Hello and welcome to today's workshop. Can, first of all, can everyone hear me? If you can either pop in the chat, if you can hear me, that would be great, please. Great, thank you. And if I could ask for people to, in this workshop, we're gonna primarily be discussing via the chat. There will be an option to use a microphone throughout, but for now, if you could meet your microphone as we get started, that would be great. I believe I can hear someone typing. So if we could just mute that, that would be ideal. So welcome to today's workshop, practical tips, how to make your teaching more inclusive for neurodiverse and disabled students. My name is Dr. Miranda Melcher. I am representing my current employer of City University of London, as well as my previous institution, Kings College London, where a lot of this research and practice was initially developed. These tips are based on about seven years of university teaching and about four years of intensive research on pedagogy related to students with disabilities and learning differences, which is the particular lens that we are going to be focusing on today. This workshop will be obviously a workshop. It will be interactive. So I will be asking you to use the chat. We're gonna do a bit of padlet stuff towards the end, but please feel free to pop in any comments or questions as we go in the chat. You don't need to wait for a particular point. So you are welcome to if you would like. We're gonna be talking about practical tips today. So there's gonna be a bit of theory, but how we apply it is the key thing and is applicable for any kind of teaching that you do, whether it's online, face-to-face, something in the middle, whether you are the one creating the content or whether you're just the one delivering it. So this is equally applicable to lectures, creating modules, to GTAs delivering it, to professional services, creating events. These are widely applicable tips that you should be able to implement in your teaching pretty much immediately. So why are we looking to improve inclusivity in our teaching? Now, presumably the fact that you are here in this workshop today, you already have some ideas about why this is, but let's go over a few general principles to give you some context for what we're looking at today and what my research focuses on. So the first one is that we have actually quite a huge population of students with learning differences or disabilities in the UK. Our last official government data from before the pandemic in 2019 suggests that it's probably a minimum of 17% of students coming into higher education in the UK with some kinds of declared disability. Within the different types of disability that can be declared, specific learning differences is the largest category, but 17% is likely much lower than the actual number. And it is already quite high. So even if we don't necessarily know whether our students have disabilities or not, the chances are statistically, it's quite likely. And so this is a provision that we need to be thinking about and ensuring our teaching is accessible to. The second is this idea of the social model of disability. Traditionally, we have a medical model of disability, which is that the body is meant to function in XYZ way. And if your body cannot do those things, you are somehow deficient. It's essentially on the individual and it's something that is lacking. The social model of disability, however, focuses on how we as a society and as institutions can make things accessible for people. And so the idea is that you shouldn't have to fit one particular model of how your body works in order to participate in society. So one really great example that we're all familiar with are wheelchair ramps. It used to be thought of of if you're in a wheelchair, well, you have to sort out how you're to get around and if you can't get into a building, well, that's your problem. We as a society have moved on to thinking that actually it's the institution's job, it's society's job, it's transport's job to ensure that we have ramps and accessible options for wheelchair users. And that's the idea of switching from the medical to the social model. And in our teaching, we can be part of this transition as well. So instead of saying that our students have to fit into our particular mold, it's about making our teaching more broadly available to people. There are also legal requirements. We have the 2010 Equality Act and we also have the more recent Web Accessibility Act, both of which have pretty high standards for anti-discrimination and accessibility for disability. And so there are some pretty strict legal requirements that I'm sure we're all pretty aware of at this point. This also fits into the wider equality, diversity and inclusion agenda, which of course covers many different issues. But disability and neurodiversity is often one that we don't maybe think as much about. In many ways, this is because they can often be more invisible. And so there's still quite significant societal stigma for people to come forward and disclose. And this is something we can work to address in our teaching. And then finally, what a great reason to improve inclusivity in our teaching is that it's actually applicable to all of our students. All of the tips that we're gonna be talking about today benefit students with disabilities and neurodiversities, but also benefit many other kinds of students as well. So some of the things we're gonna be talking about, for example, make a huge impact for students for whom English is not their first language, for example. So that just adds to the number of students that can be helped through these things. And the beautiful thing about these tips is that no students are disadvantaged by making these tweaks to your teaching. So there's lots of great reasons to improve inclusivity. So we are going to be focusing on three practical principles of inclusive teaching. We're going to be working through what they all mean, how you've encountered them, some specific scenarios, asking questions, and where everything's gonna kind of revolve around these three principles of inclusive teaching. They're very simple. The first is be specific. The second is be transparent. And the third is be mindful. We're gonna go through what those mean, and I'm gonna ask you to have a think and contribute a bit of how you might encounter these in your teaching practice. So how can we be specific? Being specific means clarifying a lot of information that we probably already know as teachers. To give you an example, we know when we ask students to do something, generally what we expect the outcome to be. If we're asking students to participate in a discussion seminar, we have an idea of what participation looks like. We probably have something like, well, we want students to be asking questions, to be participating in group work, but participation doesn't mean that you have to be the one who speaks the most or always have the right answer, right? We know that participation means a bunch of different things and we generally know what that means in our particular context. But if we simply say things like, we want you to participate in this discussion, our students may not fully understand what that means in this context. And so by being specific, we can make it easier for students to participate, to have a go once they know what's expected of them. Similarly, if you're giving, for example, a writing assignment, I'm sure I'm not the only one who field seemingly endless questions from students about things like word count. We know what the word count is, we say it, we make it clear, but students really appreciate that specificity so they know what to expect. Similarly, things like, I'm sure a lot of you have used forums in the last year. It's one thing to say, post in a forum. Okay, when? Add a deadline, post in a forum every Monday. Great, but when we say post in a forum, do we mean 100 words? Do we mean one sentence? Hopefully, what are we expecting from our students here? And this is the sort of thing that can be really, really powerful. It reduces, if students understand what to expect, it's not about giving them the answers, but it allows it reduces anxiety. It enables them to manage their time and their energy more effectively, both of which can be particularly difficult for students with disabilities and our diversities. And again, benefits all of our students. So this being specific is a really key principle for inclusive practice. It is a workshop, it is interactive. So I am gonna ask you at the moment, if I can get three ideas in the chat please, what other ways can you think of to be specific? I've mentioned forums, I've mentioned word count, what other things can we think of where specificity might be helpful in the teaching context? I'd like to get three separate ideas in the chat please. Familiarity with grading rubrics, great, thank you Vicki. We're actually gonna talk about that in a minute, that's a good one. How long each activity should take, Tilly, that's incredibly important. Thank you for raising that. So again, it's not a hard and fast rule, what writing something might take one amount of time for one person, different for another, but giving students an idea of in general, this should take about this long is incredibly helpful. Q&A, provide examples, yep, those are great. Number of examples you'd like to give, great, thank you Lynn. This is exactly the kind of idea of specificity. How students can get feedback, examples of models, yeah. So these are all great ideas of being specific. Now, they're obviously gonna be different depending on your particular teaching context, but some basics to keep in mind. When are they meant to do the thing and how long is it going to take? Making sure that the instructions are available somewhere they can go back and look at them. So this can be posting it on Moodle, putting it in an email, even during a live session, for example, if you send students into breakout rooms, having the instructions on an attachment in the chat if it's Zoom or keeping it in the chat if it's Teams, so that students can go back to it, it can be incredibly powerful and massively reduces the number of repetitive questions you get. Who needs to do what? What are the roles here? So especially in group work, this can be really important. So clarifying, for example, this group of five is gonna go off and discuss this and they come back and one person needs to report back. You've now clarified that one person needs to be the one presenting, which makes it much more possible, for example, for anxious students, maybe autistic students or maybe less confident students generally to feel willing to participate in the group knowing they won't have to present back to the whole class. Depth and lengths, are you expecting, for example, in a forum post a sentence, 500 words? Is it a discussion question? Is it something where you're expecting them to have citations? So these are the kinds of things that can really help inclusive teaching. And again, the great thing is that you already have all this information in your head. So it's not about changing how you teach or what you teach, just adding a bit of extra information. Our second principle, how can we be transparent? So transparency is about explaining explicitly the norms and expectations that we have on our teaching. We often, by the time we get to a position of teaching in higher education, we've been in academia for a while one way or another. And we often forget how alien some of our expectations and norms can be if you're coming in from the beginning. So this can be what Vicki mentioned earlier, marking criteria. I'm sure that my department is not the only one that requires all of us who mark to go through training to make sure that we're all interpreting the marking criteria in the same way. If we have to go through training for the marking criteria, our students don't. Do they understand what it means? Even if we give them access to the marking criteria, which is a great first step, that may or may not be enough. We all, I'm sure, have come across marking criteria that are incredibly vaguely worded, for example. One instance of this that can be especially powerful is it's worth thinking about whether you're marking criteria, how much it cares about English as a language, grammar, punctuation, typos, et cetera. Maybe in your discipline, that does really matter. But I'm guessing that in a lot of disciplines, my own included, actually, that's not a huge priority. In my discipline, we care about sources. We care about depth of understanding. We care about structure. But if you have a few typos, we don't even take off marks of that. It's literally not in the marking criteria. As long as we can understand what students are trying to say, that's fine. You would be astonished by how much more willing all kinds of students are to get drafts, to try something out, once they know that perfect grammar is not an expectation. This is not just for written assignments. This also is things like the chat in a live session. So simply saying something like, I want everyone to put something in the chat. Don't worry about your English. A few typos won't matter.  