Okay. This evening we have three counselors attending remotely, therefore let the record reflect that counselors Darcy Dumont, Evan Ross and Andy Steinberg are attending remotely via speakerphone for the meeting on September 17, 2019 at 5.30 p.m. because it would be unreasonably difficult, which is permissible under 940 CMR 2900 for them to be here in person. So Darcy, can you hear me? I'm sorry. Darcy, can you hear me? Darcy, can you hear me? We could just drill it in. Hello. Darcy, can you hear me? Thank you. Can the other counselors hear Darcy? Okay. Evan, can you hear me? Evan, can you hear me? Evan, this is Lynn. Can you hear me? You have to be quiet, please, so I can hear Evan. Evan, can you hear me? Okay. You have to be very quiet, but if you are, can you hear Evan? Can you make yourself louder? Can I assume Darcy is still on the line? Yes, I'm still here. Thank you. Okay. Andy, can you hear me? Yes, I can. Can other people hear Andy? Yes. Thank you. Evan, we're going to try again. Evan, can you hear me? And can you hear Evan? Sort of. Okay. All he has to do is be able to do a roll call. Okay. So we've tested that out and let the record reflect that Councillor Damont Ross and Steinberg's attendance via speakerphone can be heard by all present at the meeting. All votes taken during the meeting with a remote participation shall be by roll call vote. Therefore, at all votes, the town clerk, I'm sorry, the clerk of the council will ask for the individual votes. If technical difficulties arise as a result of utilizing remote participation, I will have to suspend discussion while reasonable efforts are made to correct the problem. However, I just want to warn you that given the time limited nature of this meeting, we won't spend a lot of time trying to do that. If remote participation is disconnected, that fact and time, it shall be recorded and the remote councillors when you speak should state your name. And meantime, our technical assistants who are back here will indicate that a councilor participating remotely wishes to speak. Evan, basically that means you're going to have to just speak up and say, I'd like to speak since I understand you're on a cell phone. Okay. Seeing that we have a quorum in the room, which is important, we therefore can convene this meeting and I call to order on September 17, 2019 at 5.30 p.m. We have one action item on the agenda. It is a motion that is already on the floor and has been seconded. That motion is to move that the sum total of $90,000 be appropriated for purchase of a school bus and to meet such appropriation and to meet such appropriation transfer $90,000 from repurposed prior capital appropriations. The motion has been made and seconded. Is there any comment at this time? Oh, Mandy Jo. I moved to postpone to September 23, 2019 at 9 p.m. Is there a second? Could you just repeat exactly when you're postponing to? I didn't hear it. September 23, 2019, so next Monday at 9 p.m. 900. We have several other items on the agenda that are known. We have two forums. We have two and then we have a lengthy presentation on parking. So that's our answer. I will second the motion. Okay. The motion has been made and seconded. Does anybody like to speak to the motion? Alyssa? I'm totally lost us where we're at. I don't believe a postpone, if it's the postpone motion that was made last time, doesn't need a second. That's a completely different kind of motion. So I'm just trying to be clear on what we're doing tonight and what we're doing on the 23rd. And if we're not doing this item tonight with an official nuclear postponement like we had last time, then I don't believe we can take public comment on the item either because discussion is over and I don't know why we would take public comment on something we're not allowed to discuss. I wasn't asking for public comment. I was asking for councillors comments. And my understanding is that a motion can be made to postpone to a date certain. And that's the motion that's been made. The motion was made to postpone this vote until September 23rd at 9 o'clock. Right? Yes, please. Thank you. I'm making this motion because when Councillor Dumont invoked her right to postpone, the charter required that it be heard at the next council meeting, which happened because of our quirk and scheduling to not be a regular council meeting. It is my preference that votes like this be heard at regular council meetings. And if we had not had two special meetings scheduled between last meeting when she invoked her right and this meeting, that's a September 23rd is when it would have been heard. In addition, the person who invoked her right, Councillor Dumont, is not here in person tonight and all of us know participating remotely can be very hard to actually participate. We have a town council rule that when a person, when a councilor sponsors a motion or sponsors a measure, that if they cannot be at the meeting, that we postpone the discussion of that measure until a meeting they can be postponed, they can be at. And I realize she's attending remotely here and that she is not a sponsor, but she did invoke her right and I think it would be courteous for us to postpone this discussion until a time at which she can be present. A slightly further delay is not going to affect the vote and if it passes the purchase of a bus, but it would also allow time for Councillor Dumont to gather the information that she was seeking on the motion to refer that failed last week, a little more time to be able to be present here and discuss that information as it relates to the motion on the main motion on the floor. Lastly, there is a secondary right to postpone in the charter that if four councillors invoke that right tonight, the meeting cannot take place for at least five days. It has to be under the charter postponed for not less than five days. If that right is invoked and if that happens five days under the charter is not, is after the 23rd. And so doing this this way would allow us to hear the main motion quicker than if that right happens to be invoked tonight. Sean, Councillor Dumont, please speak. Yes, we can. I'd like to say that what Mandy Jo is requesting us is very simple. As the maker of the motion to postpone, I would just like to be present for this, what I think is an important discussion. It would be very simple just to postpone it to the 23rd. I'd like to do that for, I would do that for any of you. I really feel like collegiality is a value we honor as well as concern for the climate. I move to postpone not to be an obstructionist, but because I actually really feel deeply about the urgency to act on climate change immediately.  So I would hope that you give me that. I can't effectively defend my motion while participating remotely. The motion's been made. I feel like simply delaying to the 23rd would allow us to get some more really valuable information about the issues that could affect the vote and result in the whole council feeling better about the outcome. So I hope my colleagues will respectfully vote for this just simple delay of a few days until the 23rd. Thank you, Darcy. The motion has been made and seconded to postpone to a date certain that date certain being September 23rd at nine o'clock p.m. Are there additional comments? Alyssa? So I appreciate the technical explanation that Mandy Jo offered. That's very clear and it makes eminent sense to me and I don't think there's any argument here about that. My concern remains normally we don't take public comment after we vote and so will we allow for a brief period of public comment before we vote on the motion to postpone? Since the motion is to purchase the school bus or to postpone if people would like to comment about those two issues they can but this is not a motion about whether or not we're going to invoke any kind of climate change rules or anything else. It's only about purchasing a bus or postponing a meeting. I just want to be clear what what the motion is and what the discussion is. Yes. I'm sorry of all three. Okay. We only have 10 minutes left of this meeting. Are there other comments from the people here? I'm going to just continue to let them try to reconnect. Are there other comments from people here? I have a question. Dorothy? Last meeting Darcy wanted to make a motion to send this to the Finance Committee to do the research on climate change. Has that motion been withdrawn or not? That motion was defeated. Okay. And so I assume if a motion is made and defeated does it get brought up again? I'm just wondering if that's going to be brought up at the meeting on the 23rd and we have an emergency request for a school bus? I have not been informed one way or another that it would be brought up on the 23rd. Pat? Dorothy presented a series of questions that I thought were good questions. Would we have some answers to those by the 23rd? Thank you. The Council voted 8 to 3 not to ask those questions. Kathy? I am going to support this, the motion that Maddie has made to postpone it, but I wanted to know whether we could get an answer to one question I have right now, which is, is it possible to rent a school bus? And I just, and I don't need the answer to that now, but if we postpone it, because what I thought is if we could rent it, we could buy time to know whether there's a grant to buy the other school bus. And I have no way of knowing if we can rent. And Lynn, you're the one who mentioned rental might be a possibility. And I see that some of our school buses now are contracted. We don't own them all. So I'm assuming it's possible or someone would loan. So I just would like that answer next week if we could get it by the 23rd. Mr. Bachmann. I can look in if that's what the Council would like. You'll look in. For those, are you back on? Yes. Darcy, you're back on? Okay. I'm hearing Darcy, but is Andy back in? Looks like he's not. There we go. Okay. And is Evan back in? Yes. Okay. We have all three back connected. Steve. So I'm like, I'm going to vote no on the motion because I think we should deal with this issue tonight. 