Hey, good afternoon everybody Tom Stewart here with Liz Trotter. This is smart business moves.  What does that look like? Um happy birthday to hope you're having fun You set a card or I should do that. That's a great idea Tom Maybe I'll do that for the next brother. Yeah now You can do something digital, right? Like one of those singing Happy birthday things where some celebrity is like singing something and yeah The name in there. I like those so maybe I will do that. I Uh, I was thinking I would send a potato.  No, what does that what would mean potato like a real? Yeah, it's a potato Hey Denny and that's why it's funny because why would you send somebody a potato? Yeah, and and they can they'll put a message with your potato. I think they wrap wrap the message around your potato with rubber band How much does it cost to send somebody a potato like $15? We're in the wrong business, I know it's cheaper than flowers so and I think Potato would definitely be the way to go with most of the members of my family I think that they would appreciate a potato better and more than Like cookies or flowers or anything Wow, I love sending cookies I do love sending cookies too. Yeah brownies. I like sending brownies a lot of people love brownies Well, you know people get brownies or cookies. They kind of like oh boy, you know I mean there's something you can do with those you get a potatoes like Wow, how did you know? I will tell you how thoughtful.  They got conversation for a lifetime My sister sent me a potato one time when people are talking about birthday gifts. They never forget that potato They can put a stamp on a single flip-flop with an address on it and That does not shock me at all Rochelle. It just really doesn't Hey, didn't eat since it's just us on here us as growth that fellows Did you hopefully got the link to my kelin Lee I I did tag you all right So I had heard that oh we have done like public speaking exercises I've seen this done before where you have to take a potato and introduce the potato to you know, everyone in the audience and Explain what makes that potato special all its unique attributes and you know kind of what what? accomplishments and life stories and what you make that potato so awesome It takes some imagination to make a potato sound exciting well I'm I'm thinking that one of the things that we have done in the past in interviews is to tell people that they can Pick up anything in our office and Give us 20 uses for it But I might like the idea of handing them a potato and asking us how they could use that potato instead That's a kind of a good idea. I might I might try something like that To I like to mix it up a little That's a that's the exercise we do in Boy Scouts actually when we're teaching them leadership skills and we're talking leadership So, you know Nice That's a great idea about that family potato. I do like that Tom that is a Great little segue there and you can explain why we are all alone here Today, I'll let you Let's go tell a story though Richard just got back From a place out in New Mexico called Philmont. It's a I guess it's a place that Boy Scouts go go camping over the summer and for the last two weeks Ian and a bunch of his friends were like wandering around the wilderness out in the deserts and mountains of New Mexico and they survived Thunder storms and hail storms and the temperature gets, you know, really hot in the day and it gets really cold at night and We flew back Saturday and my my daughter, you know, she's been home And we went over to pick him up at the airport and we're driving back and he's telling all his stories about getting pelted with hail and so forth and My daughter and I took the dogs out for a walk Saturday and it rained on us and we were telling that story and Wasn't very he started off like it was really sympathetic And my daughter was telling the rain story with the dogs and she figured out that he was really punking her I can totally picture that happening in your family Yeah, you're not getting anything in that family Bears and all kinds of weird stories Oh, yeah, it was awesome. I mean your teenage boy. I mean it couldn't be any better, you know, I Passed on on that experience. I was given more than one opportunity to go and it's like, you know, I'll set out They were seeking referee refuge one night with a really bad storm in some abandoned building That was full of rats Oh, it's a trying to survive Yeah, I know you do a lot of the weekend things the three-day weekend things with the Boy Scout, which I Yeah, that's that's not as quite as rugged, you know And I'm within a cell phone call away from, you know, some emergency medical help if necessary You're not having to eat rats No I mean, I have I've taken like wilderness first aid classes, which are really I guess they're okay But they really don't prepare you to do wilderness first aid All they really do is bring you to a level of understanding of all the really bad things that can happen when you're more than two hours away from medical help that You know, you don't you don't finish that feeling better you just have more things to worry about you're like, oh great. I'm so glad we're going now. Yeah No, they have places where they like throw food at them and a lot of it's like the it's all the dehydrated stuff and Like like I you don't understand the visual that's going on in my head right now No, they use the dehydrated stuff and you have to get like water out of streams and cook it in little, you know Boil it in little stoves and you know a lot of like granola bars and stuff like that You know a lot of junk food actually a lot of candy and cookies and whatnot because you're supposed to have At least like 3,500 calories a day when you're doing stuff like that.  I do was thinking when you were talking about that Tom I do remember Derek talking about the story where he went I can't remember where he went so and you'll probably remember you're the where guy and He ate his Dehydrated foods with no water and He had to be taken to the hospital. Do you remember that? Yeah? Yeah, I do and he was by himself Hiking and he was tired. He was hungry and didn't feel like Preparing his dehydrated hydrated meal properly. So he's probably dehydrated himself and Eating the food with no water and he had to like He had his phone with him and he was able to find out where the closest road was and he had to make his way Like a mile through the woods if I recall the story correctly to make it to a road that EMS could pick him up Yeah, but he actually had to be taken out of there by ambulance. I was like, oh my gosh That's scary and all by himself and he'd been out there all day long was like nine hours or something You know as those big five gallon pales now the dehydrated foods, I don't know if everybody's seen them Like for the proper kits. Yeah. Yeah. And so Tim is loving these things. I think we've got like six of them. I'm like We're feeding everyone apparently if something happens Do you know something that the rest of us don't I mean should we all go out? I do not nothing zero my brother is always Heavily prepared for anything and so I think that's what happened plus You know opportunity Costco has these these kits and they're really inexpensive. It's like wow Does Costco know something? All right, we're buying the we're buying the kids They know if they make them people will buy them and that's kind of what they exist so true fact We are talking leadership this this month and We had a guest scheduled King Carfagno, and he is really an awesome guest. He's Done smart business moves at least one other time. I Were calling he's twice. That's what he did like back to back didn't he was so good He had to come back and finish the the topic and He's fun and and informative and He's like a solo cleaner and his business model is He actually Performed he doesn't really have an employee is the last I talked to him. He cleans himself, but he gets wacky He has started Hiring people and then teaching them his solo cleaning method So that he can or even more money. Yeah But you know he it's it's it's it's a it's a different model and he gets a price point that most of us could only dream about But there's a whole methodology goes through and He was going to be sharing some some some leadership secrets with us, but You know my bad I guess I didn't Get the link out to the show to him until about I don't know five minutes before we were going live and Liz forgot to remind him. So I Didn't forget. I don't ever remind anyone. I just plan on that He that text going out the reminder, but maybe I should We're gonna try it We're gonna see if we can move some things around and try to get him here Wednesday. If we could That being said, you know part of part of leadership is planning and looking ahead and preparing for for the unknown and Here we are with the unknown because we didn't know that we were gonna be doing this without what Robin is out here But this hand did know what the other hand was doing hot this hand was just wrong Followership session. Thank you David. What is that? See ya? What is a followership session? We're not leading. So I guess we're following each other You're good David. Yes Well, what's the latest with cove it because one of the things as leaders what y'all need to be thinking about is You know is cove it gonna be impacting our business in the weeks and months ahead And if so, how and what do we need to be doing now to to plan and prepare for that? What have you been hearing Liz? Ah? Well, same thing as you. I do know they're talking about here locally They're scuttle, but again that they're gonna shut us down. Uh, I don't know how everybody else's states went But our state last time we closed we opened in phases phase one phase two phase three and Or actually not that way was the other way five four three two one and Phase one is completely open again So currently we're operating in phase one, but they're talking about shutting us back down again and taking us to phase four So that is not a fun little bit of news that is You know going around it's not that is not news that is Just scuttle, but my friends of mine that work in the state have been hearing so, huh? Hoping not fingers crossed. That was not fun last time Well, one of the things that are that that seems to be different now then say a year ago when we were doing this is The Delta variant seems to affect children Than the variant that was going around, you know previously even even you know this last winter Yeah, and there's some concern that if kids can catch this easily that that's gonna make it difficult to open the schools And if they open the schools and kids start getting sick, they might not stay open for long Which means that a lot of the folks who work for us are gonna have a hard time working because they're having to stay home to take You know, you know that whole drill so Don't know if I have any wonderful answers for that and I don't know, you know Let's hope it doesn't happen, but we need to at least need to be Aware of the possibility for that as we we plan for the for the months ahead Yep We we have a Somebody right now currently an employee that has COVID. So we're not happy to see that already Coming into the business that that wasn't the case Last time we we didn't I Think it took us a long time Maybe 11 months to get our first case and it wasn't even anybody that was working out in the field cleaning homes so Not not I'm not loving it. There'll always be a variant We need to push through too many businesses and people didn't survive. It's more kids to suicide here though We personally know then we did take COVID. Oh Lost more Yeah, I Don't think they have numbered on What's happening with the kids yet for this new variant? Have you heard? Like I've heard I've heard anecdotal some interviews of like ER doctors and you know doctor You