Can be incredibly, incredibly powerful to our students. So similarly, it's your turn to participate. What other ways or what other aspects of our teaching can we think of that maybe aren't transparent or could be more transparent for our students? Can I get three ideas, please, in the chat? Where might we need more transparency? Areas that are being assessed? Yeah, okay. So emphasizing kind of what counts and what doesn't. That's really important. Great, thank you, Sheila. Acceptable use of quotes, length and referencing. Yes, thank you, Lynn. The purpose of an activity. Tilly, this is brilliant. Yes, this is really important for transparency. Why are you asking them to do this? And why are you asking them to do it in this way, right? Why, for example, do we in academia care about footnotes and referencing? What's the basis for that requirement is really important. If students are asked to work together, explain why and what they'll get out of it. Olivia, exactly, right? Why is group work important in this instance? What is the purpose of not just the content but doing it in this manner? Breaking down jargon, like office hours, extenuating circumstances, formative summative. Yes, Vicki, this is incredibly useful. I have colleagues that have literally built-in glossaries onto their Moodle pages for exactly these kinds of things. The marking process, yeah, will it be double-marked? Will it be moderated? What does moderation mean? These are all really great ideas. So, transferable skills generated by tasks, yep. So some general things to remember, right? What counts, what doesn't count, and why? Where are you coming from? What are your preferences? So things, for example, like yes, in my module, I'm assigning a lot of group work. This is because I think group work is really helpful for teamwork, which improves your employability, right? We all know as teachers, we have different preferences. It can often be really powerful to just acknowledge and explain that, not in a defensive manner, but to help students understand more of why you're asking them to do things. Another important one about transparency is to indicate openness for accessibility improvements. Simply saying, in the first few sessions of your module, if you have any accessibility needs, please feel free to come let me know. It's incredibly powerful. Students may not need to come forward. That would obviously be the ideal that everything's already accessible for them, but simply the fact that they know they can can be incredibly helpful. In fact, a lot of the feedback that I've gotten from my students has been around this. Thank you for saying that I did need this or I didn't need this, but it was good to know I could. And again, that just takes a sentence. And then the last one, as we've mentioned in the chat, explaining how does academia work and why? So things that often seem second nature to us, but may not be for our students. So transparency is incredibly key. Finally, we have a third principle of being mindful. And this is actually a skill that we've all hugely developed in the last year. So things that maybe two years ago we would have had trouble with, like, oh yeah, maybe this event is not going to be able to include people in this time zone. We've gotten a lot better at that.  We've gotten quite good at being mindful in a lot of ways, but for all of us, this is an ever-evolving journey and process. So things that may be easy for you may not be for others. Things that may be hard for you may not be for others. And the idea of being mindful is making sure that especially in what can often be sort of throw away chit chat comments, making sure that we don't sort of put across biases that we don't mean to.  Really wish we could be in person, but like, fine, let's get on with the session. Fair enough. I'm sure of people it was really difficult. However, for a lot of students with disabilities and neurodiversities, but also for distant students, for example, students with care and responsibilities, the fact that so much more was available online was actually a huge benefit to their education and learning. And so they comment at the beginning of a session from the person on the other end of the power dynamic saying online learning is bad and I don't like it, immediately creates an atmosphere that is exclusionary to those people for whom they do like it. Now, it's not to say you have to deny what you think, but even balancing it out just a bit by saying things like, I know that I'm still adjusting to online learning and it may not be my favorite thing, but I'm sure we can make it work and have this be a good experience, right? You're not denying what you feel, but making sure that you're not assuming everyone is in the same situation. So that's one particular example. As before, can I get three ideas in the chat please of other ways in which we can continue to improve our mindfulness? What other things should we be considering? Providing ample time for students to answer questions. Emma, this is great. I was actually gonna mention this. So giving people thinking time and response time is really key. Breaks for online sessions, for stretches, eye relaxation, Lynn, great idea. Thank you. Allowing space for learners to reflect and ask questions. Yeah, Mark, that's a great one. These are all really good ideas for being mindful. We're gonna come across some other ones later. Prompting students to ask for help. Yep, great, Olivia. Allowing responses by a different format. Yes, some would prefer a mic, others prefer to type. Pat, that's a great point. At the moment, I am not doing that. I'm making you all reply via chat. But later on, we will have that option. And considering ways of learning. Yeah, so great. So that's what being mindful entails. And some things to keep in mind. Assume best intent. So we're gonna come across this later on in one of our scenarios. But quite often, especially with transitions, whether it's a new module, whether it's a different mode of delivery, we often, we're under stress. Our students might be under stress, but we as teachers can be under stress. And it can often be easy to assume that if something's not going the way you want, it's on purpose. And yet the vast majority of the time, it's not. So one thing to kind of keep it the forefront is always just assume the best intent, right? We can't possibly know everything that's going on. And it's probably not malicious. Assume that there are invisible disabilities in your class. As we talked about, it's a minimum of 17% of our students have some kind of disability. It's probably a lot higher than that. So even if you haven't received any sort of notice about this particular student or that particular student, in general, assume that inclusivity is going to be important for at least some of your students. This is one that was brought up, I believe, by Tilly. Apologies if I got that name wrong from the chat. I'm not looking at it right this second. But giving response times and time estimates. So for example, I'd like everyone to take a minute to think about this. And then I set a timer for a minute. Or if I'm asking someone to do a task, it should take you about this long. Consider caring responsibilities. This is really key. We know that there are a lot of them, and particularly right now. And those are not trivial things. And those are not things that people, that they do have to take into account with their learning. Consider behavioral norms and expectations. This is really key for some of the learning differences in our diversity. So for example, autism. The stereotype is relatively true that some people with autism do not like eye contact. And so either in in-person class or an online class, making eye contact with other students or the lecture can be incredibly uncomfortable. And yet we do have this norm that one is meant to make eye contact. This goes back to assuming best intent. Do they need to be making? Do all of your students need to be making eye contact in this situation? How would you feel if they don't? Is that feeling in the spirit of assuming best intent? So we now have gone through our three principles and we've come up with some great examples of areas that we can think about this in our teaching and learning. We are now going to go through four scenarios that I'm gonna ask you to take a bit of time to think about and then using either the chat or your microphone entirely up to you. I'm gonna ask for some ideas about responses. Now I'm not gonna say that everyone needs to give a response. This is, you know, feel free to participate as much or as little as you feel comfortable. We are gonna have four scenarios and we're gonna go through them one at a time. So our first scenario, you ask all of your students to present on a set topic for 10 minutes to develop their presentation skills. You're explaining why they're doing it in this way. Two students approach you and say they have a social anxiety disorder and ask if there's an alternative option. How might you respond? I'm going to be silent for a minute and just ask you to think and then I'm going to ask for people to either put responses in the chat or raise their hands. So I'm setting a timer for a minute. Okay, can I please ask for ideas in the chat or if you'd rather use your microphone please feel free to raise your hand and I can then call on you. Sheila, they can do it at the end of the session just you and maybe students, they feel comfortable. Yeah, that could really work, right? Enabling them to still do the important bit but maybe they don't have to do it in front of a big audience. Kat, a good alternative would ask them to record it if they're comfortable and play the recording class. Yeah, so they're definitely still presenting but again, without that live audience. Option to pre-report. Yep, Tilly, you have the same idea, lovely. Narrated PowerPoint, so they don't even have the video aspect. That can be an important thing to assess whether it's a live audience. If that's the thing that's making them nervous or being on video or both, pre-record, work with the students, co-author an alternative. So Mark, this is really, really key, right? Mark's highlighted this point that there are many different ways of going about this. There's Narrated PowerPoint, pre-recording, small audience, other formats, doing it in a shorter time period, pair up to present, there are loads of different options but the key here that Mark and Olivia have highlighted is the first step surely is to ask them, right? What are you nervous about? How can we make this better and to work with the students rather than assuming that there's only going to be one solution? But this is exactly the idea of being inclusive is that we go, hmm, didn't really think about this or haven't had to deal with this before. Let's have a discussion and see what we can do to make this work. So these are great ideas, thank you. We are going to move on to scenario two, scenario two. You notice that students seem to keep their cameras off in live online sessions. How might you consider this and respond? I'm gonna set a one minute timer again and then welcome contributions in the chat or by microphone. Okay, feel free to pop things in the chat or raise your hand. We've got some ideas already. So from Sheila, have a discussion about the group about it and set up a group decision. So this is really important, right? The idea that the group gets to decide what they want