10 of us went through a great big deal of trouble to be here on time. I think it's courteous to us to deal with this now. We discussed this date, you know, at the last Council meeting, and it seemed to be, you know, at that time there were no objections to this being the date. Another option would have been a special town. I mean, essentially this is a special town Council meeting. That simply takes the call at the call of the President or the call of any three more members. So I love the three people that are not here, but 10 of us are here. That's way more than a forum. I think that we have the agency to deal with this issue tonight. Are there any other comments to us, Shalini? So this was a question for Paul. Question for Paul. What are the consequences? I mean, how, when we say this is an emergency, what does that mean? Like, how soon does a decision need to be made and what happens if we don't make a decision within a week or two weeks? What are the consequences? And then I have the same question as Kathy. Can we rent? And while we figure out all of these things. Sean, thank you. And we'll come back to your questions. Okay. Andy, go ahead. So I have a couple of things. One is that my recollection of the last meeting is that Mark Darcy made a motion that we voted on that motion. And then she made her rightful request to postpone on the main motion, which was the motion to adopt the order as presented. And so I wanted to make sure that I have my understanding correct. But also speaking as a matter of courtesy, those of us who are participating remotely, it was not an easy thing for us to do. I'm out in Colorado and visiting friends. I'm on the edge of Rocky Mountain National Park and I abandoned my plans for what I would have done today because I wanted to be a part of this meeting. It was important to me to be there. But I think there's a courtesy to those of us who put ourselves out to participate remotely, too. Okay. And, Shalini, you had a question about whether there is a consequence by delaying the approval of this regarding the purchase of the school bus and the urgency of it. Mr. Bachman. So my understanding from the school department is that waiting one week or two weeks does not impair their ability to deliver the service. If they get much later than that, they really need the bus when the winter season comes. And they have to do field trips and the electric bus isn't available during those periods of time, really. Are there any other comments or questions? Shalini. So I pulled up the reason. We have one electric school bus in Amherst that was part of a pilot program, which included Cambridge, Concord, and Amherst. And there is actually an electric school bus pilot project evaluation which has studied the energy and costs and so forth of running this bus in Amherst. And I think there's some really relevant important data in there, which I just found that reported. I'm happy to share it with the group. If you would please advance it to Athena and myself. We will make sure it's posted. Okay. And I think. Paul. I would just like to clarify for the council that you don't have a proposal to buy an electric school bus on the table. Only the town manager can advance that. And I have not advanced that proposal. The only proposal you have is to purchase a new school bus. You can vote yes on that or you can vote no on that. Those are the options available to you right now. Or postpone the decision. So the motion on the floor is to postpone to a date certain. That date certain is September 23rd at 9 o'clock p.m. If there are any further questions. Okay. A roll call vote. Councilor Baumann. The motion, if you vote for the motion, we postpone until the 23rd at 9 o'clock. Okay. Yes. Please use your mic. Yes. Councilor Brewer. No. Councilor D'Angeles. Yes. Councilor Dumont. Yes. President Griezmer. Abstain. Councilor Haneke. Yes. Councilor Pam. No. Councilor Ross. No. Councilor Ryan. No. Councilor Shane. Yes. Councilor Schreiber. No. Councilor Steinberg. No. Councilor Swartz. Yes. That's six yeas, six and one abstention. Okay. I'm. Fails. It fails. It fails. Okay. So we are back to the original motion and the original motion is to for the sum total of $90,000 be appropriated for the purchase of a school bus and to meet such appropriation transfer $90,000 from repurposed prior capital appropriations. The motion has been made in second. Are there any further comments? Shalini. So I understand this is not regarding the electric school bus and but I would I want to share helped me think about this question of this particular one about the bus right now. And so I just wanted to share some of the thoughts that went into it. So this is based on the report of the current but it's informing my decision about what I'm going to say. And I think it's important for everyone to know that that anyway what I want to say about because this is something whether we do it now or in the future. I think this is something we all need to discuss. I totally agree with you and as president of the council have spoken with councillor DuMont about that and we agree that there will be a discussion about this at end year future meeting. Okay, but I'm a little confused. If you don't have that discussion, how do we make the decision about this particular? You either vote to purchase this bus. You vote to go ahead and do the purchase. You know allocate the money accordingly or you don't. And then the school has to either come back with a different request through the town manager or not. Okay, so that motion on the floor that we are to vote on is the motion about the 90,000 dollars. Okay, okay. And I understand there's lots of other discussion that individuals myself included would like to have about energy efficient vehicles, energy efficient equipment. Okay. Okay, I'm just going to go ahead. That's not the motion on the floor. Okay, got it. But I just wanted to anyway, I'm going to still say this that just I pulled up a lot. I spent a lot of time on the report, so I just want to share some of the results. And then the reason is that, okay, not the reason, but here's what it is, that we, what was proposed is that there are a lot of savings, but the report actually shows that the actual energy usage was higher in the electric buses because there were a lot of issues with the initial round of buses. There was lack of training. We've talked, I mean, the proposal is that the operations cost is supposed to go down, but there were a lot of breakdowns with the electric bus we had in Amherst. So all I'm saying is I'm going to share the report with everybody, but I think the report really highlights the importance that if you're going to go ahead with electric buses, which we should be planning for in the future, we do need to really pay attention to the pilot program and what they found because that talks about training the bus drivers, it talks about having the right kind of equipment, it talks about a lot of different aspects that I will share. So that's my reasoning for wanting to wait and have the ECAC and other people look out for grants and also look at all these different aspects of what goes into making this successful and do it in a thoughtful way. So for right now, that's why I'm leaning towards saying yes for what we have right now. We need to go ahead with what we have, but in the future, these are the things we should look at seriously. Thank you. Are there other comments? George? These are all good questions and need to be addressed, but winter is coming, we need a bus. And the motion before us is to either purchase this bus or to send it back and ask for another request. I don't have any confidence that these very good but complex questions, including what Shalini has mentioned, can be answered by September 23rd or even by October 23rd. These questions need to be looked at. We need to have before us some kind of plan that makes sense going forward. We're not going to have that in the next couple of weeks or even next month, I don't think. And so my feeling is that we simply need to act and I will vote yes on this motion. Are there? Yes. Darcy, please. We don't have information. We've now started. We have now run into the time for the school committee to have their meeting. Is anybody connected remotely or have they all gone down? I'm sorry. Yes, please, Dorothy. I have a question to town manager Backelman. And is there a procurement board or people who work for the town of Amherst? We have a procurement officer, but every department is aware of the laws of the common wealth which governs our procurement process. Because to my mind, it seems that we need a, I can't use the word working group, but an ad hoc bylaw committee that of some people from the town who are involved in buying things and the school and some of the people from the climate action committee to do the research to talk about how we would go forward in the future. I am for voting for this bus, which has been an emergency request. But I don't want to sit here trying to make scientific decisions on this. I think that a group could go into a report that would guide our future actions. I'm just going to be very clear. We're going to have to stick to the public issue. The issue is, are we buying the bus? The issue about future vehicles, the issue about future equipment, furnaces, et cetera, buildings. We have the ECAC committee. Dorothy and I have most recently talked about that and what will be included in not only their goals, but in their implementation plan. And this will clearly be among those things. So are we back and connected? Okay. Dorothy, you wanted to speak and then it failed. You heard before, but I was just saying that I believe that we need, we still need to find out a lot more about the availability of grant money, of which there is a huge amount in Massachusetts, which would make a lot of sense to take advantage of. But we need to find out more about that as a council, what's available and so forth. And we also need to find out what, if there's a possibility of a temporary solution, like Kathy was mentioning, of using our contracted bus service for a short period of time, pending, applying for a grant. So, and the other question that I had is, are we having public comment? I'm going to just be very clear. This is a special meeting of the council, although we posted public comment we are not required to. I also want to be completely respectful of the school committee who has scheduled a meeting for 10 minutes ago, which is one of the reasons we're all here. This is not the last time that we will be discussing energy and clean energy policy before the town of Amherst. It is one of the first times, but it sure ain't the last. And I do feel we need to bring the meeting to a reasonable closure. Steve? I'd be happy to make a motion if that moves us along. You just call the question? Well, has there been a... We have a motion on the table. I move to call the question. Okay. The question's been called. There's a roll call vote. The motion that's been made and seconded is to move the sum total of $90,000 be appropriated for the purchases of a school bus and to meet such appropriation transfer $90,000 from repurposed prior capital appropriations. The motion's been made and seconded. The question's been called. There'll be a roll call vote. Unclosing debate. Was there a second? Second. Thank you. Thank you. Councillor Brewer? Yes. Councillor D'Angeles? Yes. Councillor Dumont? No. President Griezmer? Yes. Councillor Haneke? Yes. Councillor Pam? Yes. Councillor Ross? Yes. Councillor Ryan? Yes. Councillor Shane? Abstain. Councillor Schreiber? Yes. Yes. Councillor Steinberg? Yes. Councillor Schwartz? Yes. Councillor Baumeil? Yes. 1111. 11-4, one against, one abstain. The motion has been... The vote's been called. We'll now move to immediately to the vote on the original motion and that will be a roll call vote. Councillor D'Angeles? No. Councillor Dumont? We are voting on the original motion which is to have the town take some total of $90,000 be appropriated for the purchase of a school bus and to meet such appropriation transfer $90,000 for repurpose from repurposed prior capital appropriations. But that's what we just voted on. We voted no. We voted to vote. When somebody calls the question you have to vote to close debate. So we have... Would you please start over again? Councillor D'Angeles? No. Councillor Dumont? President Griezmer? Yes. Councillor Haneke? Yes. Councillor Pam? Yes. Councillor Ross? Yes. Councillor Ryan? Yes. Councillor Shane? Abstain. Councillor Schreiber? Yes. Councillor Steinberg? Yes. Councillor Schwartz? Yes. Councillor Ball-Millen? Yes. Councillor Breuer? Yes. 10. 10-2-1. Thank you. It's 10-4-2-0 and one abstain. I move to adjourn. Is there a second? Second. Do we have... We should do a roll call vote on that. Please quickly. Dumont? Griezmer? Yes. Haneke? Yes. Pam? Yes. Ross? I think we have to assume absent at this point. Continue. Ryan? Yes. Shane? Yes. Schreiber? Yes. Steinberg? Yes. Schwartz? Yes. Ball-Millen? Yes. Breuer? Yes. D'Angeles? Yes. Thank you. The meeting is adjourned.


