when that occurs is you get hydrogen cyanide. That’s getting in people’s lungs. Basically, if you’re going to fail people for total count tests, things like total yeast and mold, they’re going to use more fungicides. When you use more fungicides, you’re going to get more myclobutanil around. When you get more myclobutanil around, you’re going to have people inhaling hydrogen cyanide more often. Aside from the issue of not being able to get what is considered, probably, harmless product to market because total count tests don’t actually distinguish between what’s pathogenic and what’s benign, you’re now also creating a public health risk because more people are spraying this stuff on their products.

Lex Pelger As far as pathogens go, can you tell us more about aspergillus?

Kyle Boyar Aspergillus is actually one of those endophytes in the cannabis plant that I was referring to earlier. The real problem with aspergillus is when it comes to immunocompromised patients or consumers of cannabis. We all know cannabis is great as a medicine for those who are dealing with cancer or have autoimmune disorders and things like that. But if an aspergillus spore happens to get into the lungs of one of these people, it can really cause some serious complications. In this case, it would produce something called aspergillosis—or it could. It doesn’t always¬—but it does have the potential to produce something called aspergillosis where [these] fungi will colonize the lungs of the patient and it can really cause some serious complications… You basically get a lung infection and it can be fatal in some people. There’s definitely a lot of documented cases of fatalities from aspergillosis and what’s really concerning as well is, it’s not just limited to the folks that are immunocompromised. There are some case studies out there showing that perfectly healthy people do get these types of lung infections. Ultimately, it’s just one of those things where it’s a public health issue. People want to use cannabis recreationally. They also want to use it as a medicine. We know this to be definitely something that’s harmful and it does reside in the plant, but not all of it is pathogenic. It’s really important to distinguish between what is potentially disease-causing… illness-causing in this case, and what is not.

Lex Pelger I’ve been writing about cannabis and the endocannabinoid system for years. I’ve traveled the world to gather people’s stories about cannabis and the history of our use of it. But at the time, the positive effects from CBD [cannabidiol] were only starting to dawn on me. Then I saw, firsthand, the impact it made in the health of my cousin and the comfort it gave my grandmother as she was passing. Since then, the many accounts I’ve heard from those using CBD from hemp, made me a believer in its potential. CV Sciences works hard to produce the highest quality hemp supplements, so people everywhere can experience the [benefits of CBD] for themselves. I’m proud to be working with them to help spread the good work. Proud because I know we lead the industry in research and education. Proud because I know we make excellent CBD supplements from true agricultural hemp. But I’m most proud because I know our products make a difference in people’s lives. At www.pluscbdoil.com, use the coupon code LEXFILES for 20% off to see for yourself.

Lex Pelger One of the things you mention in one of the papers we’ll link to in the episode notes, is that th[ese] fungi grow as well on the standard culture plates that are used out there. So, it can tend to be underreported using the methods that are usually used?

Kyle Boyar Yes, correct. It’s not to say that it won’t grow at all… this isn’t one of those things where it takes anywhere from five to seven days to completely enumerate on a plate. It is under reported in the sense that—what we get in terms of recovery. When we compare, say, a DNA-based method to a culture-plating method, the recovery is substantially reduced in hundreds of folds less. That can really generate some false negatives, in that regard. If you get false negatives and you’re dealing with someone who’s already sick and immunocompromised, then that can really lead to some serious problems down the line. With the world that we’re living in, in cannabis now, everyone’s really quick to go to litigation. We see this all the time with things like pesticides. One example I could think of off-top is, Brass Knuckles™ recently got sued for basically advertising their stuff as pesticide-free. When people spot checked them and took stuff off the shelf and tested it, it came up with loads of pesticides. In this litigious world that we live in, these testing labs really want to be 100 percent sure that if there is a microbial threat in the samples that they’re testing, that they pick it up. That’s another reason why things like enrichment are really important. For those of your listeners who aren’t really familiar with microbiology or the concept of enrichment, imagine it like this: If there’s a very small amount of something in a sample that you’re testing—let’s use the idea of a ‘needle in a haystack’. If you don’t allow the needle in the haystack to multiply, in this case what we call the enrichment is letting your sample sit in a growth medium and incubating it at the proper incubation temperature; If you’re not letting it grow and multiply enough to the point where you can hit the target then, essentially, you’re going to miss it. It’s a statistical thing. Especially if you’re taking subsamples out of a larger portion to try and do detection. You need to ensure that you give that ample time to multiply and grow in order to hit it. With aspergillus, it’s just one of those things where, if you’re not employing the proper methodology, and you’re not being careful, it can really end up being a bad situation for everyone involved. Both on the producer-end, both on the testing lab end, and for the end-user.