to do. So this can be a way forward. I would say that this tends to work best if you either do this right at the beginning to kind of kind of set a contract for the group or do it after the group knows each other pretty well. It can be tricky to do kind of in that middle period because you tend to get the kind of most dominant people speaking up rather than a more even continuation. Ask if there's anything you can do that make them feel more confident about turning their cameras on. Yeah, so that's interesting, Olivia. I will say that that does have an assumption that turning the cameras on is better. And one of the things that's worth thinking about for this is is it better? Why do you want cameras on? The first thing is to think about that, right? You notice that students seem to be keeping their cameras off. Why are you noticing that? Is it because it's bothering you? Is there some educational reason that it's better for them to have it on? Or maybe on reflection, does it actually not seem to be impacting their learning? And maybe they don't need to have them on, right? This is a really tricky one because it comes back to the idea that we have preferences as teachers. We're not neutral actors. And before we make decisions about how to go about doing something, we have to know where we are coming from so that that can be part of the discussion as well, right? So explore why in discussion or ask them privately why this may be. So exploring the discussion again can be powerful. I would say, or asking them to message you can work, though that can create some problems in terms of asking people to justify or explain themselves. See whether it's different breakout rooms, okay? Yeah, Emma. I think, Emma, you've come up with a really nice compromise, right? Say at the start of the session that it's optional, that if you feel comfortable to turn it on, you should, or to have an agreement that for certain pieces, we're all going to agree that we're gonna have them on or off. So, Emma, I think that starts a really nice balance of being inclusive, but also kind of signaling that that may be something you want. Lynn, alternatives, cameras, but tilted down, photos or avatars rather than cameras. Yep, those can be good alternatives. Right, so Kat, this is really important, right? Cameras should not be mandatory as a hard and possible. There's a lot of different reasons. And this is the key thing, right? It can be caring responsibilities. I literally have had students that have been in my sessions with toddlers on their laps or infants on their laps, and they don't want to be on camera for that reason, but are perfectly able to engage and follow along in the session, which seems pretty reasonable to me. We know, for example, there are cultural reasons. For example, students studying online at home who wear hijabs, and if they're at home, they aren't wearing their hijabs, and then we would ask them to put them on just for class, even though they're in their own home. We also have the idea of going back to behavioral expectations, things like eye contact. We know that for students with ADHD, pacing or fiddling can be incredibly helpful. It's not conducive to being on camera. Students with physical disabilities might have impaired mobility or it might be painful to be setting up for a long period of time. The way that one is expected to on camera. If we are in a situation where we're asking students to justify or explain themselves on a case-by-case basis, you can see how this might get quite problematic. So the idea of compromises of first understanding what are your reactions and why, and then trying to come up with alternatives, and the key thing is giving people options and agency. So this can be quite a tricky one, but important for inclusivity. The third one of four is scenario C. You are notified that one of your students is autistic, but you're not given any accommodations that are relevant to live teaching. So for example, the accommodations listed might be things like being able to check out library books for longer periods of time. How might you adapt to this information? Going to set a one-minute timer and then ask for contributions in the chat or by raising your hand. All right, can I please get some ideas in the chat or raising your hand? So we've got some already, asked what has worked well, not well for them on previous courses, how can you help meet their needs? Right, so that's a great idea, right? Building on what we've talked about before, the idea of actually asking the student what has worked for you, what would help in this situation. Great. Discuss the issues with them and any support they've been giving, see if there are any practical solutions. So Sheila, you're exactly on the same page. Ask the student themselves. So I like this idea of assistive technology, but also let's think about what can be wider than simply technical solutions. Provide a chance for people to reflect on their needs. Yeah, so these are some really, really great ideas. In a way, some of this is actually a trick question because especially when we talk about autism, which is one of the most common learning differences that we may come across and also one of the ones that maybe doesn't have the most accurate reputation, is that actually a lot of the things we've already talked about in terms of specificity and transparency can actually already be a lot of the pedagogical accommodation for students with autism in particular. And so one of the things that's worth thinking about is when we get notifications or accommodations, asking the student is an absolutely brilliant way to understand what they need, but also understanding that quite often what they need and want is more clarity, specificity and understanding of expectations rather than specific, please change this activity or please give me this piece of tech, right? The pedagogical and the behavioral side that can be done quite simply with some of the tips we've already discussed can actually sometimes make an even bigger difference. And the final scenario that we're going to be discussing today, you notice that during discussion seminars, either online or in person, students rarely participate freely and there are a lot of awkward silences when you ask the group for thoughts and opinions. How can you improve responsiveness in your class? Same as before, I'm going to set a one minute timer. Okay, what ideas do people have in response to this scenario? Allow discussions to be through the chat rather than cameras or microphones. Sheila, this is great, especially for online learning. We have this wonderful function called the chat, why not use it? Use a variety of approaches such as getting students to react with emojis or the waterfall techniques where they type their answer ahead of time and press enter, mixing it up. Cat, these are great ideas. I in fact use both of those in my teaching and they can be incredibly effective, especially straw pulls through emojis, for example.  Mark, another low stakes one is often an icebreaker question at the beginning of a session where everyone just has a one or two word answer. But if they've said something like, what's your favorite ice, what ice cream flavor do you feel like today? That's an incredibly low stakes way to get engaged. Parent small group activities, jigsaw type where groups feedback. Yeah, one sentence chat somewhere. I really like that idea, Lynn, right? That's going back to being specific. It's not just about being in small groups. It's also having quite a small task that is clear of what you need to do at the end of it. Polls and other tools that are anonymous. Yep, great idea. Nominate roles. Yes, we talked about this in transparency, asking students to appoint a spokesperson and rotate the role, turn taking, what are the norms? Exactly, these are really, really fabulous ideas. In fact, the best set of ideas I've ever had from doing one of these workshops. So that's really, really great to see. So now that we've gone through these four scenarios and we've applied the three principles, we're gonna kind of open it up a bit and look at this idea of inclusive teaching, neurodiversity more broadly. And just building up exactly what Olivia said, we actually have an anonymous padlet that I have popped into the chat. And this is for any questions, comments you might have. So things that have worked well for you if you're willing to share them, questions or concerns you might have, tricky situations, you're not sure how to navigate. It's entirely anonymous, the padlet. And we're gonna do exactly what we've done before. So I'm gonna be quiet for three minutes to give you a time to put in whatever you want, react to people, et cetera, and then start going through and giving you my thoughts on the things put into those, that padlet. So three minutes in the padlet, please. Okay, the three minutes is up, but I can still see some typing. So I'm gonna add one extra minute. Okay, so feel free to continue adding to the padlet, but I'm gonna start responding to these questions. You will still have access to this padlet after the session if it's of interest to you. So please feel free to continue adding to it. Additionally, if you have answers or comments, please feel free to pop them in yourself or put them in the chat as you prefer. So teacher confidence. I think I often worry about asking, what do you prefer as a teacher? You don't wanna create pressure to kind of come up with a solution or be an expert on inclusion. So I think that's a really understandable concern to have. But the idea here that we can avoid is specifically asking each learner their preferences, what works for them. So it's different from saying, student, how do we improve inclusivity? That's a huge question that maybe does create some pressure and is not the most fair. But if you're asking it from the place of, what can I do to help you learn? What works for you as an individual? That actually is something that the student is quite literally the expert in and it can give them a lot of agency and empowerment to actually have the ability to contribute that. So I would agree with your comment, right? You should feel more confident in asking them, knowing that when you're framing it in that way of what works best for you, it is actually quite a different question from how do we fix this massive societal problem? Not wanting to stand out. How do you approach a student who's picked out but does not want to be pointed out as different even privately, but you feel they might have needs you're not meeting? So I agree. This is definitely a tricky situation. Now there are a few things here, right? Often it's about how well do we know that student? Is it something where we can get to a place where they might be more open if in a private situation you ask them, how are you doing in the module? Is there anything I can do to help you with this module to make, are there any needs you have or anything I can change and adapt to make it more possible for you to succeed in this course? Maybe you can get point, but maybe not. Now there is kind of a limit to some degree of what we can do. We're not trained therapists. We're not necessarily personal tutors. We don't know everything that's happening. We can't force people to do anything. But there are a number of things you can do that will make it more likely for students to be comfortable. So the first one is big statements, public statements at the beginning of every module consistently throughout of if you have any needs, please come talk to me. Repeating that over and over rather than just kind of one off a sentence on a Moodle, making clear that that is a conversation you are willing to have. And even specifying things like if there's things that you're struggling with in the module, please come speak to me. So it's not just about you have to have an official diagnosis with an official piece of paper before you can come talk to me. Similarly, if you're noticing that this student is having some issues, are there things about your teaching that you can adapt without them having to say anything? So if you're noticing, for example, that they're having trouble in group work, are there experiments you can do with tweaking the instructions you give or the way that you run the group work and see what might help more with that? If there