Alright, you guys ready for module four? Last one. Alright, so this one we're going to talk about isoform discovery and alternative expression. It kind of builds naturally on a lot of the stuff that you've already been doing with cufflinks. Mostly what we're going to be doing is rerunning some of the types of commands who are already running in cufflinks but with slightly different options that are more geared towards isoform, quantification, and discovery. So some of the learning objectives of this, the tutorial that goes along with this lecture are actually, sorry I've got the wrong presentation here. Basically we're going to use cufflinks in what's sometimes called a reference annotation based transcript assembly mode or RABT mode. And also it's a denovo assembly mode, so in so far we've been running cufflinks with a GTF file that provides an idea of what the transcriptome already looks like and we've been basically asking cufflinks to really take that model of the transcriptome very seriously and to estimate expression for the transcripts that are described in the GTF file that we specify. Now we're going to run cufflinks in two additional modes. One where we tell cufflinks to basically just use that transcriptome as a loose guide, so to try to use the information there but not be totally beholden to it. And another mode where we don't tell anything about the transcriptome. So we could be studying a species where we didn't even have any transcript to some annotations at all. And it's going to just try to assemble the transcriptome that it thinks is expressed in your RNAseq sample without any prior knowledge of what the transcripts look like. So this is a slide, just a review from the first presentation. So remember we talked through this idea of the central dogma where we have information flowing from double-stranded genomic DNA template to a single-stranded pre-mRNA molecule where the introns are in place between exons and then the splicing machinery comes along and removes the introns and assembles the exons. Again, this is mostly applicable to eukaryotes. And then this thing gets exported from the nucleus to the cytoplasm where it gets translated into a protein sequence that is then folded and various post-translational modifications occur. So what we're going to be really thinking about now is the way these mature mRNAs are structured and how there can be different versions of isoforms being expressed from each locus. And those versions come about by differences in the way splicing happens. You have a splicing machinery that comes along, it recognizes signals on the pre-mRNA molecule and it removes the introns. But for basically every human gene there are multiple ways that that happens and there's a very complex regulatory system that controls exactly which isoforms are expressed from which loci. And this is a really active and deep area of research in various species, especially the mammals. So this slide is really just to illustrate that there's a lot of complexity to the analysis of RNA-seq data with respect to splicing. So I got this slide from the RNA-seq blog which you haven't checked out already. I highly recommend it. This blog does a pretty good job of keeping on top of the latest developments in RNA-seq technology and analysis. And there'll often be reviews of new tools that have come out or collections of different tools for different types of analyses. And this is actually from a sort of pre-proof manuscript that the author of this blog seemed to be writing where there he's listed a variety of tools for different types of RNA-seq analysis starting with mapping. So there's various mappers that are used for reconstruction of isoforms and then quantification of isoforms and then ultimately comparison of different conditions of differential expression analysis. And there's lots of things that kind of fit into multiple categories or span across those boundaries. The next slide is also kind of just meant as a resource. So it's a list of some useful resources and discussion that I found over the last few years starting with discussion of best approaches for predicting novel and alternative splicing events from RNA-seq data. So there's a couple of biostars posts. So we've mentioned this forum a couple times. Again, if you haven't signed up for a Biostars account, you should do that and you should in the future when you have questions after you go home from this tutorial and you have questions about RNA-seq that would be the first place I would check or the topic area of your question. Maybe spend 5 or 10 or 15 minutes doing that. If you don't see a question that seems relevant or the questions there don't have the answers that you need then go ahead and ask the question that you have there and there's a pretty active community of bioinformatics folks that are involved in Biostars and you'll often get some really useful feedback and answers to questions. A couple more posts that are really relevant to this topic on alternative splicing detection are listed here. There's an interesting post on identifying genes that express different isoforms in cancer versus normal RNA-seq. Some discussion of how cuff links and cuff diff differ in what they're doing and then a discussion of tools that you might use for visualizing alternative splicing events from RNA-seq data to help us think about both the types of events that we're going to be looking at and the way to visualize them. There's a couple slides here in sort of cartoon depictions of the types of alternative expression that occur. So the first example at the top here is just simple transcription. This is just a reciprocation of what I showed on the central dogma slide showing a simple gene with three exons and two introns and it's being spliced into what is called the canonical isoform, which is usually a reference to the most common or representative isoform that's expressed from a particular locus. And then there are a variety of other categories in which transcription can happen differently. So for example, alternative transcript initiation is where the transcriptional complex sits down in different places and starts to transcribe at one position but may also do it at another position. So for example, we have two transcription start sites being depicted here and these could lead to two isoforms that differ by having basically a different first exon. So we get three exons and then another transcript that has the same exons except it starts at the second exon and does not use the first exon. Alternative splicing is sort of a loose term to describe stuff that happens sort of in the middle of the transcript and there's a variety of subtypes of alternative splicing. So something that's called cassette exon skipping is where you have two isoforms that differ by the exons that they include. So in the first example we have our canonical isoform with exons 1, 2 and 3 and then we have an alternative transcript where exon 2 has been skipped. You can also have alternative 5' splice site usage so in this case the 5' in this case the splice site is referring to the 5' of the intron. So you've got two alternative ends to this exon basically. So basically what's happening is the splicing machinery is coming along and it's deciding to use this donor site or this donor site and this gives you slightly different mature mRNA transcripts in this case with a longer or shorter exon 2. Similarly you can have