In this presentation, we will discuss adjustments to the standard unqualified audit report. In other words, these are going to be items that aren't going to pull us away from the unqualified opinion. We still got the unqualified opinion, but there's going to be adjustments to that unqualified opinion. And therefore the report will be altered or adjusted, although not to a qualified report, still unqualified, still the highest report, the unqualified standard report, although the format of it will be adjusted for some of these items. Just a word from our sponsor. Well, actually these are just items that we picked from the YouTube Shopping Affiliate Program, but that's actually good for you, because these aren't things that were just given to us from some large corporation which we don't even use in exchange for us selling them to you. These are things that we actually researched, purchased, and used ourselves. Bayer Dynamic? Not sure if I said that right. But this is the DT770 Pro 250 OHM Studio Reference Closed Back Headphones. I wear headphones basically every day for a large part of the day. They are important to me, therefore I've gone through many different kinds of headphones. I've had these for some time and they've worked quite well. They fit over my ears, but I'm still able to put my glasses on under the headphones. The headphones not pinching too tight on the glasses to give me a headache, which is nice. The quality of the patins is good and it has lasted for some time. I've had these for some time now and they haven't gotten all torn up on me or anything like that. I also like that I have a cord when I'm doing my recordings as opposed to a USB centered headphone because that frees up a USB port and I find the USB headphones to be less reliable. They come with an audio jack that looks like this, which is useful for me because that plugs into my audio interface, however if you want to use the headphones for some other purpose I believe it's fairly easy to get a converter to other types of audio jacks. If you would like a commercial free experience, consider subscribing to our website at accountinginstruction.com or accountinginstruction.thinkific.com where we have many different courses. You can purchase one at a time or have a subscription model given you access to all of the courses. Courses which are well organized have other resources like Excel files and PDF files to download and no commercials. Note that as we go through these, some of these, such as the going concern, if we're very concerned about a going concern issue, we may have a qualification. We may qualify the report in some type of way, so it depends on the degree as to the going concern issue as to whether we keep a unqualified report with a going concern in the language of the report or if we add some type of qualification to it giving it. Notice that going concern issue is a concern that the company is going to keep on going in the long term. If we have a concern that the company is going to go out of business in the short term, then that's going to be a going concern issue, something that we want to adjust and make people aware of. Based on part of the report of another auditor. So that could be the case that we did some audit for some of the of the audit and then some of the audit might have been done somewhere else. For example, if we had, if we could not do an audit for part of the company that was done in another location, such as another country, because we don't have an office there, then it's possible that someone else, another auditor could do the audit of that particular component and help us out to complete the audit in that way. By doing that, then of course we are independent on an outside CPA firm and that needs to be stated within the financials, within the report. Financial statements are not comparable. So if there's a comparability problem with the financial statement that should be noted where ICFR not required to be audited and auditor does not audit ICFR, that's going to be the internal control. So normally they're required to be audited. If there's some kind of issue where they're not required to be audited, then that would be a modification to the report as well. Now we're going to consider a situation for working for an opinion based in part on the report of another auditor. So we have a modification here. We didn't complete the entire audit. We had some component of the audit that needed to be done by another auditor in order for us to get that audit completed. That's going to be the modification. It is possible that parts of the audit are completed by a separate public accounting firm. Obviously if that's the case, then we can't give the full opinion on that part that wasn't done by us. And therefore we're going to have to have that modification. The auditor might express an opinion without referring to the work on the other auditors in the audit report. So we have kind of a choice here. We could say this was done by the audit of another firm. If we don't reference the other firm, in that case, the auditor accepts full responsibility for the work of the other auditor. So if we just basically say the other auditor is going to audit some component and we're not going to include them in our reports, then we are accepting full responsibility. Now typically if someone other auditor was to conduct some significant component of the audit, we would typically mention that in the audit report. So that would be the other option, probably the more common option. When the principal auditor does reference the other auditors, the principal auditor is sharing responsibility for the audit report with the other auditors. And that's typically what you would think would basically happen because you want to share responsibility for the components that you might be responsible for and limit the responsibility. However, if you take full responsibility for it, then you have more liability. Maybe you can get paid more in that way. Maybe you can bill more. But I would think typically the situation would be the situation where you would reference that. That seems to be more given full disclosure over what exactly happened as well. So here's going to be an example, standard title and address. So we'd have the same kind of standard title and address. Opinion section, it's going to be the same as the standard report. So same as the standard unqualified except for the addition of and the report of the other auditors. So we're obviously just going to include that within the opinion because it's not just our opinion, but also of the other auditors with regards to these areas and the sentence stating auditors opinion. So in this sentence, in our opinion, based on our audit and the report of the other auditors. So that's going to be the new component here. The consolidated financial statements are presented fairly in all material respects and so on and so forth. Additional paragraph would look something like this. No, just this is just going to be an example of the type of language that might be included in the additional paragraph. And of course, we'll be dependent on the circumstances. So we did not audit the financial statements of the subsidiary company. So we're going to say that, you know, in this case, we're going to say there was a subsidiary company. We didn't audit them a wholly owned subsidiary which statements reflect total assets and revenue constituting 20% and 27% respectively of the related consolidated totals. Those statements were audited by other auditors whose report has been furnished to us. And our opinion insofar as it relates to the amount included for the company is based solely on the report of the other auditor. So notice this paragraph is basically stating exactly what is going on there gives basically approximations about what component was not audited and makes that disclosure clear that it wasn't audited by us but that we're completely dependent on the audit work of another firm. Basis for opinion section, same as the standard. So the basis for the opinion section would be the same as the standard unqualified except for the addition of and the report of the other auditors in the conclusion sentence. We believe that our audit and the report of the other auditor provide a reasonable basis for our opinion. So obviously we're gonna add our report and the report by the other auditors. Here's an example of an unqualified report with an explanatory paragraph for going concern problems. So again, remember it's still unqualified. We haven't changed it from unqualified the highest standard report to a qualified report but we are modifying it for this basic going concern problem. So this is just an example of the type of language which might be included. Explanatory paragraph like the one below is added after the opinion paragraph in the opinion section of the report. A reference to this explanatory paragraph is included in the critical audit matters section. So substantial doubt about going concern assumption. The company financial statements have been prepared assuming that the company will continue as a going concern as discussed in note eight in the financial statements. The company has suffered reoccurring losses from operations and has a net capital deficiency that raises substantial doubt about its ability to continue as a going concern. Management plans in regards to these issues are too described in note eight. The financial statements do not include any adjustments that might result from the outcome of this uncertainty. So that we're gonna state basically remember that going concerns is a concern that the entity will not basically be able to stay in a long-term within the long-term in a business setting to stay in business for a long term. So we have the explanatory or added or adjustment to the unqualified report in this case. And of course we'll reference to the note which will give more detail about what the issue is and what management's intentions about how to resolve it will be. Sometimes the auditor will want to emphasize an issue and auditor may want to draw special attention to a particular issue. The auditor may choose to include an emphasis paragraph in the auditor's report. So if there's some kind of issue that the auditor thinks is important then that might be another format or way or thing or item that the auditor will adjust the standard unqualified report for.