know just people who work at hospitals that you know having kids admitted to the hospital with COVID Previously as was unheard of but it's happening more regularly now But again, we don't we don't know You know, we need to make decisions from a from a business standpoint in terms of what we're going to be doing with our businesses and Yeah, it would be hard to shut down unless somebody was making us but if schools You know wind up not Not doing their normal thing then that could create just another degree of complication in terms of You know getting your workforce where it needs to be in an environment where it's already challenging to hire people and if the schools Don't open up. They start talking about that. We're also going to run into problems with daycare and all of the other Schoolish type options. So which is what I ran into the bulk of my work force was Mother single moms with kids. So it was It was a challenge for sure. What are you doing now Tommy doing anything currently? to in the realm of leadership with your people and helping we're Promoting the idea of everybody needs to get vaccination. We're you know paying people, you know bonuses if they get vaccinated That's what that's one thing that we can do we're Still we're constantly assessing, you know our bill rates and if we need to be raising rates, you know If the economy is wide open and COVID is a non-issue then theoretically We will have a larger work force and we'll be able to clean more homes But if we find ourselves in the situation where you know, we might even be more constrained We want to make sure that we've got prices that are high enough to Make it work, you know, we can make we can make money cleaning fewer homes if they're priced appropriately Yeah So I think I think, you know constantly taking a look at the pricing during these uncertain times and making sure that you're Getting as much as as you could hope to get And if there's like a downturn in labor You know even even go a little bit higher because you don't want to just tell people you can't clean their homes You'd rather for them to self select saying that this is more than than I can afford Yeah Well, historically we have talked a lot about I have and in my company and most of the companies that I work with When they do when people do price increases, they're looking at 2% or less people that say nope I'm canceling. I'll pass but the last this last round of Businesses that I work with in the last month have been averaging 9 to 11 percent That's quite a bit more. So that's a It's a harder pill for people to swallow And for other business owners to get behind the idea of raising raise is like, ah, but I Think that the math is still the math if you're not making money If you can't if you don't have enough employees to clean your jobs Need to be raising the rates on those lowest paying jobs that you're not making money Or not making as much money as you could with the new ones that are coming in And you know, we're still in an economic environment where you know Consumers your your clients have a ton of money in the bank Just as a rule the amount of money that that the average household has in the bank due to You know kind of the shutdowns and not being able to spend money on stuff And there's still a lot of things. There's kind of shorted, you know, you can't really find it so people have money scrolled away and And people are getting money every month in prices prices are going up and a lot of different ways a lot of different areas this whole idea of Inflation being transitory is the term at the that the Federal Reserve uses the economists use which means that At some point the prices will stop going up It doesn't necessarily mean they're going to be going down in all cases also for some commodities Maybe, you know situationally it would Nobody thinks that the labor rates are going to go down and There's there's a belief that a growing belief that they're not done going up yet that as Food and transportation and other things cost more People are going to be charging more and people are going to be able to demand more and especially on the lower end of the You know the labor market the lower wages is really what they're doing and there's more more You know speculation even people from the government are starting to Admit or at least recognize that From an overall economic standpoint, that's a good thing There was too much of a gap between you know the lower wages and the higher wages There were too many people the lower wage scales. I really couldn't Engage fully within the economy and buy stuff and the more we raise those lower-raised Wages the more money that's going to come back in the economy And you know, I don't know but I think I think our government likes the idea that those wages are going up Not because of some piece of legislation, but just because of economic forces Okay, well, I'm glad you pay attention all that stuff Tom and I can learn from you super helpful Which is which is another reason to make sure that you're constantly watching what you're charging for your service Right as long as you keep pushing that up you can afford to pay more And and we're going to have to so it doesn't look like that's changing any home soon I Cannot even envision a time where we're going to be able to start paying less People don't allow you to take their money away. That's that's not really a thing.  We can either close the business or lay You know come off or everybody takes a pay cut of You know some percentage. I mean it can happen, but it's it's a rare thing and even when they do that it's usually a very Short-term thing. It's we're taking a Pay cut for this period of time So I don't know how you would do that now. Nobody has a clue where anything's going Everything's so uncertain right now. Nobody Feels like they really Are confident about what's going on When we got the PPP money gave out more money letting technicians know that it wasn't forever Mm-hmm. Yeah Kind of a bonus COVID bonus or hazardous duty pay Yeah, letting letting them know that I'm not really giving you this extra money that Federal government's giving it to me and I'm giving it to you and they stopped giving it to me You're gonna stop getting it Yeah, they're just trying to help. Yeah Right now all that we are doing is just Having a quick daily at weekly We might we might increase it to daily if we need to if Numbers are changing but just a quick weekly like State of the Union, you know, what's going on. Here's here's what's going on. Here's what we need to be doing with COVID Yes, the mask mandate. Here's here's how it's working you guys. Yes, you have to be wearing a mask Yes in this situation Yes, whether you are vaccinated or not, you know, where so we have to keep updating that But we're not doing a whole heck of a lot more than that currently I I tend to like to Be more active to do to do something more than just Talk about things. So it feels a little uncomfortable not even having an idea what to do Well, all you can I mean I say all you can do I mean you can can can kind of lay the groundwork you'd probably be a good idea to take a look at your PPP PPE rather inventories and just you know, some of the some of the blocking and tackling stuff, but You know, it's a lot of us just planning at this point and you know, hoping for the best but being prepared for the worst Yeah, and and the the good thing is that right now we really do know what to prepare for because of last time We know what PPE we're going to need. We know that we need to get it now We know approximately or at least we have an idea of home.   I never even knew it. That was all about but You know, I'm sure that the whole country was in the same boat We were at different times where you would go to the grocery store and shelves were empty not just toilet paper Shelves were empty and not just cleaning supplies. Like we didn't have ketchup for a while couldn't get ketchup Certain salad dressings couldn't get there were a lot of things that were just Not like shopping in the United States Well, there was a momentary, you know drop there just because the whole Logistics side out at the trucking industry was it was was a bit off. I mean there's several things contributing to it Stuff got back on the shelves. There was a toilet paper shortage, but you know, you're in the cleaning business All you need to do is just contact the same person you get your your your cleaning products from on the commercial side They had more toilet paper. They knew what to do with because a lot of their, you know Office work and you know multi-tenant type stuff was was wasn't using a lot of toilet toilet because they weren't in the office. So You just weren't weren't looking in the right place Well, we never ran into that problem. We had plenty of toilet paper And we do get our office toilet paper from our supply distributor so we knew that there was toilet paper just because that wasn't an issue and our our Local distributor was pushing stuff All my time vaccinations for employees is a local story one of our kids They they had to close. Okay. I just read that Walmart's requiring vaccinations for employees. Yes. Yes, they are a lot of the large retailers are I think Amazon is one of the one of the airlines I heard is and I think that you're going to be hearing more of this and the in the days and weeks ahead where large employers are requiring and some of them are Requiring like their management staff to get, you know vaccinated but not necessarily the line labor and it's kind of a calculus of You know, they don't want to lose that $15 an hour person because they might quit over the vaccine But you know, the their their management would be less inclined to quit over something like that Makes sense But if they're doing it for everybody now, why that's a that's a bolder move for sure We talked about that last week. I wonder if you'd ever see it in the house cleaning industry if you see see companies Mandating that it's kind of hard to imagine at the moment because it's just one you're giving You know, your potential employees the labor pool one less reason to work for you if they were anti-vax Yeah, I I can give you another argument though that I've seen so I saw survey I think it was this morning. It was recently where like 70% of the population would like to see their employer mandate Vaccines because they would rather work in an environment where they knew that their cohorts their co-workers were Vaccinated so depending on where the sentiment goes that could actually be Something that would make you a more attractive That that's interesting. I guess that's really going to depend a lot on where you live too and What the sentiment is around where you live? I know I know some areas where It's just that the area itself is very very anti-vax in other areas where it's very pro-vax so Yeah, I guess that's So we're gonna see we're gonna see how things are gonna play out here Yeah, what are you doing Diana? We haven't heard from you in a while Are you doing anything with your? Vaccination processes Hmm. Just curious. All right anybody else David you're on her to What what have you heard or seen haven't heard from you in a while either? Actually, we're sure I haven't seen you on here in a bit. We see Danit and Robin and Linda fairly regularly although I'm not sure that you guys have shared what you're doing or if you are doing anything Curious it is leadership week. So just curious what people are doing to lead their people during these unprecedented times Now it feels a little bit more like it's uncertain than unprecedented But not really but I'm going to offer a bonus next week for the vaccinated. I believe I have about 50% vaccinated All right, so Diana are you planning on doing anything for the people that are already vaccinated? This is what a company that I know I've ran into they