alternative 3' splice site so these are where you have an alternate acceptor site being used and again this gives you isoforms that differ slightly in their length. In this case you've got a short exon 3 being used and a longer exon 3 being used in the alternative isoform. Mutually exclusive exon usage is very similar to the cassette exon usage except in this case you're going to produce two isoforms each with three exons being used but the center or second exon used is different between the two of them so you have sort of an exon 2a and an exon 2b. And then finally you can have intron retention where you have the basic scenario where you have exon 1, 2 and 3 and then another scenario where instead of exon 1, 2, 3 you have exon 1 spliced to 2 and then the entire intron is included and it continues on to exon 3 so effectively you just have a really long exon 2 here. So you're saying you have basically an an alternative gene inside another gene? Yeah so it is possible that you kind of think it happened. If you have stranded information often these kind of events are at least happening on the alternate strand which is helpful. If things like that are happening on the same strand it can be very confusing to depopulate what's going on. The good news is that those things often tend to be sort of single exon events that are within an intron so there won't be evidence for them connected to the exons on either side so you can use that information to try to tell which of them kind of go with the 5 or 10 exon gene that's being expressed and which of them are maybe a separate transcriptional unit within the intron. But yes it's possible that you could have sort of confusing situations like that where you inadvertently assemble a sort of chimeric or Frankenstein isoform that isn't really real and that kind of all comes back to this sort of caveat or warning that you have to always remember that we're inferring a lot of things here and sometimes you can put the pieces of the puzzle together wrong and it looks reasonable but you actually have a misrepresentation and hopefully you would be able to sort that out in some kind of downstream validation before you went too crazy doing functional work on this thing that you think exists but is really kind of a misunderstanding of the data. Any other questions? So this slide is just kind of a quick summary of some of the history of sequencing methods for studying alternative isoforms. This has really been an area of development over the years. People have been interested in understanding the structure of mRNAs in human and other species for many many years. Once the genome was sequenced we had this in human this amazing reference sequence to work with but then going from that to what transcripts are actually expressed and what their exact structures are was also a big task. Initially a lot of the heavy lifting was done by full length cDNA sequencing and this is really the gold standard for resolving the structure of mRNAs so if you can isolate a full length cDNA clone it and then sequence that entire cDNA across the entire insert sequence you really can tell with exquisite accuracy what the exact structure of a messenger RNA is by then mapping that complete sequence back to the reference genome and seeing where the exons and introns wind up. Unfortunately this is still really low throughput activity but if we could just somehow magically sequence full length cDNAs we would probably not do RNA seek the way we do it. If we didn't have to fragment our cDNA into all these little pieces and then kind of shotgun sequence them and try to put the pieces back together after the fact the analysis would be way way simpler. So one of the sort of dreams of the sort of next next gen sequencing is nanopore sequencing where one could imagine feeding RNAs through a pore and reading the sequence off as those RNAs are fed through that pore and not having to fragment the RNAs just read the sequence off from the beginning to the end and we would do that millions and millions of times and we would get both quantification and the complete structure of every RNA that was in your sample. But we're just definitely not there right now and we've kind of gone through these stages of low throughput full length cDNA sequencing and then to try to get at cDNAs that were missing there was sort of targeted or sequencing time primers to amplify and then we sequenced many products from those amplifications to try to identify alternative splice patterns. A lot of EST sequencing was done where we generated cDNA clones in a high throughput fashion and then just sequenced the ends of those in a fairly roboticized way and were able to accrue a lot of data. And these top three things are really where the bulk of the annotation of the transcriptome that we're using today comes from. Most of the transcripts that you're seeing in Ensemble or RefSeq came from these projects where teams of people running robots were sequencing full length cDNAs and ESTs. And that data quality is really high but it's incredibly slow and expensive to produce on even one sample or a series of samples so it's very difficult to do functional biology with those kind of platforms because we just can't afford to go back and do it for our two conditions of interest or our drug treated versus untreated or all of the tissues of an individual or a particular tumor or whatever. So then we moved into a stage where there was much more cost effective and high throughput methods for getting at this kind of information. So there was a variety of really small tag sequenced based approaches where we have some kind of enzymatic approach that gathers little pieces of RNAs, concatenates them together and then allows us to sequence dozens or hundreds of these little tags at once. So some of these are SAGE, PAGE and GIS and those basically differ in terms of whether they go after the three prime end of transcripts or capture the five prime end of transcripts or the five prime end three prime end of transcripts. And those methods are really good for looking at alternative transcript initiation and alternative polyamidolation sites. And then shortly after those technologies became well established the sort of next gen sequencing instruments arrived on the scene. First there was 4.5.4 and then there was SELEXA which is now called Illumina and now there's also IonTorrent and there are other platforms being developed and these really took it to the next level in terms of data throughputs. We were able to sequence basically shotgun sequence all of the RNA in a particular sample and produce an amount of data that is completely dwarfed the scale of the data production that we could do just a few years before. The only downside really is the size of the molecules that we're sequencing so we're still limited to these paired 100 MERS and we're gradually increasing that size and some people who are really interested in alternative splicing will use a different sequencing strategy where they sequence a single and 300 MERS or they try to do paired 250 MERS so pushing the limits of the Illumina platform to get longer reads so that you can do a better job of resolving where the exon and intron boundaries are and you don't have to do so much inference so much piecing all these little pieces of the puzzle together to get your full-length isoform prediction. And it's this kind of data that cufflinks is really meant for so it