Lex Pelger So, just to summarize a little bit, it’s that the cannabis plant has this microbiome that’s different on the inside of the plant, and the outside of the plant. Currently, the tests are being done on culture plates, but a lot of things don’t grow on culture plates and your company is using DNA. My practical question is, how do you see different states handling this problem of wanting to test for this stuff when it’s so complicated?

Kyle Boyar That’s where my job comes in. A lot of what I do is education, and that’s not just at the testing lab or the operator level of who’s using our test. It’s for regulators and people who are trying to get a handle on how to properly regulate the industry and make sure that what is going to market is safe. Many of these people don’t have a background in cannabis, that’s for sure. They’re still learning, just like everyone else, as we chart into the unknown. What they need to realize is that, yes, it’s a unique matrix. It has its own challenges and these challenges are something that they need to learn about in order to make the proper educated decision. Another example of exactly why you would want to go with a DNA-based method is, these plating methods won’t pick up things like endophytes. Because in order to pick up endophytes, you have to break open the plant and actually get those bacteria and fungi out. How you do that without actually breaking open plant cells, that’s going to be problematic. Furthermore, there [are] other things that we find on the cannabis microbiome that are atypical compared to some of the other analyses that you see. Things like, endofungal pathogens… Around this time last year, we embarked on a sequencing project where we were trying to figure out: We see differences between culture and plating methods and qPCR. What are the underlying differences in the microbiome where we see discordance in the samples between what’s being plated and what’s being run on qPCR? What we ended up finding was, when we actually sequenced the amplicons that had come out of this—for the listeners, let me give a little more detail there. When we’re doing something like, a total aerobic plate count—aerobic bacteria—we’re using primers that are targeting the 16S-ITS region. ITS stands for internally transcribed spacer regions. Those regions are evolutionarily conserved in bacteria, in particular, aerobic bacteria. We’ll amplify that region to look for what aerobic bacteria might be present and similarly in the context of total yeasts and molds, we have an 18S-ITS region. We’ll amplify those regions, try and figure out what exactly is growing in terms of total yeasts and molds. When you amplify these things, you get the amplicons that are generated out of them. Those amplicons, we can then sequence. When we sequence them, those amplicons act like a unique molecular barcode… They’re very variable, but they’re also very specific to different [genera] and species. Sometimes down to the species level, it’s not always perfect out of the species level. But when you upload this stuff into something like a metagenomic database, you can get a microbiome ID out of this. What we did was… we did both of those assays and we did a microbiome ID on this. We found one common underlying theme in the samples where there was discordance. That underlying theme was the presence of a bacteria called ralstonia. Ralstonia is an endofungal bacteria. This means it’s a bacteria that resides inside of fungal cells. So, good luck detecting an endofungal bacteria with a plating method, because you’re never going to be able to see it, because the cells aren’t being broken open in order to actually get them to grow. Just another reason why molecular methods are going to be helpful in this scenario. Ralsonia is a pathogen to both plants and humans. Specifically, it’s been shown to cause lung infections in certain patient populations—people with cystic fibrosis, in particular. Other immunocompromised populations could certainly be susceptible to this type of infection as well.

Lex Pelger Good. Thank you for sharing on that. The other part I wanted to make sure we had time to get to, was the work you’ve bee doing with the Jamaican Lion genome.