offered a hundred dollar bonus for anybody that got vaccinated and They didn't think whoever was parsing the message Did not get the memo that everybody who was already vaccinated will also get the hundred dollars and they said no You already got No, it's just for the people that are going to get vaccinated and they had a little bit of a kerfuffle as I think you can all imagine So but some people I do know another company that if you're already vaccinated They're they're giving you $50 and if you agree to get vaccinated. They'll give you a hundred dollars So that's interesting too Strongly suggest the shot wear the mask in the meantime Demand vaccine cell have considered bonus. Just don't know how to go about it. Feel my key players don't want to get vaccinated They will get the bonus. We talked about money or offering and pay day off. Hmm. I like the pay day off thing if I had labor I feel like I'd rather give money right now $100 incentive for vaccine no takers yet staff wearing masks. No takers, huh Linda? How many people do you have that you? Would like to get vaccinated that aren't taking Hmm Yeah, Diana, are you I are you over staffed you can afford to give the payday off? Or is it like for the future you can take it in a month or something like that. Just curious. It's interesting I always like giving people time off, but no, I don't have a lover Barely making it overstaffed Nice nice Diana. Tell us tell us your secrets Do you want to come on smart business moves and talk about your leadership strategies for being overstaffed? Poor Diana. She keeps posting it. She's super excited. She's proud Stop bragging I love when Facebook does that Yeah, we didn't think you were really bragging Diana Well, I have six staff one vaccinated. Oh everyone vaccinated some think it is a government conspiracy Yes, I have heard about the conspiracy. Tom. Did you hear anything more? Did you see anything more about the rhetoric of The vaccine is sterilizing people Yeah, I did see a little bit that over the weekend. I mean, I was actually Googled it and was was you know There's a lot of theories I Think a lot. I mean, I don't care where you land on this I think everybody would agree that social media Definitely is is playing a part in shaping how people feel and believe and are behaving about You know, COVID and and and everything surrounding it regardless of your opinion Which am I am I safe in saying it that everybody could agree that social media is playing a role in Helping us look at look at the situation from whatever perspective we're looking at it Yeah, I can't imagine that that isn't the case. I Am only on it's not true. I am rarely on open Facebook Facebook that is outside of groups But when I do hit it usually once maybe twice a week I get pretty much Plan on seeing something having to do with COVID and that's new for my feed, I don't tend to have friends acquaintances that are Very Political so I don't tend to see a lot of those posts, but even I'm seeing that now So I'm thinking it probably does have Impact in some way. I'm most everybody if it's it me Thinking it's it and everybody it if you're on social media. I Don't know the Facebook is the least The least visited Social media site, I believe for the younger group that seems to be having more of a problem So I don't know how that plays out. I don't know but there's a lot of other channels that people are getting their Information and I don't know. I don't know but Posing interesting challenges for us. No doubt. Absolutely Any late breaking developments are particular strategies that you hear people are using on the recruiting side. I mean You know getting back to David's thought, you know, if you don't have people to lead you really aren't leading and obviously the Labor market is still a really big Challenge here Diana, how do you look right now? You said that you'll help with this. I don't know if you're ready to go Live right now if you are Let me know and we can pull you into the show real quick I don't know has anybody received the second idle request Do you know Tom, have you heard of anybody receiving it? um Yeah, I I received round two and in one particular case and a few other cases It's still being being processed, but for one company, you know, well, there you go No, mascara is not required Diana Absolutely not. All right Tom Why don't you shoot Diana that link and I'd be great Diana. Come on and share what you're doing some stuff's working Anybody that has stuff that's working. Let's let's hear more about that Anybody that's over staffed awesome, I Know in the strategic success groups we had and Libby what's your name delusion from whoop recruit on on Friday and She had a lot of good little tips. They boy that that company knows indeed inside and out I'll tell you that oh Anybody else have any ideas love to hear that too your ideas about Attracting and getting people to show up to interview That's the big thing. I'm hearing is people can't get Anyone to show up They say they'll come and then they don't or they don't show up for the first day After the interview if there is an interview or they don't show up for the first day Hey Hey, there's an interview Yeah, you're gonna have to they don't show those down the Facebook Whenever we have a third person I always need to have my To I think so let me do that So Diana what world's going on in your world good to see it's been a while It's everything's good. We're busy growing That's good. I feel okay Yeah, we I think we got kind of lucky recently With the staffing For one I Still do all the interviewing so I do a hundred percent of the interviewing and when we need people I interview every day Usually if not four days a week Oh You know a couple months ago when it was really bad I just opened up my schedule and put all of my attention on hiring I know you guys know this but I'm pretty nice. So when I have an interview Usually I have successful interviews I have successful interviews I present the job. Well, I send them a video I've been much more particular. I've been lucky that I've been able to get more particular about who we're hiring You know, I just try to zero in on good manners and stuff and I think the last bunch We hired a couple of weeks ago. I can't remember four or five people. They all they've all stuck so far The last couple of months of hiring I have one person that's on the fence. It's been with me a few months Because the job is physically, you know, she has a hard time with that part of it but we have great trainers too, but you know, but but I also Not to I also will you know weigh up my budget for indeed for just for a short period So I'll just spend one week up in my budget and just process process process the applications the second they come in And then then then I'm full with with interviews I send reminders text messages. I reach out to everybody. I put them in my It's not called infusions up anymore keep I put them in a thing And you know, I put them straight in there if I like them so they get text email We mark it to them How many of the people you market to Diana? Do you see Coming through later That's a good question. I wish I knew I know I have 50 show rate typically Around that And that's for for a zoom meeting. I do zoom or facetime. They choose I have better I have a better show rate with facetime than I do zoom for whatever reason Um, I have a standard, you know, job form question. I ask every single person ever the same, you know I'm very consistent. I have a That I don't want to veer from them at all. I adjust my questions as things come up like since it's zoom now I need to say a new question is um So where are your tattoos? And I see them I hired somebody with a little something You know that maybe I wouldn't know You know, we would have had a conversation about it So, uh, although I hear it's it's I haven't I haven't even seen this person I haven't seen him. So I don't even know what Candice says. It's fine. It's fine mom So, you know, that really is interesting Diana because I know Candice has tattoos It's everywhere right now and I don't I don't care about tattoos.  So that that that's the one thing about zoom and honestly, I hired somebody that was missing me I did I was what? Oh They won't say Yeah, I Right. Yeah, this might be more challenging. This is how we clean Yeah, I might definitely can be yeah, interesting. I wouldn't even have thought of that I've got like three different things that I'm wrestling with and I'm just not going to say anything Now my question at the end is We lift this we do this we do that we do that Are you physically capable of doing all aspects of this job well and safely? So, you know that person's still going to say yes, for sure. She gave her she gave her effort She really did so yeah Did she did she come to work? Yeah, she came to work one day maybe two I can't remember and the trainer said she was huffing and puffing and really just doing you know Really trying? Yeah, really trying hard and of course if she could have done the job Welcomeed her with open arms. Of course. Of course, of course. It's a hard job though. Yeah, that's a good job That's really definitely with Yeah, and not having an appendage. That's definitely something we want to talk about it's hard enough Right everything going on. You know what else I've done that I think is helping is Well, we've raised our pay of course like everybody else three times the last 12 months We've raised our pay and a lot and I get comments during the interview process that okay I'm really happy with your pay your pay is good. So that so it's making a difference but We offer part-time hours And I I quite frankly am shocked We just did a poll last week Friday, I think and I haven't really looked at it but the poll in our group me was I'm happy Basically with the hours or I want more hours or less hours, you know or but it was nicely written like I'll take more hours If given the choice I'd like more hours Because we're ever staffed so everybody's getting off around two or three or I'm happy getting off early if given the opportunity And it was 50 50 there too So now instead of we have a long day for us we start at 7 45 They have to be solos have to be at their home between 8 and 8 15 and then they get out up to 5 30 So Now they can choose to get off at two Or And work five days or work fewer days or work from nine to two Kind of that, you know, yeah So it's pretty much more flexible with the solos. We're flexible. Yeah, it's easy to be flexible with solos for sure It is we're flexible and so that's really That's helping a lot because the minute I opened that app I hired three or four people right off the bat getting off at two So do you have that in your title then diana? Part-time or full-time or do you run two ads or I don't need to my title But it's in my description for sure You know, they're kind of put persnickety about the titles Yeah, so do you have that? What do you have as like a line item part-time available or full and part-time or full-time or part-time? Yeah, okay easy You know, they're making our staffs making good money I'm sure that yeah, I mean They are you ours and they're making good money. So How are you talking about pay in your ads? Are you talking per hour per week per year? Um, I present it as Uh hourly And then I send them the video that explains it fully because as you know, we have that two dollar an hour weekly bonus so trainees or 13 plus two dollars an hour Uh, once they're finished with training it's 14 plus they have that opportunity for two dollars an hour bonus So, you know, they're 16 an hour fully fully half get the bonus Um, and then the trainers make another two dollars an hour and we raise all of our prices So our labor cost is a little higher but not Uh, it's not not making me sweat. I'm okay. How do you earn? How do you earn the two dollar an hour bonus? quality