In this video, we want to explore categorization and classification, particularly in the context of science. The definitions for categorization and classification, depending on what dictionary you use, you might have slightly different wordings, but they basically overlap. It's a process or an act of distributing or placing individual things into categories or groups of the same type of thing.  So that's what we're going to focus in here a little bit. Now I'm using this as one of my college classes. So let's look at our first example as college students. We could classify or categorize college students in a lot of different ways. And these four are just a few of the examples, not all of the possible ones. So let's look at residency status. That's actually a pretty easy one to implement. Students are either in-state, out-of-state, or international students. And again, this is the way we classify them in the United States. And it's useful for tuition categories. And that's partly based on a rationalization, based on taxes paid. So determining the residency status depends on home address. It's useful for tuition categories, but really isn't used for anything outside of that tuition classification. Major and class are a lot more useful when you're talking about college students. Because all the students in a particular major are going to have a common set of course requirements. They'll have to study the same sorts of things. And there's some classes that all of them will have to take. But students from other majors don't take. Class standing, and by that I mean the freshman, sophomore, junior, is an indication of how far you've progressed in that major. So how much have you probably studied already related to that major? Now there are some complications with this. For example, you could have a student who's a double major. So which category do you put them into? They're going to have a common set of courses with each of those different majors. And what about a student who changes their major? If you change your major, you might be classified as a senior based on the total number of courses you've taken. But you might just be starting out in the particular courses needed for your new major. So separating students by major and class standing is definitely useful for determining their course requirements and getting an estimate for how far they've progressed. But it also has some complications and doesn't fully tell you what all they've studied and learned. Age is another thing that we often use for students, and we could classify them based on year, 18, 19, 20, etc. Difficulty with that is that the category keeps changing. You're 18 one year, but you're 19 the next year. And it doesn't change for everybody at the same time. It changes on their birthday. The other thing is that there's not a lot of difference between a student who's 18 about to turn 19 and a student who's just turned 19. They'd be in different categories, but really not that much difference between them. If you use bigger bins, you might be able to get a little bit of those difficulties to put aside. You could say the students who are in their 20s, the students who are in their 30s, etc. And you would expect that students in their 20s are going to be different from students in their 50s. Each group is going to kind of have some things that are really sort of common to that group, but different from the other groups.  So there's going to be exceptions. And again, there's not a lot of difference between a 29 year old and a 30 year old. So exactly where you put the break is a little bit arbitrary. So let's talk about some other examples. So common things you're going to see in terms of classifications and categorizations. If you type into a search engine something about classifications and biology, it's going to all be about the different classifications of living organisms into all the different kingdoms and phylums and their very formalized structure for classifying and categorizing living things. We've already seen it in my course here on Earth Science where we separated things out into the four spheres, biosphere, hydrosphere, atmosphere, and geosphere. And again, we had some difficulties with that with our clouds part of the atmosphere or they part of the hydrosphere because it's got water in the clouds, but yet it's up in the sky. Astronomy is our focus right now for this week and looking at how do we classify the planets or how do we classify other small bodies in our solar system. So you can imagine that again, as with any sort of categorization, there's going to be some benefits and there's going to be some difficulties. The benefits always relate back to being able to use the common characteristics as a simplified way of examining all the objects in that category. So you don't have to learn the density of each planet. You don't have to learn the size of each planet. If you know what general category they're in, you have a little bit of an idea of which ones are bigger or smaller. But there's always going to be difficulties as well. Sometimes it's arbitrary in terms of where you put a boundary between two different ones and then you might discover some new object that doesn't quite fit either of the existing categories and you have to decide you expand the category or do you create a new category, which is what they did with Pluto when they started discovering other dwarf planets. So while this video is about categorization and classification in general, I do want you to think about how that applies to our topic for this week with the solar system. But this video should also be useful for students thinking about classification and categorization in any aspect of life.


So I'm Jared Simpson, I'm a principal investigator here at OICR, I work just upstairs. I work on algorithms and data structures for sequence analysis. So I design the types of methods that we're going to hear about today for mapping sequence reads to reference genomes, doing de novo assembly, that sort of thing. So this module is mapping and genome rearrangements. So I'll talk in two parts. I'll first give a short lecture about what we talk about, where we talk about mapping sequence reads to a reference genome. And then we'll do a tutorial, then we'll have a coffee break, then I'll talk about what genome rearrangements are, how we can find genome rearrangements, and then we'll do a tutorial on that as well. So please just ask questions as they come up. If anything's not clear, just feel free to put your hand up. And ask me to clarify something, or go over something in more detail. So the objectives of this module are really to understand what we talk about when we talk about mapping reads to a reference genome. So as you know, this is the cancer bind, informatics course, or cancer genomics course. So we want to, often our goal is to sequence a cancer and understand what mutations are present in that cancer genome. And the way that we do that, well, first we'll sequence an individual's tumor, we'll sequence the individual's normal genome from, say, a blood sample, we'll then compare all of those reads to the reference genome to see where mutations are. And mapping reads to the reference genome, which just means taking a read and finding where it might have come from in the human reference genome, is the first step of all of those processes. So I'm going to talk about what we're talking about, mapping reads in detail, also I want you to understand different file formats that we work with in high throughput sequencing. So I'll talk about the FASTQ format, the SAM and BAM formats, which are the common ways of working with sequence data. Also throughout this lecture I'll talk about common terminology that we use to describe alignments like base quality scores, mapping quality scores, paired end reads, that sort of thing. And then later on we'll find out how to find genome rearrangements using read pairs, and you'll have the chance to run a mapper, BWA, and rearrangement caller during the tutorials. So I'll talk a little bit about sequencing platforms to start. So I think Francis talked about this a little bit yesterday in the introduction lecture. So, John did? Okay, thanks. So since about 10 years we've had high throughput sequencers, which can just produce massive volumes of data, and this is just a figure of different sequencing platforms in the time that they run for and the amount of data you get per run. And up here at sort of the top is the Illumina high-seq platform, which is by far the most common way of sequencing human genomes these days, and I'm going to really focus on just data from that platform, the Illumina platform, but just understand and understand from John's talk yesterday that there are alternative sequencing platforms that give different types of data, like much longer reads from the Pac-Bio or the Oxford Nanopore system, or you have an iron-torn system which produces somewhat longer reads than Illumina, but at different throughput. But because Illumina is the dominant player in sequencing, we'll be focusing on that here today. Okay, so Illumina sequencing works cycle by cycle. So you start with a single strand of DNA fixed to a slide, and then labeled bases are added one by one, and as the base is added, a camera records a fluorescent signal from each base, and each one of the four bases is colored with a unique color, and the sequencing process just records which color of base, so I need to be on both sides, which color of base is added to each set data? Will that record too? I see. Okay, fantastic. Yeah, so here in the first step of the sequencer, this T was added, and then it gave off a blue fluorescent signal, and that was measured by the camera over here. Then the sequencing reaction proceeds to the next base. It was a G, and it was recorded here as a green color, and then it was a C, which was recorded as orange, then blue, purple, orange again. So the sequencer is just adding bases, base by base, and recording the color that was incorporated into the growing strand of DNA at each step. Now, Illumina Software translates these images, which are these incredibly large images where each dot is an individual sequencing read, it translates that by looking at color by color to decode that into a nucleotide sequence. So to look at the stack of images, one taken for each cycle of the sequencing process, and it will change that into TTT, GCA, and so on for one of these dots. So we've got a file, and this is a fast Q file, which records a sequence of every one of your reads, and then an estimation of whether the base at that position is correct or not, and that's called a quality score, and I'll talk about that in just a few minutes. Now, the sequencing is very reliable. You get very high accuracy data from Illumina Sequencers, but various things can go wrong that cause the machine to record the wrong base certain times, and when you have these strands of DNA fixed through a microscope slide, they come in a cluster of thousands of molecules all with the same sequence, and this cycle by cycle sequencing reaction is happening in the same order for every one of those molecules in the cluster, but sometimes molecules can lag behind where corporations didn't happen, or sometimes they can jump ahead where too many bases have been incorporated, so they get out of sync, and they call that phasing when some of the strands in this reaction are lagging behind or pre-phasing when some of the strands are too far ahead, and the problem with this is that it then causes a mixed signal. Instead of having a pure color coming out of each one, like for example, we record a gray one here, now we've got one signal that's red at this step and three that are gray, so now the base color, when it's trying to translate these images back to base calls, it needs to try to deconvolve the fact that some of those strands might be out of sync, and that's the source of errors within Illumina data, it's just that the signal becomes essentially diluted by strands getting out of sync during the sequencing process. Now the Illumina software can measure this and estimate whether bases are reliable or not, and the way that it indicates that to the user is by outputting what are called base quality scores, and this is just a log-scaled probability that the sequencer estimates the base call is incorrect, so if you have a quality score of 10, it thinks that there's a 10% chance that the base that it out-portons out of FastQ file is incorrect. Now if you have a quality score of 30, it's about one in a thousand chance that it's incorrect, and most of the data will fall into this Q30 to Q40 range, which is quite reliable. Often, if you see what reads at how these very low quality scores, less than 10, you might want to just discard that data, and most software that uses the sequencing