Kyle Boyar That was actually a really cool project. Last year when we had the cryptocurrency boom that happened, suddenly, a lot of these crypto-companies had a lot of money to throw around. One of the companies that we felt was doing a service to the community was called Dash. Dash is, essentially, digital cash. What they do is, they have grant proposals. This is a[n]… autonomously governed—basically the stakeholders in the currency. They govern themselves and they vote on who gets funds to go to a certain project. We applied for one of these grants and the proposal was to sequence a type two plant. A plant that has both CBD and THC producing genes, and deeply sequence it so that we can understand the genetics of the plant better. After a lot of nail-biting and getting down to the wire, we needed a certain amount of votes to get this grant and we ended up getting it. We took the funds and we decided to embark on this project using a combination of different techniques… The first part of the project was isolating high molecular weight DNA in order to actually get good enough quality DNA to do the sequencing work, which is no easy task in cannabis. Especially because it’s a really complex matrix that expresses up to 30 percent cannabinoids and terpenes and all these things that can really be problematic for molecular biology. So, the first challenge was to get good enough quality DNA, in order to do the sequencing. Then once we got good enough quality DNA, then we applied sequencing technologies like, PacBio®. Now, there [are] two big players in DNA sequencing… two of the ones that stand out are the most well-known are Illumina® and PacBio®. We didn’t use Illumina®. That is what we call short-read sequencing and there’s a reason for that. Basically, cannabis is extremely polymorphic. That means, that there’s a lot of variation within the genome. To give you some context, the human genome has a snip or a single nucleotide polymorphism, every 1 in 100 base pairs, roughly. Now, the cannabis genome has a snip every 1 in 40 base pairs. The areas under selection, you’ve got a snip every 1 in 25 base pairs. So, that’s four-fold more complex than the human genome is. With all that different variation and you’re applying some thing like short-read technology where you get these little stretches of DNA, it’s really hard to actually assemble that into a picture if there’s so much variability and repeatability within that genome. Short-read technology without a reference or more complete picture, which we didn’t have at the time, is really hard to make use of anything. So, we applied PacBio® technology, which is long read sequencing. When you have longer reads, essentially, you’re able to get a much better picture of what’s going on and these things actually map a lot better as a result. So, the first step was getting a ton of long reads with PacBio® data. Then the next step that we applied was a technology called, Hi-C from Phase Genomics®. What Hi-C does is, it creates maps of where things link, in terms of chromosomes. There’s what they call, bisulfite conversion, which is another… method that people use to try and do this. But it’s wrought with false positives, it’s not exactly the cleanest way of doing it, and it really “beats the hell out of the DNA,” as one of my colleagues says. It makes it really tricky to get the stuff to work exactly perfectly. But with Hi-C, this technology from Phase Genomics®, you get these structures in a much more precise manner. Which, basically, allows you to take all these long-read sequencing data and create chromosomal structures out of it. Now that we have chromosomal structures—in cannabis there are 10 chromosomes—we can actually see all the little details that we weren’t able to before. Where we were failing the pieces of the puzzle together because they were just tiny little fragments that are—a gazillion of them. You had no idea where they really went. Now, we know where they go and where they all map. What was really cool about this project was, it was done in under 120 days. I can’t take much credit for it. That is definitely the hard work of our sequencing team, our R&D [research and development] team, and Kevin for championing this whole Dash project. It’s really been quite the history in the making to watch. There [are] new improvements happening right now. We’re doing something with PacBio® called, the Cannabis Pangenome Project, where we’re taking this now and we’re doing this to a whole family of different cannabis genomes. By doing that, we can really tease out… more of the intricacies and regulatory elements and structures associated with the cannabis genome and figure out what does all this stuff do?

Lex Pelger It’s fascinating. You get to go so deep into genome of this stuff. The last part I wanted to ask before we let you go was about your more public work. Because you’re working with the American Chemical Society as their Vice Chair for Cannabis Chemistry Subdivision [CANN], which I think is really impressive. The American Chemical Society is fairly conservative and for them to be getting into cannabis, I think it takes leaders like you pushing this forward. So, I was wondering what it’s like to be working with them?

Kyle Boyar Well, first and foremost, I definitely cannot take all the credit for getting the American Chemical Society [ACS] to accept us as a subdivision within the [Division] of Chemical Health and Safety. That came from some pioneers whom I really have a lot of respect for. Ezra Pryor has been a mentor of mine for a number of years now, and he’s the one who actually championed this. Along with Jahan Marcu, Melissa Wilcox, Mark Scialdone, and I’m sure there are a couple others that I am missing here. I got brought into the ACS around 2015 and I actually started as their social media coordinator. I was just trying to raise awareness within the community that there’s this conservative organization that is really trying to include us… Cannabis—it’s still chemistry, right? This is the world’s biggest chemistry society, and we deserve a seat at the table, too. Now, granted, we’re a subdivision. We aren’t a full technical division. We’re housed within the [Division] of Chemical Health and Safety. So, we are still governed by the [Division] of Chemical Health and Safety. We have to play by the rules, and we need to ensure that we are doing everything in alignment with what their mission is, as well. It’s been really cool to meet a lot of the budding cannabis chemists in this industry and offer just a scientific forum for people to exchange ideas, to network, and to learn more about the best practices, because, as I mentioned, there [are] a lot of people out there cutting corners right now that aren’t necessarily doing it the right way. It’s because, not necessarily out of ignorance, but a lack of guidance. I’m sure that there are some smart people that are trying to cut corners that are doing it because [they are] trying to save money. But most people are doing it simply because they don’t have resources available to them to actually do it the right way. That’s what CANN is all about fostering. It’s making sure that people interact with the scientific community, get to know their peers, and share their work so that we can all move forward together, collectively, and do this the right way. We have a lot of problems out there currently with lab testing. From be it, inconsistent test results or bad methodology or simply bad actors, where people are dry labbing. But I think, if we come together as a community and we all get to know each other and what each other is doing, we can really raise the tide for all boats by doing this. It’s been really awesome to work with the team at CANN and… I’m now Vice Chair of the organization so it’s been really awesome to watch all these cannabis chemists grow into the scientists that they are today and to just mentor the new generation. There [are] tons of fresh graduates that are coming out of school and seeing cannabis testing as this emerging new field and they want to get involved but there really isn’t a whole lot of knowledge base or resources out there so we’d like to be that resource for them. It’s been a really great opportunity to showcase the work that’s been done by others. We all stand on the shoulders of giants, right? Being able to pass the torch of knowledge on to this new generation has just been really awesome and it’s been a real pleasure to do it.