and attendance Okay, uh-huh combination. Yeah I've considered the attendance thing dropping it because we're not having attendance issues because Waiting work on that too Yeah, that's to be helping and that is they call out in our group So I feel like that's super easy Or at least much easier Yeah, and yeah, it is I look in the morning and I'll see somebody go. I'm so sorry. I just stop feeling good today Sorry team Yeah, so it's not bad Yay, so you're not using it as a um a uh shame shaming um No, I'm Not typically your style, but I know that that's that's something that's going around right now, right if people are Are you using it that way? Yeah, that's pretty cute. No, I'm um more of a uh, we're all adults Don't I don't want to tell you at least on least on paper Yeah, I've got I'm not really interested in parenting anybody else. So Yeah, I have to give kandace a lot of uh credit for keeping our staff She's really good at um You know, I know she I know she's probably a part-time therapist in there Oh, yeah, a lot of part-time therapy going on. She's she's very good at engaging and She really is yeah, I think And she's fun too. She's fun. I think four of them went out this weekend and got matching tattoos Okay, yeah, so how many of your employees are getting tattoos? That's your next strategy All right I know what Diana you're you're bringing up a great point, you know, everybody right now is focused on Hiring and how they get more people in and how much money you have to pay But if you got them going out the back door as fast as you have them coming in the front door That that's just so much wasted energy So finding a way to keep keep the people getting them to stay Is a a huge piece of it. So you're thinking That a big piece of getting people to stay is kandace's connectivity her individual connection So we start the conversation with the applicants Saying hey look like we expect really great communication on your part On what you need if you need more time at home if you need to take off whatever we expect great communication We'll we're definitely going to do the same for you You know, we do that thing I heard you talk about many years ago Uh where every day at one o'clock we check in how's everybody doing so they help each other So there's that connection. Hey, uh, I'm getting off at six. Oh, no, I'm getting off at two Let me come help you. I have a handful of people every day that say I'm finished for the day. Does anybody need my help? So there's a lot of that But it starts with You know, we kind of presented as we don't leave anybody out in the field Uh, so if somebody's out in the field, we're not we're not done with the day So I think the last time somebody complained about Well, it's three o'clock. I've already done my two houses. I don't want to go help Was about six months ago Nice Oh, yeah, they just it's it's I think when you have that group dynamic Dean uh other people saying oh, I'm getting off who needs who needs my help Yeah What platform are you using to foster this level of communication? We have group me and so we have like You know Different groups in it. We use the time variants Which is where they're asking for time and they know why you know Uh, they'll ask hey, um, this is happening and they have to tell us client issue tech issue This is how much time I need for this home because we have to keep our eyeballs on that And we explained it to them. We're paying more. We want to continue to pay you more We want to continue to raise our Pay so we have to really watch our efficiency. It's super duper important So we actually we actually manager meeting every morning. Look at the previous day So we're intel it's how that's exactly how we need it to be. We're talking about it every day And so they we have that time variance group. We have our call out or schedule impact group You know, so I'm running Tenants like and you know what I love At least six times in the last six months. Somebody's posted Um, I I need a new tire, but I'll be there when I'm done. So there's a lot of that. I'll be there when I'm done Good example, right? Yeah, that's fine. I love that So you kind of have the no man left behind mentality Do we do Right and I even hesitate to say like Well, it's a family it's a family business, but we're fake We definitely do want you to go home to your family And I in they see my grandkids and the kids at the office and you know, it we really are focused on Uh, you know, like one of the ladies that took the part time. Well, my daughters have gymnastics twice a week So I'd like to be able to get off to take them to gymnastics.  Yes, awesome I think that's a completely. I know we are out of time. Tom I know you're watching the time But I just want to point out one thing that's different about what you said diana than what most people say One thing uh, what what most people say is yes, okay We can accommodate that for you what you say is not only are we accommodating it We're actively seeking people like you.  That's a completely different mindset. You know, that's 180 from you if that's part of your culture everybody is working to help each other or accomplish that goal Yeah, and honestly I have to say Probably moving to the solos helps that Yeah Right Just because we were just at a loss of how to communicate and how to engage and so We just kept You know and then I really like doing the interviews. I like the way I do the interviews I can't seem to give up anytime soon.  No, no, I'm good. I've got it You know, you know what I would really love tom is someday if we could bring diana on here And have her interview me Do a live interview right Like exactly like you would interview anybody else and you can talk the way you would normally and give all of the normal stuff I think people could really learn a lot from that because it's It's um easy to hear the information but seeing it at together is like, oh to a a deeper level There is a guy that there was a guy at the car wash place where we take our you know We took her oil change oil chain place where we took the cars to get oil change and he's like, oh, it's okay And as my mom applied at natural care and she didn't take the job, but she said she was the nicest lady That is not surprising I really do like people Yeah, that is that is not at all surprising Well, diana, thanks for coming on here today. Hey you guys, I know we're running just a hair late. Who would have thought but diana you're you're sparkling We're without mascara diana. Oh, thank you Still got the hair Sure All right, well, thanks everybody thanks again diana I'll see you guys Wednesday five o'clock. Bye. Bye. Bye. Bye y'all


Hello, everybody. So, my name is Andrei Turinsky. I'm also from SickKids, as some of the presenters from yesterday. And we will continue with the topic of epigenetics in this session as well. So, the learning objectives is I'll try to hammer again the idea that epigenetics is very important in addition to genetics. We will then do the practical where we will explore some of the DNA methylation data sets. We'll try to classify new samples, see which ones could be classified as pathogenic or benign, and how to visualize the results of that. So, let's start with this topic. So, I have to say that epigenetics has been exploding in the last, I would say, about 15 years, even in popular imagination. So, people who are far removed from biology or genetics or bioinformatics are realizing that well, as the Time Magazine cover from 2010 says, DNA isn't your destiny. So, not everything is packed into your just genome itself, as people understand it, the double helix with the nucleic acids. Because if you think about it, my body, for example, has cells that are almost, well, virtually identical genetically, but obviously my brain cells are different from my liver cells, different from my blood cells, and so on and so on. And the same thing happens across diseases. And on the right side of the slide, you see two mice, they are genetically identical, but they obviously look very different. And that's due to just one gene and one promoter of that gene, a kooty gene that controls, well, these things, but also the hair color, the diabetes predisposition, some of the cancers that are more prevalent in the yellow mice compared to the brown mice. And again, this is genetically identical mice. Now, there's this common theme that GNA loads the gun and environment pulls the trigger, so people can have, you know, the same genome and depending on their lifestyle, things can go horribly wrong. Now, epigenetic provides the actual mechanism for that in many cases. So the way that actually occurs is through epigenetic mechanisms that impact how the body develops and how the phenotypes come about. Now, when we say epigenetics, people mean different things. So for some people, epigenetics is all about DNA methylation. So I will try to point my mouse now, maybe not so easy. Here, right, so you can have your DNA little metal groups and they would be attached to your DNA cytosines and that will impact translation because some of the cytosines may like physically block the transcriptional mechanisms. Now, for some other people, epigenetics is all about histone marks. So there are all kinds of histone marks. And you saw the ENCODE project before that. It's mostly about cheap, seek experiments. And a lot of that is histone marks of all kinds. There is histone methylation, dimethylation, trimethylation, acetylation, phosphorylation, ubytylation, and all kinds of things. And histones are the proteins that make up the nucleosomes and their little tails could be marked with various chemical tags. For other people, epigenetics is about nucleosome positioning. Nucleosomes are positioned dynamically so they can move around a little bit. And bigger and newer studies are studying more general and higher level looping of DNA. So DNA confirmation studies, they would look for big blocks where the DNA come in contact with each other and linearly very distant pieces of DNA may have major impact upon each other because in three dimensions they loop and they become very close. Now, in DNA studies, in genetic studies, the ones we talked about yesterday, the mechanics of it is more or less understood in the sense that you can have a genomic variant that could like physically stop the protein. You know, you have a non-sense mutation, the protein is too short, and disease follows.  