was really designed with these short sequences in mind and all of this sort of mathematical wizardry of it is about trying to make these inferences and piece together all these pieces and try to predict what the full-length isoforms really look like and there's a simple depiction here which is actually a very simple scenario relative to what is happening at most in most human genes but even this very simplified sort of toy example gets very complicated when you start to really think about what's going on so what's shown at the top here this is from the cufflinks manuscript are three hypothetical transcripts the first two share the same transcription start site and the third one has a different transcription start site and then they also differ in different ways so two of them share the same 3 prime exon the first one has this distinct exon that's being skipped in the second one and so forth so there's all these little subtle differences between these three but they also share certain features and I don't know if we've talked about this before but this is a very common depiction where you have a transcript that's drawn with sort of a narrow rectangle and then a wider rectangle and usually what that's indicating is the portion of the transcript that is coding so the portion that becomes translated into protein and then the narrow part is in this case the 5 prime UTR then the coding portion and then the 3 prime UTR at the right here so you can also think about in each of these isoforms what would the open reading frame or protein coding portion look like so you can see that B and C share the same short open reading frame here and A has a different coding sequence A and B share the same promoter sequence and C has a different and so forth so you've got all these different ways you can think about how these isoforms are different or similar to each other and the first thing cufflinks does is try to estimate the relative abundance of each of these 3 isoforms and then in this sort of splicing analysis component of cufflinks it tries to break down the isoforms into these different categories so for example it will compare within the transcription splicing group so it divides your samples into those that have transcription the start site 1 and then it looks just within those how well expressed there are so we have 2 isoforms the blue and the yellow sorry the blue and the yellow that share the same transcription start site so we can ask for that transcription start site how do those 2 isoforms how do the expression of those 2 isoforms differ and then when we're doing differential analysis between our 2 conditions we can look at the ratio between those 2 isoforms that use the same transcription start site in condition A versus condition B similarly we can now look at differential promoter usage so in this case binning the isoforms that share the same transcription start site so A and B both use this transcription start site and C uses the second transcription start site so basically kind of pooling the data from A and B and comparing them to C and again in our differential scenario we would look at the ratio of use of transcription start site 1 to transcription start site 2 in our first condition and our second condition and then finally you can do a similar kind of thing with the coding sequence so maybe what we really care about is the expression of isoforms that have a particular predicted open reading frame or coding sequence so in that case we would bin isoforms B and C together because they share the same protein coding section and A is what has a different protein coding section so in that case we would compare A to B and C and in our differential analysis we would look at the ratio of B plus C to A in condition A and B so that's like a very bewildering amount of numbers and letters but all of this ties back to the output that we're going to get from cufflinks when we run it in the splicing mode where we look at the files that are really aimed at investigating splicing so this first scenario the output that corresponds to these kinds of comparisons is going to be in this file called splicing.diff and then when we're looking at the usage of different transcription start sites that output will be in the promoters.diff and then we're really focusing on the differential expression of coding sequences that will be in this cds.diff file any questions on that so we've been gradually working our way through this flow chart of the different steps what we're going to do now is actually kind of circle back to what we've done before we're going to go back to running cufflinks but we're going to change up the options so cufflinks has many many options and you can run it in different modes with different goals in mind with sort of multiple tools built into one but we're going to do a sort of similar pattern where we do a transcript compilation with cufflinks and then we're going to merge our transcripts together and compare them to known transcripts and then we're going to use cuffdiff except instead of just doing simple differential gene expression we're going to do differential splicing analysis or alternative expression analysis so before we go on I just had this slide that's sort of randomly thrown in here that we I thought we would just address this because this is a question that has come up a few times already here and it comes up every time we talk to any crowd of people doing RNA-seq analysis and that's for the question of what do I do if I don't have a reference genome for my species and the bottom line is that we don't really have time to get into a lot of the details of that you really have to take different analytical approaches but one of the things that I often ask people to consider is why don't they have a reference genome and in some cases are you sure you don't have a reference genome and the purpose of that question is just to sort of explore the idea of in some cases genome sequencing has actually become quite cheap and if you're really struggling to do RNA-seq analysis without a reference genome and it actually might not be that hard to generate the reference genome for your bizarre critter that only you and five other people in the world are studying the whole genome and assembly analysis methods have really come a long way and it may actually be cost effective and a reasonable place to start to actually try to build your own reference genome sort of a related piece to that is that a really actually a bad reference genome is better than none at all and there's nothing stopping you from doing a reference free analysis but if you can quickly and relatively cheaply produce some kind of reference genome by sequencing the DNA of your species you should think about doing that now there's definitely some legitimate reasons why you may not already have a reference genome and why making it might be impractical some of the common reasons that we here are the genome of my species is too large or too complex so this is something that the plant people often have to deal with where they have these just truly massive genomes that are hexaploid or worse and it really probably would be quite computationally expensive and difficult to produce even a bad reference genome another place where you see this is metagenomics so where you're actually not studying you don't actually know what is in your sample there are multiple species and they're all mixed together