are issues with, for example, writing, can you experiment with the kinds of guidance and instruction you're giving around written assignments? So are there things that you can experiment with, try out in your teaching, to see if they might help those needs, even if you don't have the full picture or the student doesn't want to talk about it? But there might be a limit. We can't force people to come talk to you. And that's perhaps unfortunate, but there is sometimes a limit. Sometimes lecturers confuse being too specific with being patronizing or too low level. Yep, definitely a thing that I've come across, unfortunately with some of my colleagues. The key thing here is the use of the principles, being specific, transparent and mindful. If you notice when we explain those principles, it's not about the content, it's not about giving the answers, it's about clarifying what you are actually expecting from the student. And in fact, a key thing that seems to work when working with some colleagues I've come across that have this issue is helping them understand that actually the things the students want specificity on are things that the lecturer already knows and actually would be perfectly happy to give the answer if asked. It's just never occurred to them to give that answer freely. So things for example, like so colleague lecture, when you have students create reading responses, is that like half a page, is that one page, what are you looking for there? And they go, oh, well of course I want it to be one page. It's like, great, how do you told them that? So working through kind of specific things to help the lecture understand where those particular issues might be. And every single time I've seen this done or done this myself, it's actually been quite quick for my colleagues to realize that the theoretical concept of, oh, I'm giving them all the answers, it's too patronizing. When they actually work through the particular areas that this might be relevant to their teaching, they realize that actually, they already have all the information, it doesn't dump anything down and it takes them five seconds to give it. So it's about kind of moving things from this theoretical idea of, oh no, but to the like, wait a second, what would this look like in practice? Tens in my experience to be the most effective way of getting over that particular issue. It can sometimes be tricky to encourage students to be understanding of each other. Unfortunately, I don't think you're the only one who's experienced this. Perhaps others have different recommendations, I'd love to hear them. In my experience, this is where the importance of kind of the teacher, the chair, the moderator, setting the norms of understanding can actually be really important. So I forget who said it in the chat, but saying things like, having your cameras on is optional. There might be very good reasons you don't want to have it on, but you should feel free to put it on if you want to. Making sure that that's incredibly clear from you at the beginning can reduce students getting annoyed at each other for cameras on or off, for example. So making clear that you, what your expectations are can be quite powerful. What practices do you think will remain from our digital transition to improve inclusivity when we turn to face-to-face teaching? Not to be a total downer, but I'm actually not particularly optimistic that the inclusive improvements we've made in the last year will actually stay, unfortunately. But I do think it depends quite significantly institution by institution. So maybe there are areas for optimism. Sometimes people aren't aware they have a specific disability, but can surreflect them how they learn what they need to help them. Yeah, no, that's a great point. And that's why this whole idea of inclusive teaching is applicable to all of our students. It doesn't rely on a student having to go through a whole process of diagnosis and disclosure. We can actually, for example, making things more specific helps a huge number of students, including in this particular instance, students who might be autistic, but don't know it. And so this is where kind of the applicability generally can be incredibly powerful. So I don't see any raised hands or additional comments in the chat. I do see a new one that's just gone in. Learning Journal. Yeah, so Learning Journals can be really powerful tools. So great, thank you. That's a great example to pop in there. I appreciate that. So as I said, you can continue adding to this or put it in the chat, it's entirely up to you. And now that we have all these ideas, these principles, these scenarios, lots of answers, I'm going to ask you to do some sort of thinking, reflecting and planning about what you might take from this for your teaching in future. So I've popped another link in the chat for a two and a half question, entirely anonymous form. And I'm going to now be quiet and ask you all to fill it out. It should take you about a minute. And I'm looking, there are eight people in the chat. So I'm looking for eight responses. It's entirely anonymous. So I will only know the number of responses. But if you could now take two minutes during which I will shut up to have a little reflection in this form. Great, I can see the form is working. We've got two, three responses. Thank you. Great, we have just the two minutes and we have eight responses. Thank you very much for filling that out. Hopefully it will be a useful reflection for you to think about for your teaching going forward. So the last thing is just a thank you for being such great participants in this workshop. I really do appreciate it. And a few further resources. So a lot of this is based on my inclusive teaching guide, which is a much longer document that has a lot of different examples of instances of ways you can improve your teaching. So specific ideas of phrasing, for example. Podcasting article about utilizing the chat. This can be a really, really powerful way of being inclusive in online learning. And if you are ever interested in talking more about these issues, please do feel free to get in touch. So I will go ahead and stop sharing and pop these links in the chat if you're interested. And thank you again for participating in such a great workshop. I will stay on for a few minutes if people have any questions or comments. But yeah, no, if you need to leave, please go for it and thank you very much. Thanks, Miranda. That was a really good workshop, really interesting, seeing everyone getting involved as well. We've actually got a resources channel in the Discord. If you'd like to put the links and stuff into there, that would be really helpful. Sure. I might still put them in the chat because I've not been able to access the Discord. Ah, okay. You could put them in any way. Yeah, I can help you access Discord if you want some assistance with that as well. Yeah, I select the link and it just says,


Transcribed. Ladies and gentlemen, the railroad hour. And here comes our star-studded show train. Despite the association of American railroads, brings you Paramount Pictures' great Irving Berlin musical success, Holiday Inn starring Gordon MacRae and his guest from the metropolitan opera Dorothy Kirsten. Our choir is out of the direction of Norman Luboff and our music is prepared and conducted by Carmen Dragon.  And now, here is our star, Gordon MacRae. Good evening, ladies and gentlemen. Well, sir, Dorothy Kirsten and I have a wonderful invitation for you. It's inscribed with the musical magic of Mr. Irving Berlin and you're all invited. Happy Holiday! We're heading down which road, down the road and comes a holiday. The traffic noise affects you like a squeaky violin. Take your care. Happy Holiday! Can you tell me where I can find the owner of Holiday Inn, Mr. Jim Hardy? My best friend. He's a wonderful guy, really. Would you tell him that Linda Mason is here to see him? Oh, he'll be delighted to see you. I know, because I'm Jim Hardy. Oh, forgive me.  Yeah, sure is.  Brilliant idea, don't you think? Dreamed up by this brilliant friend of yours, Jim Hardy. Yep. But tell me, what are you doing way up in the wilds of Connecticut on Christmas? Oh, I heard about Holiday Inn and I wanted to see it. Well, I'm afraid it's not open yet. I spent all my money on lumber and I couldn't talk any performers into working up here for nothing. Oh, I'd perform for you, for nothing. Oh, what do you do? Oh, sing a little and dance a little. Well, come on over to the piano. You know, I've written some extra music for each holiday and this sort of gives me a chance to keep a little promise I made to myself. I said I was going to sing this song at the end of the night and it looks like I'm going to. I love a white Christmas just like the ones I used to know. I'll prompt you. Three years eve after all. By the way, there's only one provisor in this verbal contract. Do you ever hear of Ted Hanover? Famous dancer? Sure. Well, it seems every time I find a girl she's suddenly the only girl in the world for Ted Hanover. So I just want you to be clear of Mr. H. Don't worry.  The place is packed. Oh, who said holiday and wouldn't work? Ready for your number, sweetie? Sure. Look at the clock. Oh, goodness, 11.59. Ta-ta, old year. Ha-ha, it's almost time. Practically straight up. One minute to midnight One minute to go One minute to say goodbye Before we say Well, let's start the new Happy new year, Jim. I'll see you out on the floor. All right. Will you dance with me? I beg your pardon, Mr. Hanover. Oh, you're Ted Hanover. Yeah, my dancing partner left me, so I'm slumming in the halo of Connecticut. Come on, let's dance. Oh, Mr. Hanover, I can't. Music, music, please. Oh, please, Mr. Hanover. But it's in my contract. I just, I can't. Hey, Ted Hanover and her new partner. He surprised us all, Mr. Hardy. Imagine letting us see Ted Hanover and his new partner. Well, here... Good morning. Oh, morning. You're at holiday in Ted, and the holiday is over. Oh, no. Look, Jim, something happened last night. I met a girl, and I've got to havers my dancing partner. Girl? Girl? Yeah, everything was so blurred in the New Year's Eve excitement, I didn't even get her name. Jim, you've got to help me out. Who was she? Who? I think I remember. It must have been that girl with an evening gown on with a belt in the back. Yeah, it built just like a girlfriend of mine, Miss Consuela Schlepkis. I remember Consuela used to play the pinball machine down at the corner drug store. High man, three weeks in a row. Oh, no. Hey, look, wait a minute. She'll be here at holiday in the next time you're open. She will? Sure, sure. When's the next holiday? Let me see, January? January, nothing more than January, February. February 12, Lincoln's birthday. I'm sure to find her here then. Mr. Lincoln, why was you born? Can I come in your dressing room? Come in, Jim. Happy Lincoln's birthday. Yeah, yeah. Say, Linda, I've decided our number will go better if we do it in blackface. Oh. What's the matter with that? Well, for a month and a half, I've been dreaming about how pretty I was going to look tonight. Sweetheart, you'll have plenty of times to be pretty. Here, I hope you put it on. I broke in as a boot black, you know. Well, nobody's going to recognize me. Ain't it the truth? I mean, it's the 12th of February. On the February morning, the tiny baby boy was born. Rown up this tiny baby, the folks all called him Honest Dave Abraham. Back to dinner. That is why we celebrate this blessed February date, Abraham. To Washington. Abraham. This blessed February.  Oh, Danny. Danny, just hand over. Now look, as my agent, you've got to help me find this gal. Lord knows how long I'll have to wait for another Jim's blasted holiday. Huh? When? When? Day after tomorrow's another holiday? Sure, sure. I'll stick around. Happy Valentine's Day.  