reads, when the quality scores are very low, they'll just ignore those reads. But quite often, the quality scores are above 30, and the data is quite reliable. Now, different sequencing platforms have different error profiles, so the Illumina sequencer has a fairly low error rate, usually on average, about one in 200 bases, and they're mainly substitution errors, where the sequencer has said maybe there's a T in this position when there's actually a C. The 454 and the Ionetorrent sequencers are mainly insertions and deletions in homopolymer runs, and the Pac-Bio sequencer, one of these long-read sequencers has a much higher error rate of about 15%, and the errors are a mixture of insertions, deletions, and substitutions. And when you're working with your sequencing data, it's very good to be aware of these different error models or different error profiles for the platforms that you're working with, because when you're, say, looking at your read the line to a reference genome in IGV, you want to be aware of whether the differences that you're seeing with respect to the reference are due to errors or if they're due to true mutations or, say, SNPs. Sure, I should have brought one. It's about the size of a USB stick, and I've got one in my office. But the extra nanopore, it works by feeding a single strand of DNA through a nanopore, and it measures the blockage of electrical current flow that's going across the pore. And from that, it can try to predict what sequence which was in the pore at the time of the measurements were made. And because the current measurements are so small, they're on the Pico ramp scale, it's very difficult to convolve what sequence passed through the pore. So it has a fairly high error rate, roughly equivalent to the Pac-Bio of about 80 to 85%. It's still very early in the development of oxygen nanopores sequencer, so it's gone up from about 70%, sorry, 70% accuracy, 30% error rate was about six months ago, and now current data is about 15% error rate. How does the aluminum system infer the quality of scores based on the intensity of light? Really intense being light that would have a better quality score for example? Yeah, that's right. It's essentially the signal-to-noise ratio, so it's taking four pictures, one per color channel. It's got some filter on there, a particular laser that excites across some wavelength. And one of the signals will be very strong, and then the other three signals will be weaker, and that's the background, and it's essentially a ratio between the peak and all the background signal. And these artifacts you have for molecules going out of phase and not, they contribute to the background, so that's why if molecules are getting out of phase, then you have lower quality scores and errors start to creep in. How is it able to read one dot kind of the influence by all the other fluorescence around it, if it gives a concentration of spot? So they've spent a lot of time, so how do they base call off the individual dots here? Like each one of these is a sequencing reaction without being influenced by the neighboring ones around it. Is that right? Yeah, so they've spent a lot of time tweaking the chemistry of how many clusters they put in the flow cell to make sure they're well separated. And right now the latest deluminas, the high-seq X10s, actually have a patterned array where the clusters only form in certain positions, so that they're well separated from each other. What's the maximum you could actually put on an instil link That'd be a good one for John. Yeah, I think you get something like on the order of 50 million reads per lane, so 50 million clusters. Yeah, but if they do overlap and they can overlap by chance, the software will detect that and then not output reads. Okay, so here's what the Illumina Air profile looks like. Here we're looking at the error rate as a function of position within the read. So these are six different sequencing runs that I used as part of an assembly project. And you can see that at the far prime end of the read, at the zeroth base, the first base, the error rate is very low. And it continually goes up towards the end of the read, whereas if we're looking at this green line here, the error rate goes up to about two and a half percent at the last base of the read. And this is just an effect of the signal-to-noise ratio that we just talked about going up as the sequencing reaction which is signal-to-noise going down as the sequencing reaction proceeds. It's harder to measure the last base than it is the first base. Yup. This one? No, these were different runs. These were six completely separate data sets. It was all Illumina sequencing, but it was even done by different centers. It's different different institutes. It wasn't the same sequence or it wasn't the same run. Yeah, I'm wondering why some keep up so early, I mean 100 positions for people 150. Yeah, so you can tune how your read length, how long you want the sequencing reaction to go for. So this one was about 150 bases, but the common Illumina's are about 100 to 125 now. But they are more actually in the Q40 range. Yeah, yeah, yeah, especially down here at this end. On average it's Q30 to Q40. Okay, so the raw output that a user will get from an Illumina sequencer is a fast Q file. So this is just a file format that stores sequence reads and our qualities. So I'll briefly walk through the different fields in this file. So the first one is the read ID. So the sequencer will align a unique identifier to every read. There's some structured information here. The there's this first field which is HWI isn't the identifier of the sequence. And then I believe it's the fourth lane. And these numbers are the X and Y coordinates of the cluster on the machine. You don't actually need to know that information commonly. Sometimes it's good to look at which sequencer produced the data. But often you just need to use this as a unique ID. Now the slash one at the end means that this is the first read in a pair. So something I haven't talked about yet is that reads can come in a pair where you can sequence one end of a DNA molecule and then sequence the other end of it. We'll be using that in the genome rearrangement part later. But I'll just remark at this point that the slash one indicates the first read and a slash two would indicate the second read. Question? There are many definitions of pair read versus made pair. Right. Is there any difference between it or they are synonyms? Yeah. The difference is how they're prepared. So made pairs are typically much further distance apart. But I'm going to leave that because I have a slide about this later. So we'll come back to made pairs and paired ends in a little bit. Is it advisable to store images from a lemur? No. Not anymore. Is it advisable to store the images from a lumina? Used to, there was a lot of debates around this when luminous sequencing started to take off. Because people would argue that that's the raw data of the experiments and you have to store that and you have to archive it. But it's just impractical. The images are very, very large. They're terabytes per run. So basically the community decided that... Can you pre-process an image? Because it's in a jpeg file. It is, yeah. If they were stored, you could. But it's just too much data to keep around. So usually it's just to rese... If there's a problem with Iran, you just resequence it. It's cheaper to resequence than store all the images. So the second line in the FASTQ file is the actual nucleotide sequence. So if the sequence are red, that's fairly straightforward. Does that include the tag or anything like that? This should be the actual sequence you want to use. So in here, there's this ggctac in the read ID. That was the barcode that was read. Then it was stripped from the sequence. So this should be usable sequence and you shouldn't have to process it further. Yeah.  These days, because within Illumina sequencing, you don't have a really big range to choose from. You can maybe do 100 to 125. So often it's just whatever you're sequencing facility or whoever you have access to that's going to provide the data, whatever they're most comfortable generating, which is usually 100 base in this case. More generally, longer reads are better for things like structure variation. If you can get very long reads, say you're doing de novo assembly of something, then you want to think about going to Pac-Bio or Oxford Nanopore, because they give you much better genome assemblies.  The third line is again the ID. It's technically the quality ID. Oftentimes this field isn't filled in and there will just be a plus sign in the beginning of the line because this is essentially redundant information as it's the same as the first line of the file. Yeah. That's separate from the quality score. It's cool. Yeah, it's separate from the quality score. The fourth line here is the quality score, which I've got it with an arrow here. So these, I talk about quality scores numerically, say Q30 to Q40. So this is just an ASCII representation of quality scores. You can represent the quality of each base in a single character. So there's ways to convert these characters to new air quality scores online. Essentially, you just take the ASCII character code for the symbol and subtract 33 from it. Okay, so that's fast Q. I'll now talk about mapping reads. The reference genomes and the goal is simply now that we have this huge set of reads that the Illumina Sequencer gave us. We want to find out where each one of them came from. So the issues here and something that people like me who work on algorithms for sequence analysis had to deal with in the last 10 years is that the human genome is incredibly large and it's very repetitive. Over half of the genome is made up of transposons and other type of repeats. And finding where a very short 100 base pair read came from in that repetitive genome is quite challenging. So there's been a lot of work in designing data structures and algorithms that can accurately find the placement of an individual 100 base pair read on the reference genome. And of course, high throughput sequencers produce a huge amount of data and there's a lot of differences between any individual and the reference genome. For example, one in a thousand positions will be different due to SNPs than you have insertions and deletions and structural radiation. So the mapper needs to consider all of this and needs to consider differences between the reads and the reference genome. It needs to not place reads at the wrong copy of repeats and it needs to be fast enough to handle all of the data that Illumina will produce. So here's really the three criteria we look for in a mapper. It needs to be accurate. If a mapper aligns reads to the wrong place in the genome, for example, if it gets confused by the repeats, this is the main source of false positive variant calls when you go to find mutations from your sequencing data. Just the mapper putting the read in the wrong place. The mapper needs to be sensitive. It needs to account for the differences between the individual that we sequenced and the reference genome and it needs to be fast. We need to be able to do this and within a reasonable timeframe so you can finish your project and the informatics cost doesn't outweigh the cost of sequencing the genome. So there have been many, many mappers published in the last 10 years. That's probably an understatement. I think there's at least 70 at last count. Luckily, you don't need to go and pick from one of 70 to use the field of standardized around a few very popular mappers. But if you're keen to understand the differences between mappers and how they address these three different criteria, I listed on the last slide, you can look at this review paper that was in Bioinformatics a few years ago which fairly comprehensively lists the strengths and weaknesses of each one of the mapping programs. That said, we're going to be using a mapping program called BWA, particularly the BWA MEM variant of this program this is a very popular program written by Hang Lee and it's largely the standard way of mapping Illumina reads to a reference genome. So we'll just take that and run with it without having to worry too much about picking a specific mapper for the problem. Okay, so here's sort of a cartoon of what a mapper does. It's taking a single sequencing read which is depicted by this red bar at the bottom and then trying to predict where on the reference genome that read came from. So it'll do that by computing an alignment between the read and all the different places it could have come from on the reference genome, scoring them in some way and then picking the one with the best score. So in this example here, we have three different alternatives alignments. It could have aligned here with no