in the same sample so there isn't really the simple concept of oh I have a human cell line therefore I'm going to compare to the human reference genome sequence or I know it's a mouse because I can see the mouse in the cage and then I kill the mouse and I get its DNA it's a very different situation from I isolated RNA from a gut sample from someone after they ate some Mexican food or something whatever your experiment is so it might not be practical but basically one of the answers to what you do if you don't have a reference genome is to do transcriptome assembly which will work without having a reference genome the bad news is that de novo transcriptome assembly it's beyond the scope of this workshop and one of the reasons for that is it's fairly complex and the tools that do this are quite elaborate but the good news is there's a lot of commonalities between running one bioinformatics tool suite and another so a lot of the sort of basic skills that you learn here would be applicable to installing a transcriptome assembler and gathering input files and running a series of commands and then filtering the output dealing with the output files and so forth if you go to this link you'll probably have an updated version of this even by now there's a couple de novo genome based assemblers there and a variety of other de novo transcriptome assembly programs the ones that I'm most familiar with are transibis and trinity and I've heard very good things about both of those I've been involved in some projects that involve transibis in Vancouver and they yielded very good results the thing that I've heard about transibis that really attracts from it is that it's it's difficult to install difficult to run seems to be some black magic to getting it to run and I haven't seen a lot of success stories for people outside of the group that developed it actually using it which is obviously not good trinity though I have seen a lot more use just in the community so people just picking it up and using it and there's a course in cold spring harbour in the fall that has an RNA-seq component where they walk you through the process of doing analysis of RNA-seq data with trinity I've seen that a few times and it seemed to go fairly smoothly so I guess if I had to recommend one tool that you might check out first I guess that would be my recommendation this is developed at the brode trinity ok so as usual we're going to now switch over to a few slides about the tutorial itself and then we're going to dive into the tutorial and that will be the last tutorial of the day and we're doing pretty good for time so we may even be able to end a little bit early and then be able to spend some time talking to each of you about particular questions you had or challenges you have with your own experiments and so forth and do the survey spend a lot of time really thinking hard about the survey can do that too so the learning objectives of this tutorial we're going to run cufflinks again but now we're going to learn how to run it in what we call reference only mode so that's what we've already learned we're going to extend our use of it to the reference guided mode and to the de novo modes so remember this is where we use our gtf file to guide cufflinks instead of really forcing it to consider those transcripts and no others and then de novo mode is where we don't tell anything about what the transcripts look like just tell cufflinks to tell us what the transcripts look like without any cheating or hints and that's really making quite a demand of cufflinks then we're going to learn how to use cuff merge to combine transcript domes from multiple cufflinks runs so we're going to run cufflinks on each of our samples our two normal replicates and quotations and our two tumor replicates and then we're going to combine the transcript domes with all of that those four runs to give us sort of a common reference point so that we can then go back and develop a unified set of expression estimates across the four samples then we're going to learn how to perform differential splicing analysis with cuff diff so this is basically going to involve running cuff diff on the output from cufflinks where cufflinks was being run in sort of splicing aware or splicing tailored modes we're also going to go back actually a little bit further and take a closer look at the top hat junctions counts file and also dig into some of the cufflinks differential splicing files of the command line so this top hat junctions file actually can be quite revealing I mentioned it earlier I think in the first lecture where you can use summaries of how many reads span across junctions together a sense of the quality of your library another thing that you can do is just take this junctions file that comes out of top hat and look for particular interesting splicing events so this is a much more segmented or focused analysis where cufflinks hasn't been run yet all you've done is align your reads to the genome and some of them have spanned across introns that represent X on X on junctions and this junctions file is automatically produced to summarize how many reads span across each junction so you basically get a readout of every connection between two X ons that was observed in the transcript dome that you sequenced in your RNA-seq experiment and you can use that to identify interesting splicing events you don't necessarily know what the whole transcript looks like but you can still use it to find candidate alternative splicing events and then once you find those candidates you can sort of back up and say what might this isoform look like and it's sort of a very quick and focused way to look at the splicing output in your RNA-seq experiment without even having to run cufflinks and you can visualize this file in IGV and it can be quite revealing just on its own and that's what we're going to do so we're going to visualize this top hat junction count file in IGV and also the little transcripts that come out of cufflinks when we run in reference guided and de novo mode so the first thing we're going to do is start rerunning cufflinks and refguided in de novo mode as I said in module 3 we were running cufflinks in this reference only mode and the nice thing about the reference only mode is that it gives you kind of a simple output you get one estimate for every transcript that you fed into the tool which is kind of nice you get this sort of microarray style output I know I have 25,000 transcripts and I've got these 10 samples and I just want to read out that has 10 columns, one for each sample and it's got 25,000 rows one for each known isoform and you can really quickly get into differential expression analysis without having to do a lot of complicated downstream processing of files so it gives you this really microarray style output but that's really an underutilization of the richness of RNA-seq data so we're going to try to do a better job of taking advantage of that richness now so in order to do that we're going to play around these options so we're going to talk a lot more about these dash G, dash little G and dash big G options in both top hat and cufflinks and cuff diff and it gets a bit confusing because they, you know, these the same letter keeps getting used over again in slightly different but related contexts so just to review them now and then we'll do this again when we're actually running the commands so top hat has a