For over the myriad arteries of our many forms of transportation flow all the things we need and use at home and at work. And forming the foundation of this great transportation network are our railroads, operating thousands of trains over some 400,000 miles of track extending into every section of the country. Carrying the very life blood of our commerce, these railroads are doing the biggest and most essential of all transportation jobs. According to calculations of the Bureau of Transport Economics and Statistics of the Interstate Commerce Commission, which take account of both tons loaded and distances hauled, railroads are moving more tons of freight more miles between our cities than all other forms of transportation combined. Last year, for example, railroads produced nearly five times as much interstate freight transportation service as all the motor trucks in the country. Unlike other transport agencies, railroads provide an all commodity, all season and continent wide service. And it's done with an economy in the use of manpower, fuel and materials which no other form of transportation offering anything resembling a general and all round service even approaches. Yes, America has a great transportation system, a system in which no other medium of transport nor all of them combined can take the place of railroads in doing the essential transportation job that our industry, agriculture and commerce need in time of peace and must have in time of national emergency. Railroads are and will continue to be the transportation cornerstone on which is built the amazingly productive economy of our nation. Here is Act 2 of Paramount Pictures Holiday Inn starring Gordon Macrae as Jim Hardy and Dorothy Kirsten as Linda Mason. Oh, Jim, the inn looks so romantic with all those Valentine hearts. Well, I pose for those cupids myself. Here's something for you, Linda. Oh, Jim, it's the most beautiful Valentine. I've never seen such a big heart. Come on, open it up. Oh, it's music. Yes, sir, dedicated with love to Miss Linda Mason. Read what it says. Be careful. It's my heart. It's not the book. Why do things like this always have to happen to me? Oh, lady, lady, where have you been? I've been looking at you since New Year's Eve. For me, Mr. Hanover? Sure, sure. You're my new dancing partner. Where would you like to go? Havana, Rio, Hollywood, huh? I'd like to stay right here at Holiday Inn. Well, you can be here for the holidays and be my partner between times. I'll make you famous. Jim? Oh, go ahead. I don't want to hold up your career. But come home soon, huh? Straight wire to Mr. Jim Hardy, Holiday Inn, Midville, Connecticut. Regret booking in Florida for our new act makes it impossible to be there for Washington's birthday. See you Easter, best Linda. There for Mr. Jim Hardy. Thank you. Oh, fine. Regret booking in New York makes it impossible to get there for Easter. Bye, golly. If she won't come here, I'm going there. Hello, Linda. Jim. Oh, Jim. I couldn't spend another holiday without you, honey. Would you take Maram and join me for the day? Will I?  Come on, parade's ready to start. Never saw you look quite so pretty before Never saw you dressed Quite so lovely what's more Mr. Bonnet With all's upon it This lady We brought Mr. Dunbar to Holiday Inn, Jimmy Boy. He's one of Hollywood's top directors and he's offering all of us the opportunity of our lifetime. It does sound awfully good, Jim. Well, I don't know. I don't know, Mr. Dunbar. Well, the whole idea of Holiday Inn would make a wonderful picture, Hardy. And we'd like to start this sensational new dance team, Hanover and Mason. You see, he didn't even give you top billing. Oh, come on, Jimmy Boy. How about it? We're all heading for Hollywood, the land of milk and honey. Oh, no, I'd like to keep this little place just the way it is, Ted. Oh, no, you're not going to be selfish and keep Linda from getting a big break, are you? Okay. Take the place. Take the idea, take the music. Take the whole darn thing. But what about you, Jim? I want you to write the music. I'll send it to him. But I'll work here. Is that the deal you wanted? Or should I have thrown in my shirt, too? Dear Mr. Dunbar, I just finished the last of the Holiday Inn songs. It's one for Thanksgiving. And here it is, turkey time, sure enough. I've made a record of this little hunk of cranberry sauce. I pause in this letter to give it another spin just to make sure all the notes are in the right place. To be thankful for, are you kidding? I haven't got a great big yard to sail from shore to shore Still I've got plenty to be thankful for You're really loaded, Dad. I've got plenty to be thankful for Like what? No private car, caviar No carpet on the floor Still I've got plenty to be thankful for Who is this boy? I've got eyes to see with You need glasses Ears to hear with Arms to hunk with Someone to adore You're a little flat, too Or how could anybody ask for more The needs are small I'll buy them all at the 5 and 10 cent store Oh, sing it, kid! I've got plenty to be thankful for I'm not going to send this record to Hollywood I'm going to send me Miss Mason, we'll take the final shots in the set of Holiday Inn Now I think you have the mood Your Hollywood success was empty You've lost the one man you loved You know, the usual hope All right for rehearsal Let's shoot it, please All right, it's a take Lights up on the Christmas tree and dolly in for a close-up of this Linda Mason in Holiday Inn Ready? Roll on!  Every one, every holiday of the year Not to mention all the wonderful days in between Dorothy Kirsten, we'll be back in just a moment Our thanks to Tony Barrett, Boris Taylor and our entire company Holiday Inn was dramatized for the Railroad Hour by Lawrence and Lee The Railroad Hour is brought to you each week at this time by the American Railroads America's steel industry is operating at record levels turning out more steel than any other country in the world But today there's a serious shortage of scrap which makes up about half the content of new steel Every business and industry can help keep the nation's steel production high by searching out every extra pound of iron and steel scrap of all kinds and starting it toward the mills through a scrap dealer The Railroads, for years one of the largest sources of scrap metal are also intensifying their efforts to turn still more scrap back to the steel industry And here again is lovely Dorothy Kirsten What's on the show train next week, Gordon? Well, Rosemary, and guess who our guest is? Dorothy Kirsten Gee, how'd you get her? Well, I don't know I'll see you next week, Dorothy All aboard! Well, sir, it looks as though we're ready to pull out Until next week, this is Gordon Macrae saying goodbye Holiday Inn was presented to the courtesy of Paramount Pictures whose current release is Submarine Command starring William Holden, William Bendix, and Nancy Olson Gordon Macrae can soon be seen in Warner Bros. Starlet Our choir is under the direction of Norman Luboff and our music is prepared and conducted by Carmen Dragon This is Marvin Miller saying goodbye until next week for the American Railroads Now, heat tune for your Monday night of music on NBC Proceeding was transcribed Next, it's the telephone hour on NBC


Okay. Good afternoon, everyone. So my name is Andrew McPherson. I'm going to be teaching you the module five, which is the SC DNA seek module, the single cell genomics on the DNA side. I'll tell you a little bit about my myself so I started my graduate studies in BC. Thank you for BC in Canada. I was working with Dr. Saurab Shah and Dr. Aparishio at first on computational methods for bulk whole genome sequencing, trying to deconvolute the multiple clones that were evident in that type of data. And I was happy when I could start to work on single cell techniques because they really allowed us to look at clonal structure in a way that didn't require us to to overcome the issues of bulk sequencing. So then with Dr. Shaw and Dr. Aparishio, I, we developed the platform to direct library preparation and collaboration with them. And I worked mainly on the computational side. And from that work, I guess it from my experience with DLP and single cell, I was then offered a position at MSK in New York and that's where I am right now. So I'll just jump right into it. So this is the Creative Commons license. This basically means that you are free to share, copy distribute, transmit the work here in to a mix if you like and adapt in your own work under the conditions that you attribute the work in a manner specific specified by the author or the licensor and that you, if you alter this work then you share it under the same license I believe that's the stipulations. Okay, so I'm sure you've already been introduced to this somewhat but and probably know this already but cancer is definitely driven by an evolutionary process. And that evolutionary process involves mutational, mutational processes acting on the genome together within perfect DNA replication, or DNA repair that is malfunctioned in some way that promotes genetic changes that transform cells from a phenotype that favors the organism survival to a selfish malignant phenotype that favors unbounded cell division. And increased cell division together with disabled repair mechanisms promote genome instability resulting in further clonal heterogeneity. And then from that heterogeneity will be selected populations that are able to invade adjacent tissue evade the immune system of a treatment and the past size. And really cancer evolution is the reason why we're interested in in heterogeneity because by being able to look at heterogeneity we're able to reconstruct and understand the evolutionary patterns that led to that heterogeneity and then we're able to predict potentially by a complete complete understanding of that heterogeneity would be able to predict what will happen in the future clinically to a patient in which we've observed that cellular heterogeneity. So every single cell talk I think has to have a slide about maybe smoothies or some analogy like this. I think that the picture we'd like to see is this colorful picture of all of these different types of varies. Of course, the traditional techniques that we apply to the sequencing of genomes. So let's take that very interesting mixture and blend it up. And then we perhaps have this difficult process of trying to deconvolute, deconvolute what went into this mixture using the that produce the bulk genome sequencing. At the end of this lecture, I would hope that you have learned or have a knowledge of single cell DNA sequencing techniques. You understand how se DNA data is analyzed for copy number changes, single cell nucleotide or single nucleotide variants, specific for specifically for understanding the evolutionary patterns of single nucleotide variants and the copy number changes, and perhaps be aware of the pitfalls of each type of analysis and know how to se DNA has been used thus far to study cancer. So single cell sequencing in effect is multiplex sequencing of single cells using DNA barcodes I think that actually describes all of them, the many various techniques that we can call a single cell DNA sequencing. The basic steps on the molecular biology side are dissociation into single cells, suspensions, isolation of single cells cell lysis and cell lysis and also removing bound proteins from the DNA so cleaning the DNA, followed by indexing or barcoding the cells so they can be identified later and downstream analysis, and then amplification and library preparation. And so, most of the protocols involve some variation on these steps. Single cell dissociation is the first step that involves dissection rinsing and mincing. The digestive enzymes and incubation these these steps are definitely tricky and very important in terms of optimization, especially for fragile cell populations from difficult to sequence tumor specimens. So it's an important step. It's the next step in the process and this there's definitely a variety of different techniques that have been used such as limiting dilution so that's where we dilute to the point where statistically speaking. We know that if we spot a certain quantity into a set of wells, then by chance, it's very unlikely there'll be two cells in each well of course, often there's zero cells, which is one problem with that solution. There's techniques like capillary pipette that are somewhat laborious. So capture micro dissection to some extent can be think I thought of as a single cell technique micro fluidics I think are one of the more important techniques as they allow this this platform to scale. And facts and blood collection are also techniques that have been used. So once we have been able to isolate the cells perhaps into into into an array of wells, then the next step is to index those two types of indexing that have been shown here the one that I'm most familiar with is the combinatorial indexing. And that type of indexing involves having a unique barcode per row, and then another unique barcode per column, possibly with duplication of those barcode sequences, but the done in such a way that each unique pair of barcodes uniquely implies the location within this within the set of wells that the cell would have come from. And then there's also non redundant indexing in which every well has a completely unique pair. So the early days of whole genome single cell whole genome sequencing or cells single cell DNA sequencing these are the three techniques. But still somewhat relevant today dot PCR was a technique that used primarily used PCR MDA uses an isothermal amplification technique using a more accurate polymerase.  