Now, in epigenetics, the mechanism is slightly different, but the theme of it could be similar. You can have some exposure on a promoter of a gene, for example, and the gene promoter could be heavily methylated and that stops the transcriptional machinery physically from, you know, coming in contact and doing the transcription. So the gene could be silenced that way. Or the opposite case, you can have low methylation and so the transcriptional machinery activates and the gene gets transcribed. Now, environment affects those outcomes as well. So environmental agents can act either on the proteins that actually change the methylation or some other marks or on the modulator proteins that then in turn, you know, cascade into changing other proteins and so on. So there could be quite complicated epigenetic machinery from environment down the line all the way to the transcriptional, etc., to the phenotype. And transcription could be affected by both genetic and epigenetic components. So you can very well have a SNP that affects the methylation. And that is actually one of the good filters for looking for significance of SNPs. Some SNP may not be obviously related to the mechanics of a gene that is at fault, but it acts through an intermediate layer of methylation. So the SNP can affect the methylation of a certain promoter or could be disted enhancer. And then that enhancer in turn either activates or somehow represses the transcription of the gene. And again, environment comes into effect as well. So you can have the same promoter acted upon from a SNP and also from environmental agents. So altogether I have to say that methylation is complicated. Epigenetics is complicated. Histone marks are very complicated. There are lots of them and they all do their various things. So not to say that genetics is simple, but epigenetics is more complex. It has more data types. It has more agents acting upon each other and it's not entirely understood yet, so which is why we are working on it. Now in terms of diseases, some diseases famously cancer are very much epigenetically driven, not at the initial stages. So you can have driver mutations that initiate cancers, but the progress of cancer apparently in many cases is epigenetically driven. So you can have driver mutations to initiate the cancer, but cancer kills typically through metastatic progression. So like once you're at the stage where there are metastases, that's where things become very, very bad for the patient. And the development of metastatic cancer is primarily through epigenetic, well, it's not mutations necessarily, but some kind of variability. So things are going horribly wrong on the epigenetic stages of things, even though you may not find the mutations that are responsible for progressing from stage three to stage four. Now the same thing applies to diseases that have very strong environmental component, such as autoimmune diseases, various allergies, also metabolic diseases. You can think of diabetes, you can think of other problems, body mass indices, and so on. So if you're thinking of a disease that has especially environmental component or it's a kind of cancer, think about epigenetics. It should be impressed upon you that epigenetics plays a major role, especially in some of these diseases and especially the later stages of them. Now it could also affect diseases such as neurodegenerative diseases and neurodevelopmental diseases. Now here is a paper that describes Alzheimer's and Parkinson's and Huntington and other other sclerosis. Obviously there are physical manifestations, but also if you're looking at epigenetic studies, they would tell you that there is modified DNA methylation, there is modified agents that act upon DNA through epigenetic mechanisms, such as mental transferases in the case of sclerosis in the last one. So there is a complicated machinery usually acting upon different aspects of the metallome and the changes could be either major or subtle. In cancer usually changes are very major. In neurodevelopmental diseases like autism the differences in methylation could be minor and harder to find and also diseases themselves could be quite heterogeneous because different genes act upon similar pathways and so the mutations may come from different kinds of genes, so in autism there would be over 100 genes acting upon this disease, but ultimately they all somehow end up through some kind of funnel effect to result in a various degrees of autism spectrum disorder. Now that said, I will progress to DNA methylation. Now why methylation? For several reasons. Now number one, methylation of DNA is a very stable mark. You can actually keep the same sample for decades in the frozen state and methylation is preserved DNA methylation you can study it much later and compare cohorts and so on and so forth. Now there are also sort of logistical reasons behind it. So there's a company Lumina which has created wonderful microarrays cheap enough and they can scan almost well the full genome. So you can have genome-wide methylation study of your patient cohort for cheap something around $400 something like that per sample of course. So here is a snapshot of genontology, sorry, gene expression omnibus resource from a couple of weeks ago and these are the famous 450k methylation arrays. So the reason they're called 450k is they have roughly 450,000 probes actually more than that it's 488,000. They became vastly popular and as you see there is a point with my mouse right here so there's an over 1,000 studies using these particular arrays of all sorts of diseases. So you can see various kinds of cancers there's esophagus carcinoma here and there are interesting things like humanized mice even that you know is scanned on the same microarray and I've seen primates and gorillas scanned on a human DNA methylation array but for the most part I mean here I chose homo sapiens as the species and you know all sorts of diseases could be scanned on these arrays and they are a number of samples also very quite dramatically so some studies would show you you know six or two or is it nine or five I can also see from here so just very few samples and some studies are into hundreds or potentially even thousands of samples so you can have a lot of samples scanned on the same technology it's widely available it's comparable across studies there are you know protocols that make things easy enough to compare to each other and so people have been using these arrays quite extensively there was a previous array 27k which was small mostly mostly the probes were in the promoter regions but ever since the 450 array came out it became very very popular now in the last few years it's been superseded by the next big thing it's an epic array it's a double the size for 850 probes or so the labs are switching to epic now the number of studies already published is not so great yet so currently as I looked at it a couple of weeks ago and I made the slides it was only 49 samples available at the gene expression omnibus 49 studies now of course they will come out because it'll take you know it will take people a year or two or three to actually publish this study if it's a big one but you know not there yet but growing and the good thing about the epic and the 450 is they overlap to a very large extent so most of the probe over 90% of the probes that were available on the 450 platform are also available on the epic platform so in principle they are very much comparable and studies done on 450 could be easily enough checked on the epic and vice versa you can reduce your epic array to the previous 450 if you want to compare the study down epic to a previous study down the 450 so this is same company same technology behind it and large overlap between the two types of probes now for all sorts of reasons which we can list people are also doing sequencing studies so here is a list of whole genome bisulfide sequencing so of course there are multiple advantages of doing sequencing well you have a much wider coverage you're not stuck with only the 850 000 probes that Illumina defines for you you can potentially have millions and millions of cytosine scan for their methylation status useful for single cell studies useful for all kinds of things of course more complicated in terms of coverage you have to worry about whether your cytosine in this one sample is well covered if you have hundred samples you want to know whether that cytosine is covered in all of them well whether you have you know 10 reads or 30 reads or 50 reads available for your one cytosine across the spectrum of your samples so if you are trying to overlap those things it could be you know the number of cytosines would go from millions to perhaps you know hundreds of thousands or even tens of thousands depending on your coverage across the spectrum which is why people switch to regions and analysis of regions now as Guillain was showing you the iHAC consortium is compiling primarily sequencing based studies so there is whole genome bisulfide sequencing we can point my mouse to it here so WGB seek that is the bisulfide sequencing component of DNA methylation so resources are available now different technologies of course require different methodologies and 450 array has been very popular and there is a whole spectrum of pipelines and tools available to study it so here's a paper from 2015 that lists the top five and they're still fairly popular so the pipelines are in bioconductor in R so those are the famous ones such as minfi such as rnbids and this is if you see the color as well minfi which is the kind of light brownish one is available for all steps of your pipelines almost up to interpretation so the only thing they're missing is they don't do the copy number variation analysis and of course no pipeline would do would do biological interpretation for you that's why that's why we would never be replaced by artificial intelligence for example so you know artificial intelligence could do everything up to the interpretation perhaps so same thing for some of these other pipelines the rnbids would probably do most of your steps up to the interpretation on perhaps copy number variation some other pipelines are more specific to pre-processing and they leave the the juicy parts for other pipelines or for analysis that that is done manually or through some other considerations so I have to say that the thing people look for in most cases are these two calculation of differential and methylated positions and identification of differential and methylated regions so that is the juice of the analysis those are the pieces which will tell you which genes or which promoters or which enhancers are responsible for your differences between well let me say cancer cohort versus controls or disease versus unaffected siblings or something like that so the essential outcome of the pipelines typically comes as a listing of your differential methylated patterns of some kind or another so for arrays it's typical to report positions and then maybe regions for sequencing studies the regions are more essential because first of all you have enough cytosines to to merge into regions and secondly coverage issues may may be an obstacle for specific cytosines but they will be you know you will have enough coverage for a region for a tiling window for example so the analysis across regions and tiling windows becomes more of a driving sort of driving force behind all these differential methylated patterns all right so as we go into data representations what do we see essentially so we try to think regardless of whether it's an array study microwave study or sequencing study we try to think of a table so in a typical table you would have your subjects or samples or tissues as your columns and your genes or probes or cytosines as your rows and to the dismay of many data analysts the number of features which is to say probes is vastly vastly larger than the number of subjects that you're working with so the number of data samples are small the number of features is very huge so you have to somehow you know reduce your dimensionality and work with the highly highly dimensional spaces and try to make sense of them in the lower dimensions as you go so based on this mock table your two genes x and y would define a space x and y and depending on what methylation or some other measure you have you would position your samples accordingly and then try to see what patterns exist across the samples okay so typical questions that come up is the following are you doing a supervised analysis or unsupervised analysis so in again in a mock study unsupervised would be the one where you are trying to find out the groupings among your samples so perhaps you have one kind of cohort or you're not quite sure which labels are applied to which sample so you're just looking for some clustering and you know in this case you'd see two clusters and you're happy with them until you discover more data in this case you see that yes first of all your clusters were not ideally positioned you'll have to rethink about the boundaries and you know recalculate them and perhaps there's a new cluster atop which was not evident initially because you just didn't have enough data so there's a new cluster of samples there in the unsupervised scenario that is all