dash big G or GTF option and this is, remember, is used to supply the transcriptome GTF file during the alignment so we're not trying to assemble transcripts but we're trying to help the aligner do the best job it can figuring out where every read should go and reference genome and we're allowing it to use the transcriptome independent of what you do in the downstream step with cufflinks you might decide to use the GTF file when you're doing your alignment or not and then as an independent choice when you're running cufflinks you might decide to use the GTF file to basically force cufflinks to give you estimates for those transcripts or use it as a guide or not use it in the de novo mode or some combination of these things so just to maximize the confusion top hat has both a big G and a small G and in this case the small G is really an unrelated option that's used to specify the maximum number of multiple mappings for a single gene I don't know why they chose G for that they probably should have chosen something else but that option we're not really doing anything with but we did use the big G to give the GTF file to top hat and to tell it to use that information now cufflinks has a big G option and this is used again to supply our transcriptome GTF and if you specify dash G with a transcriptome GTF file cufflinks is going to quantitate against those reference transcript annotations and that's what we've been calling the reference only mode that's what we did already so big G is what we did in the module 3 we switched to using the little G option again you supply the same GTF file transcriptome GTF file that we've been using but this time it's going to use that to guide the assembly rather than really being strict about what transcripts are being used and this is what we're going to call the reference guided analysis mode and then if you run cufflinks without the big G or the little G this is what we're going to call de novo analysis mode so there's no GTF file even being specified and then finally cuffdiff it requires a GTF file but it is not specified with a big G or a little G option it's simply supplied as a path when you're constructing your cuffdiff command so I mentioned this so I realize that's very confusing we're going to go over it again when we're running each of these commands we're going to go over this top head junctions bed file as well but just to give you a brief introduction of what I mean by junctions so after our alignments you get top hat creates a summary of all the reads that happen to have supported exon exon junctions or spanned across an intron so for example you might get a readout that says for a particular gene that exon one two has five reads and a connection of exon one to three has nine reads so this exon one three is sort of implying that an exon two was skipped so this is sort of exon skipping junction and this file just has a very simple format and it reports all of the unique exon exon junctions so every line in it is a unique coordinate combination and then the fifth column simply contains the junction read counts the number of reads that supported that exon exon junction and this is what it looks like what it looks like if we view this file in IGB you get these sort of arc little red arcs that span across exon exon junctions so for example the one that's being shown here is going from exon one to exon two that's what this arc is here and then two to three and three to four and so forth and then the sort of darkness or thickness of this arc is a representative of how many reads supported that so you could also view the individual reads by loading your BAM file and you could see that all of the individual reads that supported this junction and you could start to correlate how the increasing number of those gives you a sort of a fatter arc and less of them gives you a sort of thinner arc a quick introduction to cuff merge so I think we've already used cuff merge a few times but basically the idea here combines transcripts predicted from multiple RNA-seq data sets into one view of the transcript dome and we run this before running cuff diffs so that we can compare across multiple conditions and have this sort of a unified reference set to compare to and then you can also ask cuff merge to simultaneously compare transcripts to our known transcript GTF file from Ensemble so when we run cuff cufflinks in the fully de novo mode we're going to predict potentially novel transcripts from each of our four samples and then we're going to run cuff merge to merge those together into sort of a unified transcript dome but it's still totally predicted we don't really know how it relates to what the known transcripts from human so when we run cuff merge we can also say here now at the end of everything here's our known GTF file just tell me how the transcripts you predicted correlate with what was known about the transcript dome but now that the assembly is all done so it's not going to influence what transcripts get assembled or don't get assembled it's just a sort of annotation after the fact this is a simple comparison of merged GTFs from each cufflinks mode so just to give you sort of a visual example of some of the results what's shown on the top here is a track of UCSC genes and then we're seeing output from cufflinks being run in three different modes here 1, 2, 3 and then this is the ensemble gene track that would correspond to our GTF file so you can see a number of things are going on here first UCSC and ensemble don't agree about what genes or transcripts are expressed at this region of the genome so you can see UCSC as a single gene here and ensemble has 1, 2, 3 and then when we ran cufflinks we got transcripts that appear to correspond to the known ensemble transcripts but when we ran in both the reference guided mode and the noble mode we've now predicted a novel transcript that doesn't appear to correspond to any of the genes in either ensemble or UCSC but this is potentially a novel gene that's been predicted straight from our RNA-seq data it's possible to infer false positives if a gene that's it you definitely don't know that it's real just because it was predicted it could be a false positive but the question is how do you know there are definitely pieces of information that might make you more or less confident so one thing you could do is look sort of cast the net wider in terms of what other types of data you compare to so in that case I was comparing to UCSC and ensemble but there are many, many other transcriptome databases out there that you could compare to so you could say well there's some evidence from some prior experiments or from DBEST or libraries of CDNA sequences there might be other things also about the nature of the sequence itself that made it more or less believable so for example if it looks like it's in a region that would be difficult to align to like say a repetitive element that might make it more likely to be just the result of a mapping artifact and there's also sort of a built in prior based on what species you're working with and how well annotated that species is so for something like human it really has been pretty heavily annotated so I think you need to just inherently be a little bit more skeptical about the prediction of novel genes because there's sort of a question of well how come it hasn't been found so far when we've been looking for these things so exhaustively for so