I think the main takeaway is that there are differences between these two, three techniques with respect to sort of the error profile and what you would be able to do with these techniques so dot PCR has a high rate of errors that are introduced by the polymerase whereas MDA has a low rate of errors that are introduced that allow it to be used comparatively better for single single nucleotide variation deep dot PCR has a relatively higher uniformity sort of the double negative in the slide. So dot PCR because of the uniformity of coverage across genome is better for copy number calling. And alternative, which is the technique that we use in our lab for DLP is direct tagmentation of an unamplified genome prior to any kind of amplification that's applied after the fact. This uses a transpose on a TN five transposes loaded with library adapters to basically fragment the DNA and also add adapters for sequencing to that DNA. What happens is the DNA is is targeted so these these TN fives they cut the DNA and introduce these primers and then the primers are used for dual barcoding in which we do a minimal amount of PCR, just enough to introduce the barcodes into the into the amplified sequences so that the DNA is barcoded for identity and identifying the cell of origin of the sequences later. Another technique that is I think is quite interesting and and novel is single cell combinatorial index sequencing. And this uses a split and repooling approach to ensure that for the most part cells are uniquely barcoded, even though they're never isolated into individual wells for one for each cell. So what happens is in a first in a first part of the experiment, the first barcode set first set of barcodes is introduced into the nuclei, keeping the nuclei intact using a TN five transposes. And so you have pools of nuclei that are uniquely barcoded, each set of nuclei is uniquely barcoded. And then these sets of uniquely barcode coded nuclei are pooled together and then redistributed and during a second barcoding step using PCR they're barcode with a second set of barcodes. And the hope is that it's statistically unlikely that a cell or nuclei would would end up in the same set of wells across these two grouping and splitting and redistributing steps as another cell so no two cells hopefully would end up in this in this the same well as in the top and the same well in the bottom and therefore all cells would be uniquely barcoded and so this doesn't, there is some error rate to the, to this process so I think there's maybe five to 10% of the of the barcode pairs end up being for two or more cells. And finally the method that was developed in our lab, and that we now have at MSK is called direct library preparation. This uses cell isolation using a piezoelectric dispenser so this is a droplet dispenser that dispenses into a nano wall array. And by effectively using a small, a small acoustic pulse to create small droplets and together with an onboard camera. We are able to able to observe as we're dispensing droplets when there is a cell within the dispensation region. And when we observe a cell, or when the machine observes a cell it automatically takes the nozzle and dispenses the next droplet into the, the well in the nano wall array. And it's followed by steps that do additional imaging, add lysis buffers do TN five tagmentation to additional additional reagents steps and then pool the resulting tagged DNA, and do regular sequencing on aluminum sequencing machine. And maybe I'll mention but unfortunately it was discontinued. This technique uses uses uses an emulsion of droplets. There's two steps here where in first step in which droplets are and droplets are sorry cells or nuclei are placed into droplets using this microfluidic system. And then following that those, those droplets with cells in them are run through another drop microfluidics device that incorporates these gel beads which have the additional reagents and barcodes that are needed it for independent for the next steps which involve amplification within each of these droplets, followed by pooling and then regular illuminate sequencing. And I think that the final type of single cell sequencing I'll mention just because now it has a commercial platform and we're starting to see more publications using this platform is the tapestry platform for mission bio. This is also droplet based. This is a very similar set of steps where cells are nuclei are incorporated into into droplets of oil in an emulsion. And then within that those droplets independently. We have distinct barcode I five and I seven barcodes and together with gene specific primers. We do a PCR to amplify specific genes and also add cell specific barcodes. So this is a very this is a target approach that involves developing a panel, but it can be very high throughput. Okay, so in the next part of the talk I'll, I'll describe a CDNA data and analysis. The analysis that are possible using DNA seek are to uncover clonal substructure. So looking at different populations of cells and how they differ in their, perhaps driver genes, or mutational processes. And then also looking at clonal lineages so understanding basically the afflution relationships between those clones. Looking at tumor evolution, perhaps over time where we have time series measurements of of clones, from which we can understand changes in the clonal abundance and then also looking at mutation co occurrence. So the first look at one of the data types to data types. I'll talk about the most in this set of slides, which is low coverage single cell whole genome sequencing. This encompasses DLP. 10x CNV would have also been a low coverage single cell technique and some of the other techniques like dot PCR a bit higher coverage but they're also much less lower much lower coverage than a regular whole genome sequencing so this is what you would first see when you open up one of the BAM files in IGV. As we zoom out a bit we can see that hopefully we get even coverage on average and that coverage. When we look at any two cells isn't biased to particular regions in the genome and that's what we've optimized for in DLP and others have optimized for with say dot PCR or 10x CNV. I think given that the coverage is low for these types of sequencing assays. These really are copy number primary assays and so the first step is a copy number analysis almost certainly. Copy number calling in single cell whole genome. It's, I think, Serena will have introduced you to this and it's quite similar. The bin reads across the genome are binned into regular or regular length bins. We have GC correction and mapability correction as the next steps, removal of outlier bins and then removal of outlier cells, followed by segmentation and copy number calling sometimes those two are done jointly. So this is an idea of how important GC correction is. This is one of the cells that we see constant with DLP. If you overlay the GC of each bin, the average GC of each bin, you can see that there's a strong relationship and then if you look at that relationship as a scatterplot between reads on the y-axis and GC on the x-axis, sort of Nike swoosh shape that shows up the sort of bias that you get characteristic of sequencing. Going back here, this bias is usually corrected by some kind of polynomial or low S regression that's fit to this curve and then we effectively regress out the contribution of GC to the biases in read depth coverage. Segmentation involves a few different approaches. So we can have a sliding window approach. CBS is one of these types of sliding window approaches. CBS stands for circular binary segmentation. This is a procedure where we iteratively look for intervals of the genome that have a that are outliers with respect to coverage and then we iteratively build up a list of regions that have differential signal between their neighboring regions. A hidden mark of models are another option for this type of analysis and that's, these are what we use primarily in our lab. They allow joint calling of the segments and the copy number state through this Markov process at the bottom, right here, which basically describes the different probabilities of going from one copy state to another, as you move across the genome, you may, you may sort of, I guess, you know, some of the intuition you have can be encoded as a prior into this kind of transition structure such that for, for instance, you don't often transition into a homozygous deletion state because you don't expect many homozygous deletions in your typical genome and so learning that transition matrix allows you to see where what kind of transitions are more likely and then at the same time infer the actual states for different regions of the genome and what the boundaries are for those, the states within the genome. So there's another approach called an objective function based approach that doesn't do copy number calling at the same time but does does effectively a segmentation with a specific like mathematical objective function that it's trying to optimize for say the objective function approach is similar to the sliding window CVS approach but instead it's trying to optimize a specific a specific objective. These are nicer approaches and. Yeah, I think they're a good option also. Excuse me. Yeah. In the, in the previous sessions, they talked about the different positions in the genome have a relationship to each other. They called it linkage or something like that. There is a Markov model. The assumption of Markov as far as I remember is that the probability of going to state the next state doesn't have any relationship to the previous state, but here if two states are related you can't go to the other state. How do you assume this kind of assumption. Which, can I ask which module that was so I think linkage that would probably refer to genetic linkage and. These are genomic positions aren't they. So, yeah, doesn't mean that the copy number in different positions should have a relationship to each other. Yeah, that is a very good point. I think maybe not. Maybe I misunderstand your use of the term linkage but what I will say is you're absolutely correct because one thing that I'm not talking about this very much in this set of slides. One thing that we're missing here is the different connections between the segments that are implied by genomic rearrangement. There's certainly, you know, there's certainly violate the assumption that we have in the Markov process that a bin is a bin's copy number is only dependent on its adjacent bins not on this and bins in the genome, because, you know, we could have instances where a bin has been rearranged by these very complex processes that take different parts of other chromosomes and juxtapose them with different positions and in the different chromosome. And so that in those situations yeah I mean this is a very much a simplification and a model that you know is more useful than it is. Absolutely realistic. That makes sense. So we assume it but it's not a realistic model. Yeah, it's useful more than realistic for sure, like most models. And, and can we assume it like for, I didn't understand that rearrangement that you talked about. Can, can you please explain that a little more. Sure. Can you see my cursor on the slide. Yeah, I guess. Okay. So, you know we're saying that this. These bins here that I'm circling that in red are only really dependent their state is only dependent on the neighboring ones or the ones perhaps