you can do so you're looking for grouping in a supervised scenario you actually have labels so you would be asking questions of here's my disease sample let me point to it at the bottom here and there are my two controls or two normal samples where is my decision boundary so the essential question you want to ask is well how do I decide for a new sample whether it's normal or disease where you know if you place your new sample somewhere here how do you decide whether it's closer to normal or closer to disease and therefore what is the classification for your new cases and again same issues apply if you collect more data you might discover that you know you were previously missing two of the disease samples like that so they define perhaps another group of the disease it could be a heterogeneous disease so your decision boundary can become more complex than you thought initially or you have to redraw it through some other way depending on which the which model you're actually using for the classification right so typically this will be an example of unsupervised classification uh right so this is another approach which everybody loves principle command component analysis everybody knows what it is right raise your hand if you don't or if you have used it you used it yeah okay so you're looking for the largest variation between your groups or just generally within your data set and this is an example this is actually a figure from a study we published in 2013 so very nice to draw a decision boundary between two clouds like this you have your diseases in red on the oops uh on the left side there was a here so on the left side you have a bunch of diseases shown in red on your right side you have a bunch of controls shown in green so amazingly easy to say that you know there's a clear boundary in the first principle component where you know things on the left are diseased things on the right control now this you know the big secret this was done after the coordinate space was prepared in a special way so we first extracted only the most discriminating or the most telling the most predictive probes so we spent some time finding those differential limit related probes and then in the space defined by those probes uh or which there were 53 down from whatever array that was that was actually 27 k array so from 27 000 uh dimensions dimensions you went down to 53 dimensions in which you see this pattern fairly clearly and now you can draw the decision boundary between them and so on so uh initial data set was something like this so this is based on the entire array to it was 27 k array uh promising but not as great right you could see clearly that there is a difference between the first principle component uh diseases and control so you know diseases tend to be on the left controls tend to be on the right but the separation is not as clear and there are some borderline cases uh so you have to spend a little bit more time to actually find a very good separation so in which we can build you know classification models or use it downstream in some other analyses all right and there are some tools that do well almost entirely this so there is this uh for example Glucor omic explorer tool which you can get for yourself at glucor.com and what they do is this is a uh differential limit related signature slash principle component analysis tool so all they do is they take well i don't want to minimize what they do of course they have lots and lots of options and very useful tool but uh the essential pipeline of how they're thinking about analysis is take a data set find differentially uh methylated regions and as you keep shrinking them or making them more stringent your pca becomes more and more apparent so you start with a cloud that is not very well separate separated but then as you find differentially methylated positions your whatever yellow uh red and orange or blue points will start separating into proper clouds so you know useful tool people love to use it now there are more complicated analyses so this is a paper that came out just uh in march in nature they threw together not pca but something slightly more complex tisny it's a stochastic neighbor embedding tool but again it's a different uh dimensionality reduction tool of 91 types of brain cancers all thrown together uh the picture looks something like this and you can now define your different clusters and then based on these clusters you can build your special models and then use them for classification so a good paper similar theme now we tried uh of course differential methylation patterns in all kinds of scenarios this is another paper we published in nature communications a couple years ago three years ago uh where you can show patterns of differential methylation by you know standard methods hierarchical clustering so again you shrink your space from 450 000 probes down to in this case 7 000 probes or so and in that newly defined space where things are nicely separable yes your diseases in this case uh this was soto's syndrome one of the neurodevelopmental diseases are very nicely separated from controls so not all probes define good separation but these ones are the ones we chose do and that was easy to see and clearly there is a pattern there uh now the problem with hierarchical clustering uh it's wonderful too everybody loves using it but if you add new cohorts uh the clustering will change right so like even if you add one new point to your clustering your uh the endogram may actually change its position so here we try to do uh like i'm even ashamed to call this machine learning it's that's it's too simple for that but you know it's some kind of decision boundary so what we try to say is the following we look at our uh disease cohort we look at our controls we define two profiles and then for every new case whether it's a control or disease or a similar disease or unknown samples we try to see which whether it's closer to disease or control that's it so uh we define oops we define some kind of correlation metric and we say that you know for every new point we place it whether it's closer to the uh disease profile so it will be higher on the upper triangle of you know on the on the diagonal plot here or if it's closer to the control profile then it will be on the lower triangle of the this diagonal plot and turns out that uh if you take you know over a thousand controls from the gene expression nominee boosts throw them on this plot all of them very very nicely would go and cluster here so they have a giant cloud of controls all of them properly classified but if you throw some other cases or a similar you know clinically similar syndromes some of them will go to the proper position so like a weaver syndrome in orange it was clinically similar and it was all classified as controls so we can you know we can molecular distinguish the two uh diseases but if you have some unknown cases uh or mis-sense mutations it depends so here you would have this causative genes the one for non-sense mutations of which causes a disease but mis-sense mutation is unclear and some mis-senses will go and cluster with the disease cases and some mis-senses will cluster with control cases and so you should be able to then tell which mis-sense mutation is pathogenic or benign just using this tool in addition of course to some other predictive mechanisms now uh you know all the cool kids now do machine learning and artificial intelligence like we are not averse to that um so we uh we published another study recently so we looked at two different clinically overlapping syndromes this is charge syndrome and kabuki syndrome uh they uh they result in clinically similar but still different phenotypes so there is facial abnormality there is uh intellectual disability but the problem is uh sometimes it's hard to tell apart especially for younger children now the molecular genetic side of things is very different so in charge uh this is primarily from the chd7 gene in kabuki it's from two genes primarily kmt2d but also kdm6a so kmt2d is histone metal transferase gene and the other one is histone d metallase gene so all of them are some kind of histone modifying genes uh but they are different genes so you know the the actual genetic mutations are quite different but apparently they end up working on the same pathways and the same chromatin modifying machinery which is why you have papers like this this was a previously published paper 2014 so they say things like we report a patient that was initially diagnosed as charged but that eventually it turned out that it was a kabuki case and they say that well you know these two positive genes apparently work on the same machinery which is why you know it was hard to do it initially so what we um ended up doing is we developed a dna methylation based predictive system so in this case we did you know support vector machines and tried to plant a forest and you know a few things was what we tried here and uh basically we built well two classifiers if you think about it so we can assign a score for your charge syndrome a score for your kabuki syndrome based on DNA metallomes alone and nothing else and uh well the good thing is they were all classified correctly by themselves and also some of the replication cohorts and validation cohorts and nothing was classified here and the upper right side which means no cases or controls or any samples of unknown significance were classified highly as both diseases which means there was no ambiguity so uh even though doctors may have trouble distinguishing the phenotypical manifestations of these clinically overlapping diseases especially in small children under three where things are not quite developed yet where you can specifically clearly tell yes it's kabuki or it's charge on the molecular side of things so the dna is other things uh the mechanics of it are quite different and we were able to build models that are not ambiguous and distinguish those the g the diseases early enough so that was good and uh we also threw like all kinds of geo controls hundreds and hundreds from uh publicly available data sets and they were all classified as controls so you know things were properly validated in that sense and we're all also able to say things like well uh for gene here like it's a hoax a5 your background ventilation level across a region uh this is a promoter region as you see so hoax a5 goes that way so the background level is in green and both genes are uh gaining methylation there so the hoax a5 sorry not both genes in both syndromes hoax a5 in charge syndrome has higher methylation level shown here in red and also in uh in kabuki syndrome it's a higher methylation level shown in blue which is why perhaps these genes are sorry these two syndromes are acting and showing similar phenotypes because they affect genes like hoax a5 in a similar fashion but also there are genes like uh which one is the slit trick five where uh in one syndrome you have a loss of methylation so there's a red line below background and in the other syndrome you have a gain of methylation so there's a blue line in kabuki that is above the background so perhaps these are the genes that eventually distinguish these uh these two syndromes on the molecular level and again you have a region where methylation is acting up or down as in the case maybe all right so these are the you know high level themes of how we go about and find what's going on now there are things to be aware of and i will mention two of those things so one has to do with sand line heterogene so if you think about it um let's say you know an analogy is uh you have people in the room who are like poor students and uh then you replace them with rich professors or rich lawyers or whoever and then you start measuring the wealth of the people in the room and you discover that uh people are getting