long whereas if you're dealing with some species that there's hardly any annotation at all you might expect plenty of novel transcripts to be found and for quite a number of those to actually be real I would also say that transcripts that are multi exons that have good coverage that are longer those things are less likely to just be artifacts of mapping whereas small single exon just like a block of reeds aligns here and then cuff lengths is like oh that looks like a transcript there those things are probably more likely to be false positives than oh I found this thing that's 1.5 kb long and it's got tens of thousands of reeds of support and there's 5 exons and 4 introns it really looks like a transcript it just doesn't line up with other known transcripts so there are a bunch of things like that that you can do at the informatic level but then of course you would want to definitely validate it by some orthogonal approach but the nice thing is that cuff lengths really gives you what you would need to design that validation experiment you basically have a prediction the sequence looks like this so it's fairly straightforward to go back to your sample and say try to amplify that CDNA clone it see whether you can actually rescue that this is sort of a similar idea but this is where we've merged our GTS from each sample again showing the reference only in de novo mode so in this case we've got in the reference only mode we're just seeing one transcript being summarized by cuff lengths because that's what we told it to do by saying do this in reference only mode and then in de novo mode we're seeing at the same locus a lot of different transcripts being predicted and each of them is very similar but they have sort of subtle differences so for example you can see that this one here has a rotating intron and this isoform has a predicted alternative transcript initiation start site and a slightly longer exon than what is used in these other isoforms and so on and this is typical that isoforms that are predicted are usually very similar to each other and they have some subtle distinctive difference yes yes I'm not showing it here and I don't remember this is just a screenshot that I took at some point while I was exploring the data but we're going to look at them side by side so you'll start to get a sense of how much they differ and how much they vary depending on what data you use they might differ quite a lot or they might be very similar and it will also depend again on the reference transcriptome that you supply when you're doing the reference guided mode if it's really complete it'll have a bigger influence than one that was quite incomplete and generally just the cleanness of the data I think if your data is noisy or you have a lot of misalignments then you might tend to get more false predictions but I think generally the authors if you just ask them like I just want to run the software in one mode I don't want all these different modes I want to kind of simplify the analysis but I want to do splicing analysis they'll generally recommend that you use the reference guided mode although I think that's changed over time and they did publish I think on the wiki there's some of the papers that are listed there are sort of follow up papers by the authors of these suites of tools and one of them is another sort of published tutorial that sort of best practices for using the tuxedo suite and it will describe which modes they think are appropriate for what types of goals and that's actually related to this next slide so what if you return to your lab and you can't get to work on your own data so obviously one thing you can do is refer to the materials that we provided with this course so we're really kind of giving you a bit of a survey of a lot of these materials and there's a lot of detail there that we're having to freeze past at times if you go back there may be additional sort of annotation in the tutorial files that we didn't expressly cover or that kind of flew by and it didn't sink in at the time so that's the answer just by going back and then there's this nature protocol tutorial that Cole Trapnel wrote and it provides actually a troubleshooting guide which I have on the next slide that explains some sort of common problems that come up when you're doing this kind of analysis so you can check for resources like that I've already mentioned searching biostars and seek answers and of course there's always Google for every problem that you might have if your question's not already on it's just a good way to sort of get the question out there and you never know you might get a great answer and then if you pursue the problem further so this is sort of a best practice for these question and answer forums is that if you find the answer please come back and tell us at Biostars about what you discovered because before too long you'll be the expert not the people on Biostars so here's this troubleshooting guide it kind of reviews some common problems that people have when using top hat cuff links and cuff dips so for example the first one is top hat cannot find bow tie or samtools and the possible reason is that you've got your path variable set incorrectly so remember we went through this exercise of setting our path variable to tell top hat and basically tell Linux where to find bow tie top hat samtools and so forth so I'll leave those extra problems for you to refer to on your own time


In this game, I will be asking a popular YouTuber about five different Yu-Gi-Oh cards. Each round, the guests will have five questions about the round's chosen card. Each answer is worth a point, until the final round where they must guess that card's price. The better the answer, the more points the guest receives. Can your favorite YouTuber win the ultimate prize? You will just have to watch to find out. If you want to win the giveaway for this video, which is half of the prize money won during today's game show, just like the video, be subscribed, turn on notifications, and let me know down below who do you want to see on the next episode of Guess That Price Season 2. Welcome back to Guess That Price. This is the first episode of Season 2 and we have, of course, the man, Simo, on the other end to test his knowledge of Yu-Gi-Oh cards and their prices. How are you doing, Simo? I don't know why you have me on this show.  