neighboring in this nearby amplified set of bins. Only the adjacent bins impact the probability of your state under a Markov process. But, you know what if this purple region over on the right has been sort of cut from this right part of this chromosome and joined to some part of this chromosome of this green or perhaps that's I'm trying to come up with an example that's off the cuff but actually here's maybe a better example. So, this is amplified, but we also don't need to know the orientation of this green segment it could have been reversed and amplified. So that would mean that this these bins here are actually adjacent to these bins over here. Does that make sense. Yeah, that makes sense. Thank you. Okay, so an example of copy number in a single cell. This is the cell, I think is a lymphoblastoid cell line it's near deployed. Actually, I'm wrong. This is, this is actually the ob 2295 data, high grade serious ovarian cancer cell lines that will be working with in the lab. So this is baseline diploid. A median copy number of two but you see that there are quite a few amplifications and solutions. It's a female sample obviously so it doesn't have a white chromosome. And then this is copy number in a tetraploid cell and from the same sample. And one question is how, how can you tell from this data that it this is a good solution that it's actually tetraploid versus deployed. And the key thing to look for here is these copy number states at at a level of copy number of two or sorry three or five. And here's a copy number one segment. So, in terms of evaluating whether or not you have a good copy number solution. One of the questions you should ask is whether or not the the pointy is something is correct or makes sense given the data. So we can see that it probably does because we have these odd numbered copy number segments that help us sort of anchor to these anchor to these, this solution. Of course, we could end up with a solution like this. And here the question is, you know, there's no odd numbered copy number states that are helping us to assess whether that this or ascertain that this is a tetraploid cell. There's no significant segments at any of the odd numbered states, we could divide the entire set of copy numbers by two and get an equal equally reasonable genome that would be baseline diploid. And this is the identified viability of copy number problem that doesn't go away with single cell for sure. And is perhaps exhibited here. This is just showing how well we can fit an HMM model to data of using different ploidy solutions and you can see that this is the log likelihood so actually it's the negative log likelihood so the smaller this is the better and we can see that we could just keep in increasing the ploidy and we would keep getting a better fit according to our model for the data so unfortunately things like measuring the likelihood of your model in an HMM don't give you any idea of whether or not you should pick a tetraploid over deployed solution. Okay, so that's copy number I'm going to jump into phylogenetic analysis. And first, talk about the different models that have been used for phylogenetic analysis in SCD and AC data. I think the most prevalent is because of its simplicity is this assumption of the infinite sites that leads us to the perfect phylogeny model. The infinite sites assumptions is the assumption that mutations are only once throughout the tree. And that there's no reversions of those mutations or parallel mutation. That can be shown of the, an example of this is shown in the bottom right this is actually from next train which is about viral evolution, but the, the, the really the principles are the same here. These mutations. Once they originate you see that they propagate to all of the descendants this blue mutation is exhibited in ABCD and E. It's originated near the root of the tree. And then this this red mutation is only in C. And this yellow mutation is in all three of its descendant genomes or sequences. Oops. And for the infinite sites assumption is that the genome is quite large and mutations are although they do happen frequently they're relatively infrequent compared to the size of the genome you maybe get 1020 maybe 100,000 mutations in a very highly mutated patient sample that the genome itself is three sequences of sequence. So, relatively speaking, the, the chance of any one particular location in the or nucleotide in the genome of being mutated is low for that particular location. Of course, this is similar to an HMM. It's a useful but imperfect model. You know that there can be violations to the infinite sites assumption and including conversion evolution, where there is a strong fitness advantage to mutating a particular gene cancer. There's also the possibility that there's a back mutation. Those have been observed, for instance, in BCR, BRCA patients that are that are treated with PARP inhibitors you can get a reversion of a BRC mutation. And you can also have instances where mutations can be lost by copy number change. Two alternative models here are finite sites. This is just sort of a, I guess, a free for all model. If you could put it that way, mutations can occur, they can reoccur, they can back mutate so anything is possible in this model. Another model that I've used in my work is the DOLA model mutations can occur only once in this model. Somewhat like the infinite site assumption. So assuming that the genome is large relative to the rate of mutation. But they can be lost, for instance, through copy number change that deletes the segment that harbors the mutation. And once they're lost, they cannot reoccur. In a study published last year, and through an analysis of single cell data, the authors claim to find quite a bit of evidence for violations of the ISA in real single cell sequencing data including parallel evolution, loss of heterozygosity. So deletion of a region that contains MSNB and also back mutation. And within the context of single cell, one of the, this is not necessarily a model, but sort of a simplification in the way modeling is done, because often single cells are quite similar. So we find that, for instance, this red mutation is differentiates this population of three cells from all of the other cells, but within this population of three cells, they these cells are genetically equivalent. And what this has done is to create something called a mutation tree which is just using mutations on internal nodes to show the structure of the evolution of mutations, and then cells, these internal nodes in clusters to represent which, which mutations are in which cells. So for instance this set here of cells have all of the mutations that were acquired above in ancestral nodes. And these two blue mutations and this red mutation. You'll notice that this mutation tree on the right here is using the perfect phylogeny model. One of the methods that first methods that came out that did this inference, this type of inference where they trying to try to build a mutation tree. Noisy SNV presence absence data is called site. So the data here that's input to this, this method is just observations of presence or absence of a particular mutation in a particular cell. So the sort of upstream analysis done as part of a bioinformatics pipeline will just will give you counts of reads. For each mutation for each cell for the reference or the variant. And then, as a first step, the site authors just to steal that down as did we see I mean enough mutations, or enough mismatches in the reads that would support a particular SNV to say the SNV is present if we did mark a one in this matrix. If we didn't remark a zero if we didn't really see any reads that overlapped that particular mutation at all then we have a missing value. And unfortunately, missing values are quite prevalent in this type of data as you'll see when we do the lab. Just because you have something called a little dropout happens so in the amplification. You can have preference of amplification of one region over another resulting in very free reads coming from one region of interest in a particular cell. So site, the site authors propose a method that takes this data and produces this mutation tree and we'll go into the details of of the method. Another method is sci fi. This is more relevant because we are going to be using a sci fi inferred tree in our lab. And this is a slightly different approach because it goes from a they propose a model that incorporates both both the read level data and the tree modeling the presence of absence and absence of mutations across the set of cells. And so they in a combined fashion call the presence or absence of particular SMVs in each cell and reconstruct the tree at the same time in an integrated way and then call mutations by doing posterior inference over the inferred model. And then finally a method that my colleague and I published in 2016 doesn't do phylogeny inference but I'm showcasing here because I, it gives you some insight into one of the one of the potential pitfalls of this type of data and this type of analysis which is doublets. And I think I find the colors here a little bit confusing but so if you'll allow me to explain them a is the reference means that there's only the reference reads that are occupying this particular or covering this particular mutation in this particular cell. And AB is heterozygous and B is homozygous for the variant and so that means that we have this collection of cells down here that have essentially all of the variants that we have profiled again here that the columns are variants. And then the rows are cells. So these cells in clone three have all of the variants at least heterozygous Lee. And in comparison to these populations at the top zero and one which have just a subset of variants. A prayer we knew we happen to know from in this experiment that these particular cells were, or at least the data in these particular rows comes from multiple cells, and likely from combination of the cells in zero and the cells in clone one clone. And three is essentially cells pairs of cells from zero and one. And then of course if you infer a tree from that kind of data you get something that's much more complex than you expect. And by contrast, if you use a method, which is what we proposed in this paper that is capable of filtering doublets then you end up getting a much simpler and more realistic tree. Right. What about phylogenetics and copy number data that's certainly an important problem. And we have. There's, there's been much less work on that probably because it's quite a difficult problem, and possibly also because single cell whole genome sequencing is more quite a bit newer than techniques that look at these in single cells. And the method that we've proposed recently in a bio archive paper is called sicker. This is baby Bayesian phylogenetic inference from CWS derived copy number data. And the idea here is to use transitions and in copy number data in individual cells as as binary as phylogenetic markers, and then perform inference on those markers. And the assumptions here are that we don't have any loss of change points through deletion, which is a somewhat strong assumption in some contexts. And we also have the assumption that change point distance approximates evolutionary different distance and I'm showing at the bottom here. So what do we mean by this idea that we use copy number transitions as markers so for instance in a we have perhaps an amplification that results in two copy number transitions. One at the beginning of the amplification another at the end of the amplification stacked on top perhaps we have a subsequent amplification that introduces two more copy number transitions so two more additional markers. In the middle you hear it. Excuse me, you can see where we have an instance of a duplication overlapping a deletion that results in the removal of these change points and so this is an instance where the model is not going to sufficiently capture the changes that differentiate these different genomes. The data looks like this for SIGCA so we take a copy number matrix that we see here, rows again are cells and along the y axis in this heat map is chromosomal positions. So we take data that looks like this, and we try to infer these binary indicators of transition points and then build