Lex Pelger That’s great and we’ll link to the CANN Subdivision in the episode notes for any budding scientists out there. Which brings me to the last point… CANN has the first scholarship for cannabis scientists available, correct?

Kyle Boyar That is true… That scholarship started last year with a generous donation from Heidolph North America. They’re a manufacturer of lab equipment, things like [rotary evaporators] and alike. They gave this very generous donation for five years to give money out to people who are doing research in the space. What it does for them is it essentially provides funding for them to fly out to the ACS national meeting. This happens every spring for the scholarship. Basically, they get to present their work and share their ideas with their peers. We give away up to $1500 per recipient to reimburse those travel funds and it’s really open to anybody. You don’t necessarily have to be a degreed scientist or PhD. You don’t need to be a student. This is open to anyone who has good ideas that they feel needs to be shared with the community. It’s a great opportunity just to get your name out there. We’ve had a lot of great talks. The inception of this award, we had seven different recipients… Initially, this was not called the ElSohly Award. It has this very clunky name called, the CANNCHASHNA Award. So that was CANN, CHAS. So CANN: cannabis chemistry subdivision, CHAS: [Division] of Chemical Health and Safety, and HNA: Heidolph North America. We called it the HASH Award for short that year. But of course, that wasn’t a very sexy name now, was it? We changed it up and we felt we need to honor somebody who made big contributions to the field of cannabis science and we felt that Mahmoud ElSohly was a great candidate for that… People would sometimes give me flack because… he’s got the only research license out at Ole Miss and he grows… not the greatest cannabis. It’s used for research purposes. But, hey, at least there is somebody out there doing it. He’s done the best he can with the cards that he’s been dealt because he has to deal with the federal government. You don’t get to ‘grow the bomb’, so to speak, if you’re working with a government agency… The cards are dealt… I think, aside from all the controversy there, I think he’s done a whole lot of work to support the industry and get us to have better products. One that comes to mind is things like THC-Hemisuccinate and all the different derivatives that he’s used, all the delivery methods that he’s developed. This is taking a more pharmaceutical approach, granted. He is very well published he does have a lot of literature out there… that is substantial in terms of contributions to the field. We wanted to honor him and we’re really grateful to have the award named after him. For those who are interested in applying for the ElSohly Award, I encourage you to submit an abstract and a resume. Those are all due July 1st to elsohlyaward@gmail.com. Lex, I’m sure you’ll put that in the show notes as well?

Lex Pelger Yeah. Thanks for that callout. It’ll be great to see the array of voices. So, thank you so much for taking the time to talk to us and for your work with CANN, spreading the knowledge, and all of the learning we got to do today.

Kyle Boyar Thanks for having me, Lex. It was truly a pleasure and I look forward to hearing it.

Lex Pelger Thanks. Until next time.

Lex Pelger Thanks for tuning in. To listen to other episodes, find us at PlusCBDoil.com/lexfiles. If you have any questions, compliments, or suggestions, feel free to write me at research@cvsciences.com. If you enjoyed the program, please rate us on iTunes and share a link to your social media. It means a lot to us. The Lex Files is produced by Matt Payne. Our chief advisor is Amabelle Dela Cruz. The music is by Jake Bradford Sharp. Our sponsor is CV Sciences, maker of America’s favorite CBD oil and remember the coupon code LEXFILES. I’m Lex Pelger, signing off.