It's for entertainment, I suppose. I'm doing good, Brux. And thanks for having me. This is a new season and things have changed. So I think that you might be excited with some of the changes we have here, because a lot of this stuff, I feel like as I was going through here, I was like, Simo was going to know this kind of stuff, because it's not just prices this year. Oh. So it's Guess That Price. So the final and the most important question will be the price, but we actually have multiple other questions about the cards and you can earn points by answering those correctly. And I'm not going to reveal what the questions are just yet, because I want you to be surprised, but I think you're actually going to do a little better on these than some of the prices. As long as I beat Gage at the end of it all, that's all I care about. Well, last year, or at least last season, he destroyed you. He did, I need my retribution. All right, Simo. Are you ready for the first card? I'm going to tell you the card name, and then we're going to have a bunch of questions about that card. Okay. Let's see what we got. The first card I think you'll be familiar with, we'll see how familiar you are exactly. The first card is the Salamangreat Sunlight Wolf. Ooh, Sunlight Wolf. Okay. So what questions do you have to ask for me aside from the price? So the price is going to be last. So we'll get to that later. That the price is worth the most points based on what you guess and how close you are. The first question is a two part question. It is when did this card come out? Which year and which set did it come out of? You get two points if you answer both correctly. One if you get one and zero obviously if you get zero. So if you can name the year and the set for this card, you get two points. Okay. So this is a bit of a trick question for people who might not know because immediately I think most people are going to say, oh, duh, this came out in the Salamangreat Structure Deck. It did not. It came out in a core set that I believe came out right before the release of the Salamangreat Structure Deck because I remember doing a video on like the budget Salamangreat Structure Deck with just three structure decks and it didn't have a sunlight wolf, so the deck sucked. And we also were just like near this point in like history of Yu-Gi-Oh. So Salamangreat is part of TOS, but it was obviously like early. So now I have to remember my set releases around that time. This is why I think you might be good at this just because you've done so many of these series and you've been through all these sets a lot. I'm a bit of a mini Yu-Gi-Oh historian at this point. So where it gets a bit confusing is I'm pretty sure the Salamangreat Structure Deck came out in 2019. It was early 2019, I believe. I think it was like January or February. It's tough though because I don't remember the core sets. I'm gonna have to think about this. The core set that came out around that time is either like late 2018 or early 2019 for a way. I will remind you that you're lifeline for this set of questions because there is a lifeline for each set of questions. So if you want a lifeline for the year of the set, the lifeline option that you have is one word from the original set. It's gonna make it a lot easier if you don't know the original set. It won't necessarily help you with the year if you don't know when that came out, but it will help with that other part. But see, this is the first set. So this is the first card. I want to save this for when it's actually difficult. So I'm just reminding you that that is an option if you're not sure. Thank you, but I will politely pass on that. Okay. I'm pretty sure. I'm trying to remember like what sets came out around that time. So like Dark Neo Storm was Orcas. So it's before Dark Neo Storm. What was before that? Like Soul Fusion was around there as well. Soul Fusion was a purple set.  I'm trying to think if there was a set between Soul Fusion and Dark Neo Storm. And I think there might have been because I think Soul Fusion was the end of 2018. Dark Neo Storm was the... Dark Neo Storm was like May 2019. So I think there was like a set in between those two. Maybe there could be. Who knows? I'm trying to think of like the color because like you know how the booster packs follow the color wheel? Like they have that pattern. I'm trying to remember like the order of the colors. That would honestly be a really good lifeline. The color of the booster pack. The color of the booster pack would be good. Yeah. That's what I should do because this other one's a little bit too easy. No, you know what actually? No, Soul Fusion, what Dark Neo Storm was right after Soul Fusion I think because I think purple and then black and then white is the order. Because I'm thinking Gladiator's Assault and Phantom Darkness then Light of Destruction. So I actually think Soul Fusion was right before that. So then the question becomes is I think blue comes before purple? We've gotten into colors. Yeah. On the first question. Okay. So I have to guess the year and the set specifically. Yeah, but you get one point for each. So if you only get one, you still get a point. Okay. It's tough that it's like the end of a year. If it was like in the middle, that would be a lot easier. I'm going to give you one slight hint. Just for free here. You have not said the name of the set yet. Oh, okay. So clearly I'm missing something then. How polite of you. Thank you for the free lifeline. You're the first guess you get one bonus hint. Wow. I'm way off then if I'm forgetting it. What's, I'm trying to visualize. You're forgetting a big set. You're forgetting a big one. I have to be forgetting a big one. I remember it was a rare too. Well, that's good because that's the next question. So hold on to that. Okay, okay. Oh my God. I'm kicking myself and I can't remember the set. I'm like visualizing it too. You're going to be so mad if you don't remember this. You can use, you can use the lifeline. I don't want to waste the lifeline, Ruxin. I don't want to waste the lifeline. It's not a waste if you get the answer right when you want to missed it. But I can save it for something else. But it can only equal two points at max. Oh my God. Oh my God. So if you don't want five, you talk to me into the lifeline. I'll use it. Whatever. You get to know one of the words from the set. So it's a two word set. So I'm giving you 50% of the word here. And this might help you with the year. Do you want the first or the second word? Oh my God. You're going to make me. It's all in your hands. Second. I feel like the second is going to be more descriptive than the first. The second word is strike. Savage strike. So white was before black. Okay. So I, God. Okay. Yeah. So it's savage strike. Savage strike. Oh God. Now what was savage strike right at the end of 2018 or the beginning of 2019? Oh, this is so much more fun than I thought it was going to be. God. I feel like that made it. I think it was, I think it was, I think it was early 2019 because I'm pretty sure Soul Fusion was like right at the end of 2018. You want to lock those in? And like usually they release core sets in three month increments. And if dark neostorm was May of 2019, I want to say savage strike was like the beginning, end of January, beginning of February and the Salamangrid structure that came out like a couple weeks after that, I think. So I'm going to go 2019 savage strike. Final answer. Simo. The lifeline paid off. Okay. Two points for you. Two points. Savage strike was released on February 1st, 2019. So you're exactly right with the timeline of it. Yeah. So it was, it was very beginning of February. I was worried you were going to go 2018 for a second and talk yourself into that, but you figured it out. And fortunately for you, so you're up to two points already. The second question, what is the rarity of this original printing? You seem to already know. It's a rare answer again. It was a rare. It's a rare. You're locking that in. Yep. Boom. Three points in a row. No suspense needed. Easy. You already have three points. That's going to be every card. You're going to have those two questions. So prepare for that on the future, future cards coming. Sure. Now we get to one that's a little bit harder, I think. It's so hard that I made a multiple choice because it would have been impossible just to guess. Okay. For a lot of these cards. The next question, question number three for the Salamangreat sunlight wolf. How many