mismatches. It could have aligned here with one mismatch or could have aligned here with two mismatches. Yeah. Later, kind of figure out if there were, say, say you're doing a cancer project and you end up with a whole bunch that have the two on the bar side you know that are missing our reference. Yeah. And so you say you end up with a whole bunch of those like does it ever go back and try to actually map it there by taking into account that that's really, you know, they can have that again and again. So, yes and no. Yeah, it does make sense. So analysis pipelines are very long. They consist of 10 to 20 steps where mapping is just essentially the first step. And when you go to find mutations, often the mutation callers, which I think you'll hear about tomorrow, they'll do realignment. So they'll take putative mutations and say, okay, now that I know there might be a mutation here, do the reads fit better to the reference, if that makes sense? Yeah. So yeah, they're essentially trying to learn the differences and then remap to them. The aligner, now we're probably going to change it. Right, yeah. In the quality of that base, we know that we might actually choose one. Yeah, exactly. And it does exactly that. In the scoring function weights it. It's essentially a probabilistic model. It'll say what's the probability of the data from given this reference location or this reference location. And those calculations always incorporate the quality scores. So yeah, they will. So how do you calculate it? Yeah, that's the next slide. Yeah. So mapping quality is essentially, it's the same scaling as the base qualities. And it's an estimate of the probability that the position that the mapper chose for that alignment is incorrect. So if we go back to this, there are three different alternatives and it will give a quality score. It will choose this one, say, because it has no mismatches. But the mapping quality will be the probability that the true alignment was this one or this one. And to calculate that, it's going to use the quality score, the base quality scores, as we just talked about, to estimate this mapping quality. So if you have a mapping quality that's Q30, about one in a thousand reads with a Q30 alignment will actually be mapped incorrectly. This concept of mapping quality is incredibly important when you're trying to find mutations because the alignments have to be correct. If there might be a very strong signal of a snip at a certain position, but if the read is mapped in the wrong place, then that's not a true snip. It's just, it's some other variant. It's just an artifact of the mapping algorithm. So often when you're going to look at mutations in IGV, IGV will color the read alignments by mapping quality. A white alignment is a Q0 mapping quality, which is essentially just a randomly chosen position, which can't be trusted at all. So you want to keep this in mind when you're looking at your alignments. Even if you're short-quakes, how does it map? Even if what it does is the highest, how does it know? Yes, so the internals of the mappers, they'll usually, they'll have a data structure, which is essentially an index of the genome. And it will use that index of the genome to look up candidate locations where the read can map. And this can usually be something that's quite simple, based on just exact matching strings between the reads and the reference genome. It will then take those candidate locations, so it might say, okay, it might align to this position of chromosome one, this position of chromosome seven, et cetera. And then it will perform a more detailed alignment using dynamic programming, which then gives you this sort of picture, the exact differences between the read and the reference genome. And then it will use a statistical model to estimate the probability that the mapping is correct or not. So what's the relationship between base quality and mapping and read quality? Yes, so the base qualities are the individual probabilities that the sequencer has called the wrong base at a position. The mapping quality is for the whole read, and it's the probability that the read was placed incorrectly on the reference genome. It was mapped incorrectly to the reference genome. Sorry? No, not directly related. They're not directly related, no. The mapping quality calculation uses base qualities when it's trying to assess whether it's a true difference or just a sequencing error. But it's not direct. Yeah. If you have paired in leads, does it take the second lead into account when trying to resolve low-quality base scores? That's an excellent question back here first. No, it's good. It's good. It makes my talk feel like it flows really well together. Yeah. And how reliable is it? That's a good question. It's better than it used to be. It used to be that low-quality scores would be underestimated and high-quality scores would be overestimated, quite badly, actually. And the Broad Institute wrote a program called Base Quality Score Recalibration, which you can guess what it does. It tries to fix it by adjusting the low-quality scores upwards and the high-quality scores downwards to make it a true signal. Nowadays, if the quality scores are good enough that often you don't have to do recalibration and you just take them on faith. Did they ever publish their categorical vision about, like, reserving indels? Yeah, so there's two types of... So there's Base Quality Recalibration and there's Indel Realignment. So GATK's Indel Realignment will take a database of known insertion and deletion positions and then realign the reads around those indels to try to improve the alignments around indels. Indels are sort of a difficult case for mapping programs because if a read aligns at the end of an insertion position, it will often just introduce mismatches rather than opening up a gap at the end of a short read. So Broaden wrote this Indel Realignment program that will fix that problem and allow it to make gaps at the end of reads. Yeah, it does look at... It looks at all the possible alignment positions. So it would say... So the mapping quality would take, say, these three mapping positions, one with no mismatches, one with one mismatch, one with two mismatch, it would calculate the probability given that this is the true alignment in that this mismatch is a sequencing error. That uses the base quality. It would also calculate the probability that these two mismatches are sequencing errors using the base quality and also probability that this had no sequencing errors. And then the mapping quality would be set as a function of those three calculations. Does that make sense? No, usually, for most regions in cancer genomes, mutations are fairly well-isolated, whether it'll be a single point mutation. And mappers are usually pretty good at accounting for a single difference or just a few differences between the reads and the reference genome. If you have a very rearranged region or something where there's been some hypermutation, you can misalignments in those regions. But for most of the mutations in cancer genome, they're on their own, and the mappers are well-able to account for that. So how does this distinguish the sequencing error from heterozygous? So that's when the quality scores come in. So sequencing errors will tend to have very low quality scores. It might be a Q5 base, and the mapper will think, well, there's a 20% chance that base is a sequencing error, whereas a heterozygous position, it might be a Q40 base. So the mapper would be very unlikely to say that that's a sequencing error rather than a true head. Some of them do. BWA doesn't. BWA is called a best mapper. It will report the best alignment for the read and a mapping quality score, but it won't report alternative hits. If we go back to this review, that this will talk about different mapping strategies. So multi-mappers will be included in there. Kassava is Kassava included in this paper. Kassava is Illumina's aligner that comes bundled with the high seek. I think it might be too recent. I think Kassava, John, do you know when Kassava was introduced? It was around a while. A while? Okay, so it might be, but we don't know. Okay, so let's go ahead and read. So paradigm reads are, as I said earlier, when you sequence both sides of a DNA fragment. So you might take a 400 base piece of DNA and then sequence the first 100 bases of it and then the last 100 bases of it. So now you've got 200 bases of sequence separated by a gap of unknown sequence of length 200. And this is actually very useful when you're trying to map reads to a reference genome. So if we go back to our example here, now we're trying to map this read pair to the reference genome where we know the sequence at the start and the sequence at the end, these black bars here. And we don't know this red sequence in the middle. Now if we just map the first half of the read to the reference genome, we might find two hits that are roughly equivalent. They both have a mismatch at this position. But because we have the additional information of the second half of the read pair, when we map it to the reference genome, it might resolve what the true mapping is. So now we've mapped the second half here with no mismatches and the second half of the second alignment with six mismatches. So it's much more likely that this is the true alignment than this is. And this again will go into the mapping quality calculation and a map like BWA would certainly choose this as being the true alignment rather than this one, which has a much poorer alignment for the second half of the pair. Okay, so BWA will write all this information into what's called a SAM or a BAM file. So this is a standardized format that was developed around the 1000 Genomes Project as a way of working with alignment data. So SAM is a tab-to-limited text file. At the bottom here is one line of SAM. And BAM contains the same information, but it's a compressed binary format of the alignment. So they're inter-convertible. You can convert from SAM to BAM and BAM to SAM. And this is really an important format to understand when working with sequencing data. So I'll go through all the fields of the SAM file in order. So the first one is just the read ID. So this is the same read ID that was in the FASQ file that I showed earlier. It lets you figure out which read each line of the SAM file refers to. The second field is what we call a flag. So this flag is indicating which strand of the reference genome the read aligned to, whether it has a paired end read, whether the paired end reads are mapped in the expected orientation, and all sorts of other data that BWA has inferred from the alignment. There's a very nice document online. I should have included a slide on it, which will take one of these numerical flags and tell you what information is set for each flag. Yeah, it's a flink. Yeah, it's on Picard's webpage. Yeah, yeah. So that's a very good way, because this is just a binary flag. It's impossible to interpret. I can't look at 99 and tell you what that means. You have to essentially look up what this flag is trying to get across. So the second, so the third and fourth field are the chromosome of the reference genome that the read aligned to and the base position, that's relatively straightforward. Next field is the mapping quality that we've discussed. In this example, it was mapped with a Q60, which I believe is roughly one in a million chance that the alignment is incorrect. Often when you map 100 base pair reads with BWA, the strongest alignments will come out as Q60. These are very trustworthy alignments. Does the SAM file tell you which one? No, you could argue it should. So often you can add extra information into the header of the SAM file, and often there'll be a tag in there that says at RG and then the path to the reference genome. So when you run the SAM, when you run the mapping, yeah, so you specify extra documentation. Yeah, BWA can take in essentially an arbitrary header that will just get pasted into the output SAM file. So the next field is the cigar string. So this allows you to represent the alignment between the read and the reference genome. So it's encoded using a number and then a symbol. So 76M means that 76 bases of the read were matched to the reference genome. Now, if there are insertions and deletions with respect to the reference genome, they show up like this. So here the read has a single gap here where this T is missing, and the cigar string says that there's four matches, which is here, ACGA, and then one base deleted from the reference, and then six more matches. And then if there was an insertion in the read with respect to the reference genome, it would look like this. 4M, one I, one insertion, 4M. So this just lets programs or you decode how the read actually aligns to the reference genome. And this