a tree as you see on the left. And I think, and competing method that's also recently come out in my archive that I think is quite interesting is something called psychone, and the difference here is they look for transitions and copy number across all of the individual cells. But then what they do is from those transitions and copy number they build a segmentation of the of the genome into regions, and then they reconstruct phylogeny over the copy number changes within those regions. And in integrated fashion they take these noisy raw read counts cells by bins, you can see in panel B and simultaneously infer a tree as you see on the right and call copy number as you see on the bottom in the middle. So, another type of analysis you can do is something called suitable analysis. So, let's say that we are interested in SMV evolution in low coverage data, like DLP or 10xCNV. What we can do is first cluster the cells by their copy number profiles. And lastly, you can also use a sitka or psychone to cluster to create to generate an evolutionary tree and then cut the evolutionary tree at certain branch lengths to generate clusters so first step is to generate clusters of cells. As you can sort of see here with the left. Now we have like an annotation of the cluster across this population of cells. And then by clustering of cells, you can then merge all of the data within those clusters, and then use those as effectively as just independent WGS data sets, from which you can call SMVs call break points to any of the analysis, you would do with a regular whole genome sequencing data so long as you are able to cluster this data into groups of cells that are large enough. And then you can do something like this. Where, and this is the data that you'll be analyzing in the lab. So we've looked at SMVs and break points across these nine clones, I believe, from the OV2295 samples, and then reconstructed a phylogenetic tree across the clones, including all of the driver events annotated here. In the last part of the talk, I will go through some of the studies that have already been done with leveraging single cell DNA sequencing and talk about just in general where the field has progressed up until now. So what we've been able to study so far as clonal evolution, therapeutic resistance so understanding how clones and fitness is affected by by drug treatment. There's been studies of metastatic dissemination and the dynamic processes by which clones populate other other tissues. And there's been studies of invasion in pre-moleconcies. One of the first studies was this study published in 2011 by Nick Naven, who is now a pioneer in the field of single cell genomics. When they used .PCR to sequence 200 cells. This is, I would say the first big single cell sequencing study. And what they found was that when they reconstructed a phylogenetic tree from the copy number that they inferred for each of these cells, they described what they saw as punctuated clonal expansion with few persistent intermediates. And a subsequent study, this was also done by Nick Naven's group, who studied clonal evolution in breast cancer sequencing and using a technique called nuke-seq. So nuke-seq is effectively MDA on cells that are in G2M phase. So they have double the amount of DNA available for sequencing, which helps with things like allylic dropout. So if you have four copies of a particular loci as opposed to two, it's less likely that you will lose both, you lose an allele when you amplify from those four copies. So this, they applied nuke-seq to understand the breast cancer sequencing similar to the previous study. And they found that aneuploid rearrangements occur early and point mutations evolve gradually and they were able to do this because they combined .PCR for copy number with nuke-seq for SNVs. And this was a strategy that was used by subsequently by a few papers, combining multiple types of sequencing. One type of sequencing perhaps is strong for SNVs and one is strong for producing copy number. And so there's been quite a few studies and methods surrounding the combination of those two types of data. A study by our group that came out in 2016 used a large amount of whole genome sequencing that targeted bulk genome sequencing and targeted single cell sequencing to try to understand a clonal evolution and clonal spread in high-grade serous ovarian cancer. And so there was quite a few data modalities in this particular study, but the one that was quite useful at understanding evolution was the targeted single cell sequencing that used it, used custom echelofluidics and a custom panel is designed for each of the patients that we were studying. And we were able through using that data to be able to resolve fully resolve clonal genotypes and be able to understand which mutations were concomitant in the same cell populations and then study those cell populations more accurately as they, as we observed them in different locations within the peritoneal cavity. And what we found was was that there was polyclonal seeding in some instances, so multiple clones seeding the same metastatic site within the peritoneum. And we also found examples where mutation loss through copy number chain was was driving diversity in genomic diversity in some of the tumors. Much more recently, this paper that I'm showing a figure from here is the DLP paper. And I really like this particular result from a fine needle aspirate that we did, that we applied to which we applied DLP. We were able to extract 220 cells from like a very, very limited sample, so limited that it's possible that it wouldn't have been feasible to apply whole genome sequencing or other sequencing techniques to this amount of DNA. But using DLP, we were able to isolate for a small number of cells realistically, 220 cells from the sample, and then sequence them and then obtain very high quality copy number that allowed us to identify clonal and subclonal gene amplifications. In the same study, we looked at rare or events that happened in rare populations or in individual cells, where we see copy number changes affecting individual chromosomes or spread throughout the genome affecting many chromosomes, even I would say sort of catastrophically affecting most of the genome. And these, these are likely the result of errors during mitosis or misaggregation that happens sporadically and contribute to some extent to a small amount of heterogeneity and then maybe provide a reservoir for which selection can happen if, if by chance one of these mitotic errors happens to lead or amplify a chromosome that's has added fitness advantage. On the right here we're showing that for these top two data sets gains are more prevalent. And this is also associated with the fact that the top two are deployed. The bottom is the bottom right is tetraploid and in that sample we saw a prevalence of losses over gains. Even more recently and I had my notes to describe the number of cells that were sequenced as part of each of these studies but I forgot to mention them. The first few sets of studies that I described from Nick Damon's group and from other groups run in the hundreds, 200s. I think our nature genetic study that on high grade serious cancer was less than 1000. The DLP paper was on 50,000 and this recent paper by Nick Damon's group is on 10,000 cells so you can see that the advances in the technology that this type of an out of type of data generation is becoming much more scalable. So in this paper. This is again on breast tumor evolution. They sequenced I believe eight TNBCs and reconstructed phylogenies. They investigated the copy number structure of the of these tumors using a new method that is very similar to DLP. And they were from this data they were and so in silico simulations. They studied their model from punctuated evolution and clonal stasis which is the first model that they proposed back in 2011 to now a model in which there is some ongoing CNA evolution and some transient instability, preceded by so there's a punctuated burst that generates a lot of heterogeneity there's some transient instability that generates a small amount of additional heterogeneity and subclonal structure, and then there's ongoing CNA evolution but at a lower rate and I think the key sort of question they're trying to answer here is. Is there a mismatch between the amount of heterogeneity we see when we look at individual cells versus the amount of heterogeneity we would expect. Historically, based on a reconstruction of the evolutionary history. Is there a mismatch between essentially the historical mutation rate and the present day mutation rate contemporary mutation rate. I think this is the last few slides will finish on studies that use single cell SNB sequencing and this is a study that uses mission bio. The studies here are really starting to increase this is this is a study of 735,000 cells from 123 AML patients so huge study. They used two different types of gene driver gene panels. They investigate ML and try to understand several different aspects of the evolution of ML in this slide I'm showing mutual exclusivity between the drivers in individual patients of each of these. Heat maps and then on plots on the right are one patient at the top and one patient at the bottom.  In fact, it seems that no cells harbor the same to two or more of the same mutation. So there's a striking amount of mutual exclusivity and independent evolution of. Different drivers and presumably clonal competition between these different cell populations that are East each harboring their own driver mutations. From that data, they were able to look deeply at what happened to the different populations under under pressure from. Chemotherapeutic treatment and they found a number of different signals for instance for clonal selection here we have a large bottlenecked due to the imposition of this treatment, followed by resurgence of new populations. That are able to evade the treatment. And then in contrast we have. As far as as as failure of treatment to reduce the burden of the disease. And a significant amount of clonal clonal competition between these different populations with distinct drivers. And the last study I'll talk about is one that we recently published, in which we applied DLP to a serially, serially passage triple negative breast cancer. You can see some of the data that was generated here. I think we had 130,000 cells in this study. And this is probably about 10,000 that were in this particular data set alone, from which we constructed a phylogenetic tree that you can see on the top right and then tracked over different courses of treatment and without treatment. And how the clonal populations and which clonal populations to precedence within these xenografts and you can see that different distinct populations have higher clonal fractions at the end of this time series, but different between untreated on the left and treated on the right, as shown by these clonal fractions at the top and then these fitness. coefficients on the bottom. Oh, I didn't mean to include that slide I'll go to the next one. So challenges and future developments I think one challenge and also something that's a definite positive as I'm pretty sure that data generation will only continue to increase as we have methods that do single cell sequencing in that are more routine and more scalable. And as as sequencing becomes cheaper, it will allow us to increase the coverage per cell with the same cost and also increase the utility of this data, eventually reaching a place where we can have more accuracy for say things like SMVs in perhaps with low coverage sequencing data. Yet there's many challenges I would say this is just definitely a very short list that I've jotted down here. We're in sizes right now at the coverage levels that we have in single cell data copy number with the accuracy that is possible with some of the the assays that have been optimized for SMVs or copy number alone. So that means change at some point also. And finally I think there's quite a bit of work to do yet to be done on phylogenetic models of copy number and single cells as you as I've shown in the in these slides. There's only two methods of significance right now and they're both unpublished. So I think in the future there'll be a lot of computational development on the side.