richer uh well that would be a wrong conclusion people are not getting richer you're just replacing poor people with rich people you kicked out the poor ones you replaced them with totally new ones but the income level stayed exactly the same for all of them so the same thing may happen in these dna methylation studies where you would discover that there's a change in methylation and you say oh we have a gain in methylation no you didn't have a gain of methylation you just replaced your cells with the ones that had higher methylation to begin with so typical example well this is again taken from some paper where in cases you would have these two cell lines and let's pretend that uh you know the blue one has low methylation and the red one has higher methylation so initially uh the higher methylation was dominant and prevalent and then in the cases you suddenly observe that the level of methylation is lower so you would then start asking yourself uh is it the consequence of the disease so the disease causes the drop in methylation or is it the opposite is it that the drop in methylation causes the disease well none of the above all that happened is different populations of cells walked in and you know your methylation changes were only due to that and a lot of studies have to have to take that into account there was a nice paper by two prominent researchers Jaffa and Irisari Rafael Irisari who look at changes in blood composition with age so they look at various studies and decided that they really have to pay attention to the blood composition because as people age i'm pointing here as people age the amount of different kinds of blood sub types of cells changes a lot so eventually you have nothing but granular size for the most part so as you're studying your methylation level especially between you know comparing pediatric cohorts to older people to adults uh the apparent changes in methylation are not due to actual gain in methylation or loss of methylation but primarily due to the replacement of different cell types so this is one of the confounders that very much has to be accounted for and there are studies that actually take a kind of deeper look into different sub cell types and this is one of them by Grineus et al which look at specific sub types of blood and they you know they they they found various patterns among them and basically they decompose the blood into different profiles and these profiles could be used as references so the general theme of how we should understand methylation is there could be let me point this there could be initial cell types so think of them as purified cell types they have certain methylation levels so here are the profiles of your favorite cell types and then there are different people who have various combinations of those cell types you know these are your patients your your controls your cohorts and the weighted combination of those cell types and the weights depend on the person produce the actual methylation that you then see through the microarrays or through sequencing study or whatever the case may be unless it's a single cell sequencing where things are easier right so in general or up until now the methylation that we see is typically a result of some kind of weighted combination of profiles and we need to keep track of what those things are so these profiles could be based on either existing references or you have to discover it through some deconvolution methods that have been produced such as the one i'm quoting here reference free deconvolution method for recent enough paper now there's evaluation of all these deconvolution methods so some of them are right so this paper studies them if you want to look you know take a look so some methods are reference based as i mentioned you first produce your methylation profiles and then you see what your patients and cohorts have in terms of those references so you find essentially the weights and there are reference free profiles of which there are several so there's quite a few of them and you know the number was growing the recommended one apparently is surrogate value variable analysis package that seems to do well across the board some other ones are famous as well now we found it easier sometimes to claim our results through some kind of shortcut so you can do the full deconvolution and then use your proportions of your cells in your study that's one way of doing things or if the question is simply is your disease pattern due to cell profiles or not is it robust to changes in cell composition or not you can say the following you can take all your initial profiles and see if they cluster with controls or not so here again this is from the nature communication paper we published some time ago we found a bunch of diseases we found a bunch of controls on the pca they look like this so you take your principal commons analysis and diseases are nicely separable from controls and then you throw together all these individual cell profiles and see if any of them end up with disease so if any of them end up with disease you or someone rather your critic may claim that oh this disease pattern was entirely or perhaps entirely or in parts due to that cell type present in the disease population but not in control population so your study is confounded results are garbage and you know go go back and redraw things uh well in our case we were you know we were good so our cell subtypes all cluster with controls and of course any linear combinations of them will also be there with controls so you know that's a presentable enough case to say that our disease patterns are actually not dependent on the cell heterogeneity or cell composition effects and this is not only true for blood blood is a complex mixture of all kinds of cells granulocytes natural killer cells you know all kinds of things but so is saliva so is buckle buckle is the cheek swabs that people use in all kinds of studies so you can take a brush and you know swap someone's cheek uh tissue and that would be uh DNA for analysis so again uh cheek swabs and saliva have uh heterogeneous mixtures of cells and you have to be aware of the effects of that okay so another important aspect and technical aspect of all these methylation studies and not only is batch effects so I'll just spend a little bit more time on this everybody okay who has worked with batch effects I have heard of batch effects before all right not too many all right so typically batch effects is something like this you have one technician working on monday and another technician working next Tuesday and they have different techniques and skills and and one of them spits into the sample just for a good measure and the results are different across the technicians even though your data seems to be the same from the same distribution so results are dependent on the technical facility the skills of a person working with the samples the country in which the samples were processed the year and date on which it was done and so on and so forth so if you do your processing even in the same facility in you know january versus july you may and very well end up with different results not only that if you spread your big cohort across multiple chips like microarray chips a chip can only hold that many samples so 450 array holds 12 samples on the chip so if you have you know more than 12 samples of course it will be spread across the chips and different chips can be physically processed in a different way right they are like handled differently they are dried differently and so on and so forth so what you may discover could be differences due to chip processing the microarray chip processing rather than due to the actual disease versus your control study so these batch effects are prevalent across lots and lots of studies and more and more people should be not alarmed but concerned and this is an important issue that should not be overlooked so here's a paper from 2010 where people looked at what was 1000 genome at the time so they saw well let me see what it is so across the bottom the x axes this is a particular genome location all right and the y axes these are dates on which the chips were processed so you know days are numbered and what you see here is the coverage the genome coverage and orange is high and blue is low so for some reason you know there was a period of blues where coverage was lower and then there was a period of highs where coverage was higher and then periods of lows again so things very much depended not on anything else but on the date of processing so if you happen to have all your diseases processed on blue days and all your controls processed on high you know orange days then you will see some differences perhaps but they will be due to different dates of processing and maybe different people or different facilities or different other you know kinds of things like that on which your analysis depends and these confounding factors will well potentially ruin your study as we are about to see so the normalization doesn't always help so people say well I normalize my data well it's not good enough so even if you normalize your data your entire data set and again methylation data set can have as I said half a million probes or 80 800000 probes so your total methylation profile may very well look well aligned and normalized but you can have a hundred genes that behave like this where on day one they're all low methylation and on day two they're all high methylation or expression or whatever it is you're studying right and so even though a overall picture genome-wide seem to be okay because you did your quantile normalization and things like that you would still find enough genes with difference and then you say oh these are differential emethylated or differential expressed genes well no they were just processed in different ways and the batch effect was not accounted for and you know things should not be published so unfortunately now there are various ways of correcting batches so from simple ones to more complex ones so simple ones simply you know take the batches find what were the average levels or something like that and then try to you know equalize them and merge them down to ups down to more complex ways so the standard and you know state of the art is called imperial empirical base methods so typical example is combat function so combat function has been widely popular and everybody says we use combat don't worry so combat was used it corrected for the batch effects and so on and you know it's a powerful enough function it was initially published as a standalone r package in 2006 so this is the paper where people announced it Johnson only and it was an r package not even the package it was like an r function for many years and then more recently became a standard part of this surrogate value decomposition package sva surrogate value analysis variable analysis right so now through the sva package available through r and by conductor you can just have your power of combat with you and you can use it and we'll do that today as well now main thing to remember is bioinformatics cannot fix bad design so if your study was poorly designed a bioinformatician cannot fix it necessarily it depends so batch correction may not help actually and this is a paper again fairly recent this is march of this year where well the title almost says it all where's my mouse here adjusting for batch effects and DNA methylation study a lesson learned and that was a hard lesson so as you can imagine people did something they realized things are not great and then they had to redesign the whole you know the whole analysis so the problem here was they were looking at two variants and a reference so you you know references and what color is that yellow let's say and variants are in blue and orange spread across several chips so here