is a SNP callable where we use this to try to figure out where the variants are. This question? If it's a mismatch? Yeah, so it always uses M's for matches and mismatches. M just means that a base aligns to a reference base. There are extended tags where you can indicate the positions of mismatches. So it could be... So for example, here there's a mismatch here. You could represent this as 1M, 1X, which is a mismatch, 2M, 1I, 4M. That BWA and most mappers will use this simplified convention where M just represents align faces rather than matches and mismatches. And then if you have paired end reads, there's a couple of fields that describe the relationship between the pairs. So if there's an equals sign in this field, it means that the pair mapped to the same chromosome as the read that the sound record refers to. The second field here is the position of the pair. And then to feel after that is the insert size. And the insert size is the distance between the ends of the DNA fragment that was sequenced, which is shown here at the bottom. And then the next two fields are the actual sequence and its quality score. So that's the same as the FASTQ file. It's just duplicated here in the SAM to be self-contained. So you have the... When you sequence a pair to end read, you have this DNA molecule, you sequence from one end, you sequence from the other end. The insert size is the length of the total DNA molecule you sequenced. Period. They could be zero though, right? Well, most of your fragments would be much... Well, I think in the next part, we'll talk about the distributions of insert sizes that you get because usually you'll size select the DNA in some way, usually by running it on a gel. And then you'll get an insert size distribution, which is like a normal distribution around 300 bases, let's say 30 bases standard deviation. And most of your reads will fall within that distribution. Yeah, but I'll maybe leave that for the next part. Okay, so some resources. So SAM Tools is the toolkit that everybody uses for working with SAM BAM files. It's got a converter to go from SAM to BAM and vice versa. You can sort all the alignments either by read name or by aligned position on the reference genome, and you can extract alignments for given regions of the genome that you're interested in. We'll be working with SAM Tools in the tutorial. It's just coming up in a few minutes. This document here is the SAM specification file. So it's actually the technical document describing what each field of SAM means and how to interpret it. And if you're the author of a mapper, this is what you'd look at to try to figure out how to write SAM. If you have questions or you need help, the SAM Tools Help mailing list is very good. Hang Lee is the owner of that mailing list and he'll answer questions from users and there's a large community of people who are keen to help new users of SAM Tools. Or you can ask on Biostars, which is a Stack Overflow-like site for sequence analysis or seek answers. We're all great resources for getting help. So I just want to go over a few issues with an IGV as we'll be using that in the tutorial. So I think there was an IGV session yesterday. Is that right? Yeah. So when you're looking at alignments in IGV, you usually want to look for the mapping quality of the reads. In this case, all of these bars are colored gray, which indicates quite high mapping quality. This example is a tumor normal pair and this is what a standard somatic mutation will look like. There'll be the tumors on the bottom here. There's a consistent difference here where I can actually read what base that is. I think it's a G. Where there's a G in the tumor and at the same position of the normal, it's just the reference base. So there's no mutation here. So this is as good as mutations will look when you're sequencing a cancer genome. Since not all bases were changed, does it mean that the mutation is heterozygous or that the tumor is heterogeneous? It can be either. Yeah, it can be. And then getting at these questions is really rather difficult. But yeah, usually with the cancer mutation, it's never going to be entirely one base, right? Because unless one of the copies was lost, the reference copy would be lost. If often when the depth is equivalent and you have one base like this, just means one of the chromosomes was mutated. So I wrote this up because I'm going to show you what alignment problems look like. So as I said before, alignments are the biggest source of false positive variant calls. And a ton of work has gone into understanding where alignment goes wrong and tried to eliminate the false positives. So this is BW alignments for a reference sample called NH12878. And this is the centromere of chromosomes on two. And as you can see, all of these colored bars are differences between the reads and the reference genome. And you can see there's vast numbers of differences here. And the read depth, which is plotted in this track here, goes up and down. There's depth of well over 100,000 in this region, in the 10,000s here. And really it's just a very difficult region to understand what's going on. Now the problem here isn't that this individual is very different from the reference genome. It's just that because the centromere is so repetitive, BWA just can't reliably map reads to the centromere. So it's just placed reads wherever it best fits. And it ends up with a lot of very poor quality alignments that would cause false positive variant calls if you were to run a variant caller over that. So what you'll often do is you'll screen out these very difficult regions. If you see variants in the centromere, try to temper your excitement because they're probably not very reliable. A different way of looking at this, this is one of the figures from the 1000 Genomes paper. This is using inheritance patterns. So calculating the probability that the variant calls are in Hardy-Weinberg equilibrium as a proxy for whether the variants can be trusted or not. And we see that the variants around the centromeres, this is, I'm not sure what chromosome this is, I think 17, the variants around the centromere of chromosomes, 17 are far out of Hardy-Weinberg equilibrium. And this is just indicating that all of the variants that were found in 1000 Genomes project around this region are probably not true variants and just mapping artifacts. So that's something to watch out for. You also see at the telomeres, which tend to be quite repetitive also, that you're getting a lot of variants that are probably artifacts. Now, one weak spot of mapping 100 base pair reads, using any mapper really, is that they can't deal very well with large gaps in the alignments. So this is a real tumor genome where there's a large deletion in the tumor with respect to the normal. And BWA has only found one read, which is this one here shown with this gap, this black thin bar. And the other reads that should show evidence for this long deletion were soft clipped. So BWA just stopped aligning the read up to a certain point and gave up. So if you were to run a very colored on this, you probably wouldn't see this deletion because there's only one read that shows the evidence for it. If BWA was able to handle larger gaps in the alignments, this would come across as a very strongly supported deletion. With about 10 reads showing evidence for it. I think this will be covered in more detail in the mutation calling section tomorrow. Okay, so we'll start the read mapping exercise now. If before that, is there any more questions about mapping and alignments? Okay, welcome back. So this part of the module will be a little bit shorter. And I'm going to specifically talk about structural variation and how we can find structural variation. So other parts of this course are dedicated to different mutation types, like single nucleotide variants, which we'll hear about tomorrow and short indels. Also, Sarah will be talking about that. But here I'm going to talk about structural variation. And what I mean by structural variation are very large scale changes to chromosome structure, like large insertions and deletions, where a lot of sequences has either been added into the genome or deleted from the genome. One sequence has been inverted, so an entire stretch of the genome has been flipped. Or translocations where material has been swapped between chromosomes and copy number variation. Copy number variation is a little bit special. And again, Sarah is going to talk about that tomorrow morning. So I'll be dealing with the other three structural variation types, the large insertions and deletions, inversions, and translocations. So the way that we infer structural variations are going back to paired-end reads. And we had a question this morning about the difference between paired-end reads and made-pair reads, and I'll describe that now. So here's how we produce paired-end sequencing data, the type of data we heard about earlier today. So we start with genomic DNA. We shear it or fragment it and size select it into roughly 200 to 500 base-pair fragments. We then add sequencing adapters to either end, to rather both ends of the fragments, and then sequence from one end in and then the other end in. And we call those paired-end reads. That's what we aligned this morning. So the important thing here is that the information you're getting is fairly short-range. 500 bases is still fairly small when you compare it to the size of an entire chromosome. Now, to discover even larger structural variation, we want to increase the separation between the ends of the pairs. And to do that, we get what are called made-pairs. So made-pairs have a different preparation where, again, we take genomic DNA, we fragment it into this time one to 20,000 base pieces, and then we make this long 20kb piece a circle. We then enrich for the sequences that were correctly circulized, and then we shear the DNA at near the point where we joined the two ends. And we get a fragment that looks like this, which is where this yellow bar here is the point of circularization. We then do the same sort of sequencing as paired-end sequencing, where we sequence from either end of this fragment. But now, because this part, this blue part of DNA, was from one half of the circle, and this part of DNA was from the other half of the circle, the physical location of these can be up to 20,000 bases apart. And the circularization procedure, we call these made-pair reads, allows a much larger separation when we map the pairs to the reference genome. Is that all clear? You'll get opposite strands for this too, right? And the orientations are now different, and I'll come back to the orientations of reads in a minute. But with paired-end reads, we expect the reads to be pointing to each other like this. With made-pair reads, they point away from each other. Yeah? Why not do a size dimension of 20 kb, a set of 500? So the aluminum sequencer has a limit of how long the molecules can be when you put them on the sequencer, which is effectively about 600 bases. And that's why we need to do this molecular biology magic to get much larger separations. Okay, so going back to paired-end reads, as I just said, there's an expected orientation. The reads should be pointing to each other where one read is on the forward strand of the reference, and the other read is on the reverse strand of the reference. And this is important when we try to enforce structural radiation as the orientations may change depending on the type of variant. Now, when we perform the size selection, which is typically performed by cutting out a band on a gel, it puts a constraint on the size of the DNA fragments that we're going to sequence. So typically, they'll follow a normal distribution with a mean of about, say, 300 to 400 bases, and standard deviation of about 10%. And that is really important information when we go to try to enforce which type of variants there are, as if pairs are mapped much, much further outside of the range of insert sizes that our library preparation says they should be, it might indicate that there's a deletion. I'll give you examples of that. So when we align our read pairs to the reference genome, the ones that match the expected orientation when they're pointing to each other like this, and within this fragment size distribution are called concordant read pairs. Those are the ones that are mapping as expected. And discordant read pairs are the ones that are mapping not as expected. And those are the ones that might give evidence of structural variation. So note, though, that not all discordant read pairs necessarily indicate structural variation. As we talked about this morning, there's a lot of mapping artifacts where BWA might just put reads in the wrong place, and those can also look like discordant read pairs. So typically, we require evidence from multiple discordant read pairs to call a structural variation, and the reads must have a fairly high mapping quality, as well, to be evidence of a structural variation. So what kind of coverage would you need with, I could say, more exotically to be able to get enough read pairs to call? Yeah, that's somewhat difficult. Usually, if you're doing an integrated analysis of everything, SNPs, indels, structural variation, you'll have 30x coverage, which is more than enough to call structural variational. But you can get away with a lot less coverage. Maybe I'll ask Andrew.  What coverage would you suggest for doing an SV analysis of a genome? I only would, although I've done something... 10x, yeah. But then what we did is we leveraged the fact that we also had matching coordinates. I see. Right, so if you have other data, then you can get away with lower coverage. The standard recommendation is 30x, though, like different individuals or different sequencing runs. Yes, okay, so can you merge information from different individuals to increase the power to find structural variation? In a population context, you can. If it's a structural variation that's segregating in the population, you can sequence more individuals at low coverage and then find things that are at high frequency in the population. That's essentially the design strategy for the 1000 Genomes Project, where they sequence 1,000 individuals at 4x and then call structural variation off that. For a tumor sample, you can't, because the things that you're interested in are essentially private mutations. So you're stuck with just sequencing 1 individual at high depth. But if you're interested in polymorphic structural variation, you can merge information from different samples. There would be some, since most structural variations are in a few private... Yeah. ...if you see something across multiple variations, they're probably going to be a false positive or a germline that you have to make something different or normal. Yeah. So a good way of getting rid of those positives. Right. So just to say that in a different way, like often what we want to do is sequence the individuals' normal genome and the tumor genome, say, call structural variation in both, and then see what's only in the tumor and not the normal. And what Andrew's suggesting is if we pooled the normal samples across multiple individuals, we'd have more power to find the polymorphic germline events that we're not so interested in. So that's one strategy. Okay. So now I'll talk about patterns of paired in reads that indicate structural variation. So I'll be using this sort of diagram for the next two slides. Here we've got a reference genome, our reference genome, and a donor genome, which is the individual that we've sequenced. In this example here, the donor has a deletion with respect to the reference. So this random block here that's present in the reference is not present in the donor genome, and we've introduced a gap here, which is just depicted by these dashed lines. Now, when we sequence paired in reads, we're sequencing the donor genome, and they should look like this. Read one is pointing this direction, read two is pointing at read one, and the distance between them is roughly about 300 bases. Now, because the donor genome doesn't have this large block of sequence that's in the reference, when we go to align these reads to the reference genome, they map further apart than expected. This read would map over here, this read would map over here, and when BWA writes out the sandfile, that insert size is going to be whatever the true separation is, plus whatever the size of this red block of sequence is. So the signature of a deletion is reads mapped in the expected orientation, but much further apart than we'd expect based on the insert size distribution. The opposite of that is when there's an insertion in the donor genome, the individual's genome or sequence, with respect to the reference genome. Here, the red sequence is present in the donor, but not the reference, and when we sequence around it, the pairs will map much closer than expected because now the insert size is the true insert size minus whatever the sequence is that's not present in the reference genome. And again, the pairs are going to map in this orientation where they're pointing to each other as expected. Now we have more complicated struct variation like tandem duplications. In this case, there has been some part of the individual's donor genome that was duplicated and just copied... made a second copy in the same place. Now if we have read pairs where one read is at the end of one copy and then the other read is at the beginning of the second copy, when we map them to the reference genome, this read, which is at the end of the copy, goes here, this read, which is at the beginning of the second copy, goes here, and now the leads have mapped in the wrong orientation. They're mapping away from each other rather than towards each other. So when you look for structuration patterns, you can look for this sort of signature of tandem duplications. Now similar to that, we have signatures of inversions. So here, this blue sequence, which is running in this direction in the donor genome, has been flipped. It's been inverted, it's now running in this direction on the reference genome. So when we sequence it, we have one read here, one read here, this one gets mapped all the way over here, and now it's been reversed, this one gets mapped in the same orientation, and now the reads are pointing in the same direction, and that's the signature of an inversion, and also the reads are much further apart because this one has now been moved all the way over here. And in the exercise coming up, it's going to, we'll see an example of what a real inversion looks like in the BAM file you mapped earlier. So here's a summary of what we expect for the different types of structure variation. So when there's an insertion in the donor with respect to the reference genome, we expect the map distance to be too small, but the read pairs will be in the correct orientation. When there's a deletion, we expect the distance to be too large, again in the correct orientation. And inversions have this pattern where the reads point in the same direction, 10 duplication as a pattern, where they point in opposite directions. And if there is an interchromosomal exchange, we'll have mappings where one read maps to one chromosome, the pair maps to the other, and they can be in any orientation. Okay, now when we, if there's a very long insertion in the donor genome with respect to the reference, we might actually miss it. If this inserted sequence is longer than the insert size of our distribution, we won't have pairs that map one side on one half of the inversion and the other pair on the other side. So here we might not be able to see very large insertions. What will happen is we'll typically have one pair here, the other pair in the novel sequence, and maybe only one end of the read maps to the reference genome. So that's something to watch out for, that there's a limit to how large of insertions we can find from, say, 300 base pair paired end data. This one? Okay, so when, if, let's say we've sequenced 300 base pair paired end reads, and let's say we have a 1000 base pair insertion into the genome. Now, to be able to find this with paired end reads, we typically need one half of the pair on one side of the insertion and the other half of the pair on the other side of the insertion. But because the event is much larger than our insert size, we can't have that. There's not going to be pairs that span all the way over from one end of the insertion to the other. So this isn't a problem with deletions because deletions are spreading pairs out, and you'll always have one pair on one side of the deletion-based grade point and one side on the other. I think this would become a lot clearer when we do the exercise, which we'll go through these types of events, and we'll see the actual patterns in IGV. Do you have a question? I was just going to say, so then do these bits, the red bits, get up to where? It depends on what type of insertion it is. If it was, let's say, transposons jump around the genome. You can have transposon insertions. So if it's a transposon that just moved around the genome, then anything that aligns, there was sequence from the red bit will just align somewhere else in the genome. If it's truly novel sequence that's just not in the reference genome, it's likely that the pair just won't map at all. Yeah? Could it happen that you have a run into the insertion and then you have, like, the read that catches the halfway through and then it's actually truncated by the audience? Yeah, well, that's definitely a signature of these events. You'll have exactly as you say. For the part of the read that's the same as the reference, it will align and then BWA will truncate the read and it'll have these soft clip tags where it just says, I haven't aligned the last 30 bases of the read. And that sort of leads into the next slide, which is a different type of signatures. So so far we've talked about just using the distance and orientation between paired end reads to infer structuration, but also if you can have reads that are split, where the alignment is just broken into two places. So for example here, there's a deletion at this point. This read sequenced the deletion breakpoint and then BWA went to map it and mapped one part here, the other part here with a large gap in between indicating the deletion. Now, if this was an insertion, it might just align half of the read, as we just said. Do you have a question? Do you know? Okay. Okay, so this is cancer bar informatics course. So we're interested in sequencing tumors. So I'll talk a little bit about somatic variant calling versus germ9 variant calling. So we're almost always going to sequence both a tumor sample of an individual and the individual's normal genome. And we're typically only interested in the structure variations that occurred in the tumor, not the ones that occurred in the normal. So there's two approaches to to just finding the tumor specific structure variation. And the first approach is taking the tumor sample, running a structure variation car on just the tumor and then running a structure variation call on just the normal and then looking for things that were called the tumor and not the normal. The second approach is just finding structure variation in the tumor and then using a program that will search for evidence of the same structure variation at the read pair level in the germ9 sample. And the there are programs to implement both of these approaches. We're going to use a program called lumpy SV in the in the tutorial, which will call both tumor variants and normal variants internally and then filter out all of the germ9 variants automatically for you. But structure variation detection is very active field and different programs will implement different approaches for enriching for just the tumor mutations. On that point, I'm going to talk about the performance of structure variation programs. And as I said, this is a very active field of development. There's unlike read alignment where the field has largely settled on BWAMM as the read aligner. There's no one true structure variation car. There's a lot of programs that will perform well in certain contexts, not so well in others. And Paul Butros here at OICR is organizing essentially a benchmarking project, which benchmarks different structure variation cars. And there's a live leaderboard that you can look at to see what programs and what pipelines are currently the state of the art. So this is a good resource to look at when you're trying to select what program to use. For the tutorial, I chose lumpy as it's a very easy program to use and it's good for demonstrating how structure variation colors work. But I encourage you to sort of have a look at this leaderboard and see what alternative programs are out there. Now I'm just jumping back to this slide about gene fusions. We're going to hear about that in the afternoon. But this is a special type of rearrangement where you come where there's been a rearrangement between say different parts of a chromosome or two different chromosomes which can create a new gene which is a bit of gene X in this case and a bit of gene Y and Andrew will talk all about finding these in the afternoon. So with that said, we'll start the second exercise as before. It's on the wiki and just a list of commands that you can enter into the terminal and in this case we'll heavily rely on IGV to view the actual alignments and to look at these structure variation patterns based on these diagrams that I've just made. But before we launch into that is there any more questions about this?