are seven different microarrays are shown as I mentioned before a microarray can hold only 12 samples the problem was some of these chips like this one second one it only had one reference and two samples from that other variant but no orange variants here they only had the orange variants on chip number four but no references and no blue variants and and so on and so forth so the problem was that their groups were not equally presented in all batches so a batch here was defined by a chip let's say and not all batches contained equal number of groups and that became a big problem so they discovered that yes they went through the motion and after they applied the comeback correction which they thought will fix all their problems suddenly they saw a giant spike in the results so they had tons of differentially methylated regions and instead of publishing them they were very smart they were actually quite alarmed and went back to study what's going on and they discovered that all of that was fake so you know you can you can artificially spike your results with false discoveries and then you know hopefully you will catch it in time and another paper this is a good one methods that remove batch effects while retaining group differences may lead to exaggerated confidence and downstream analysis so a good statistical paper it shows you the mechanics of what can happen so here's a the first panel panel a in which you would say there are three groups so you know red blue and green and let's just pretend that we try to confound group a with batch one so batch one only contains this one group and batch two only contains these other groups but nothing from group one and then for some technical reasons batch one may have you know slightly higher methylation or slightly higher something some kind of measure you're studying gene expression maybe then batch two so for whatever reason you observe that batch one is different from batch two you try to correct for that so you try to centralize them and of course everything goes slightly wrong because now batch one goes all the way down to make its center equal to this whole central batch two right and therefore your you know your your pink group your red group drops down below the blue group for example and the true values were exactly the same but now you see artificially introduced differences simply due to the fact that your batch correction fails to correct so you know the batch was bad enough the correction was applied and the correction over corrected so problems like that do exist and we actually saw a number of our collaborators doing this so they would confound their study because a technician is not is not aware of the dangers of batch effects and they would place all their you know controls on chip after chip to chip and then they go to their disease cohort and place them on the sequentially next group of chips and of course this is a study stratified randomization controls better for batch effect so they did this in which case the you know the case and controls were severely confounded with batch effects with different chips they found 94,000 differential methylated positions at five percent false discovery rates so you know this was awesome after combat correction so combat correction was applied this is state-of-the-art batch correcting method and you know all of that was garbage all of that was fake discoveries so it was not five hundred it was not five percent false discoveries it was hundred percent false discoveries so zero remained after the after the chips were properly structured after the design was improved and after they randomized or in this case did the checkered placement right so the study of another group same result so this is a different paper in which you know you place all your studies in one batch and all your you know other cohort in the other batch and then you discover things and then you realize that it was all due to placement in different batches and unbalanced design of every batch so long story short batches should be populated with equal proportions of your groups so make sure your chips are populated with equal proportions of your groups if you're doing micro rates or your labs are structured in such a way that you know you you do half of your experiments on monday and half of your experiments on tuesday but it should be a mixture of your groups in on monday and also a mixture of your groups on tuesday so it's not to discover that the difference is due to monday versus tuesday rather than disease to control all right and while we're at it simply because this paper was available they said there are two potential perils in cancer studies involving things so batch effect was number one and they also discovered that what everybody loves which is the pathway analysis that can also go horribly wrong you can actually discover a lot of interesting enrichments in totally random data so they took random sets of probes and they discovered enrichment in cancer enrichment and developmental diseases enrichments in all kinds of ways so be aware that some of the pathway analysis software may present issues so if you if you're not careful you may discover enrichments in pathways that are just always there cancer development and beyond development all right so i think this is more or less at what i was trying to convey today so i'll just say that there are you know luminaries in this world much better than i am and you can listen to them so there are people like raffaella risari who leads a lot of analysis and biostatistical package development and they know a lot and they have seen a lot about batch effect and all kinds of issues like that related to DNA methylation also expression and other types of bioinformatics analysis so it's worth your time to go through a good selection of lectures on you know available on youtube from broad institute for example this is just one of them this is from 2016 but you know it's still there and still good and for about an hour you can listen to a prominent expert warning you about various pitfalls of your analysis so it's worth to be aware of all these things all right so to summarize again as giam told you in previous lecture in the previous module epigenetics plays key role in many diseases so again let me hammer down this this idea that if you're looking for cancer especially at the stages of progression not the initiation but progression it's more likely to be due to epigenetic disruptions than anything to do with genetic disruptions there is also you know there's an interplay you will probably discover some mutations as well sure but it's unlikely for you to discover too many driver mutations for your later stages of cancer so once it's already there it's progressing through epigenetic this this regulation same with any disease that you may think has environmental component asthma allergies rheumatoid arthritis things like that psoriasis perhaps also any disease that has metabolic component anything to do with your diet anything to do with diabetes things of that nature epigenetic effects are present also neurodevelopmental neurodegenerative effects you know the embryonic development is heavily affected by the epigenetic state in in the womb in the surrounding placenta and so on and so forth so there's a lot of factors that are in play beyond simply genetics so genetics is also there but epigenetics is very heavily in play now epigenetic machinery is complicated as i mentioned so again not to simplify and make fun of geneticists they do a wonderful job and there's a lot to do but epigenetic just has so much more to offer and you know think of like the difference between coding and non-coding genes so of course you know no one will want to throw away 98 percent of your genome because it's you know what used to be called junk DNA right so like it's not junk DNA anymore so people used to think of you know there are genes and there is junk DNA which is like 98 percent of your genome so like raise your hand if you want to throw away your junk DNA i don't right like i better keep it and people are realizing in the last you know five to ten years something that you know non-coding pieces of your DNA are very very very important to your transcriptional machinery and how things are actually progressing in the same fashion you can think of epigenetics genetics is good and then recently we're discovering more and more that diseases could be caused and you know strange phenotypes could be caused by variants in the non-coding part of the genome you know non-coding and enhancers in some other distant locations because you know DNA loops in a certain way and you know physically close regions could be far away in terms of the genomic coordinates same thing with epigenetics there's a lot going on not you know not everything is understood far from it and there's lots and lots of different agents acting upon the DNA so there's you know DNA methylation there are all various histone modifications of which there are too many and the combinations are affecting transcriptions and eventually translations and so on then there are all these issues related to transcription factor binding sites so some binding sites could be physically blocked by all these marks and some other binding sites are open and so the transcriptional machinery can or cannot access them so there are too many of these agents acting and for us the bioinformatician that presents a kind of combinatorial challenge there are too many things meaning there are now multiple data types multiple data formats multiple sources from which these data pieces could be could be fished out from encode from icac from go from all kinds of other places they're all different formats which means also you have to develop multiple pipelines or think how to put all these multi-omic things together and make sure all the formats match and you know the methods apply and so on and statistics is there as well and of course the you know next big thing machine learning everybody's doing it so you know lots of things will be probably explosively developed in this area simply because the area is complicated and lots of things is going on and this will probably be enough for us to do for many decades to come all right so on the technical level those are you know nice motivational things but beware of certain technicalities so beware of confounders such as cell heterogeneity as I mentioned your results could be almost entirely or to some extent due to the fact that you forgot that the cells are not a uniform mixture it could be a heterogeneous mixture and marks such as DNA methylation act as an averaging agent so unless you're doing a single cell analysis you have to be aware of the complexities of your actual DNA population and also beware of batch effect that applies well beyond epigenetics you can you know you can think of batch effects applicable to almost every area of quantitative studies in biochemistry and so on so gene expression is no you know not averse to batch effects and of course DNA methylation histone modification so on could be very much impacted by batches and just beware of how you design your study because if you spend your hundred thousand dollars on a study and then all of that is wrong bioinformatics student cannot fix it for you in two weeks so you have to go back and redesign your whole study and pay the money again unfortunately okay so on this very positive note thank you very much and right so I'll just mention that I am from sick kids I'm from center for computational medicine Michael Brunner who presented yesterday's leader of the center and we have a big and nice team Sergei sitting there and also I work with Rosanna Wexberg lab maybe some of you know her for and some clinical geneticists that sick kids as well so we do some interesting studies on developmental diseases and so on thank you