printings does this card have? Oh my God. Okay. So obviously without multiple choice, this would be really difficult. So you have four answers here. Your lifeline here is to eliminate half if you need it. So you could get down to two answers. Sure. Okay. So your first option A is three printings. Okay. Second option B is five printings. Third option C is nine printings. And your fourth option D is two printings. So it's three, five, nine and two. Correct. I can immediately eliminate two because I know for a fact it comes in rare. It comes in super because it had an OTS pack reprint which looked very clean at the time. And then it also comes in ghost randomly. I don't know who decided it needs to come in ghost, but it does. I can also eliminate three because I believe it also comes in ultra. I got like a reprint and one of like the sidesets as ultra. So that's four. And then I think it has a 25th anniversary printing as well which would put us to five. So it's between five and nine. So I don't need a lifeline. The question is are there four more printings of this card? The thing is I don't need to know if there's nine necessarily because I just have to know if there's one more than five because if I know there's six then the answer is nine because then it can't be five. That's true. It's also assuming that you're correct on all the printings. That is true. That is true. I really want to say it has a common printing at some point. I feel like it got a common printing somewhere. And I don't remember what, but I swear I've seen like a common version of it before. But if it also came in like the rarity collection or anything like that, that could have been the extra four printings right there because it'd been like like 25th anniversary and like all the different things. So like that's where it could possibly hit nine. You know what? I'll say nine. All right. You locking that in? Locking it in. Final answer. You were right about almost everything except the answer. You nailed every printing except you added the 25th anniversary which doesn't exist. Oh, okay. So there's a common printing in the 10 of lost memories. There's a ghost rare. There's a savage strike printing. There's an ultra rare from Armageddon. And then there's the OTS pack. But there's no 25th anniversary. There's no 25th. I don't know why for some reason I thought there's a 25th anniversary one. So I was right with the common which would have put us to five. And I think because I thought there's a 25th that would put us to nine then because it would have like the extra version. So and I'll give you one extra hint is that if it was in rarity collection, it would have seven more. So it would have been over. Oh, it would have been over because it has to have seven. So for a few and there may be some cards that have rarity collection printings. I don't even remember what I gave you, but okay. But that's okay. You're still at three points out of the possible four. So it's okay. Pretty good. That's okay. That's it. The multiple choice is one of the tougher ones. That's pretty hard. I'm impressed. I knew all of the other printings of somebody. Yeah, you were nailing everything, but that shows you how hard that is. Like if you just mess up one thing, you can miss the multiple choice. Where was the common from again? The common was from 2010, I believe. Oh, it was a 10. Okay. All right. The last question for this card, which is our price question. So I changed up how this works because there's no tiers and stuff like that this year. I wanted to be able to be more flexible with which prices we use and stuff like that. If you're within 10% of the card's price, you get three points. So you can get as many as you have right now. You can double it if you get within 10%. If you get within 20%, you get two points. Within 30%, you get one point. And if you nail it, you get four points. You get right on within like it has to be on the dollar to get the four. Okay. So we're asking about the same card. We're going to do the Ghost Rare printing because that's like the high rarity. First edition Ghost Rare from the Soul Burning Volcano. What is the price of this in near mid condition? Full disclosure, I have zero clue what this actually is. This may shock you as someone who hasn't played Modern Yu-Gi-Oh in a minute. I do know that Sunlight Wolf is extremely popular right now because Rescue Ace is a deck. It also is popular because Fire King is a deck and that's rising in popularity. So I know the card is being played outside of Salomon Grape. So it's going to have some value just because it's one of the best Fire Link monsters in the game. And by the way, your lifeline is lower or higher. So you can get the lower higher like in the previous game. All right. We're not going to waste more lifelines on Sunlight Wolf. I'm sorry. As much as I love Salomon Grape, we're not going to do that. We're going to go with Ghost Rare Highest Printing. It's still a Ghost Rare, right? It's got to have some value. But how much exactly is the question? Yeah. Like how much would I pay for a Ghost Rare Sunlight Wolf is the question. It's from what set again? It's from the side set, right? Yeah, Soul Burning Volcano that came out in August. All right. All right. I got my answer. I'm going to go $40. 40 big ones. Is that your final answer? Yeah. I think because it's a Ghost Rare and because like a lot of the Fire Dex wanted, it does have like some higher demand. If it's more than that, I will genuinely be shocked. So 40 bucks. Final answer. Well, you can proceed to being shocked. OK. This card is $88. $88. It's a one of. You only need one of it. The way you were talking about it, I thought you were going to guess like $200. No. And then you said, then you said it's a one of and I was like, OK, you might be coming back toward $80. And then you said $40. And I was like, OK, you're in trouble. So unfortunately. No points. Zero points on this one. I guess I it's still a Ghost Rare. At the end of the day, people like their Ghost Rares. It's performing really well compared to recent legendary duelist Ghost Rares. Like those $40 would be actually high. Some of them are like 20. You know, they're very low. So it is higher than those. But this one, I guess because of the playability and Sunlight Wolf is kind of a, I think a fan favorite for people who played the, you know, the Salamagri deck and stuff like that. Yeah. Yeah. So it's it's got that going for it. It's kind of one of the only good cards in that set. So that kind of helps too. Sure. Even then 88 is still pretty high for that card. Exactly. Like I feel like, OK, most of the decks that would play need one. If you're actually a Salamangreat fan, you do need like three. And so like, yeah, but no one's really playing Salamangreat even if people like the deck. And so I it could be something like you mentioned. You mentioned it might be a little inflated, which it could be because I remember it recently being in the 60s. So I think it's going up recently. And you know, I think if it wasn't like the 60s, that's like more believable. I would never play $90 for a ghost. Yeah. So I'm like, well, I'm sorry. That's insane. Yeah. That that's a crazy one. But that's OK. That's OK. It's OK. It's only the we got four more cards to move on to him. You're doing fine. These this was actually a little bit a little bit harder though. So let's see if you know. I was writing these and every time I put the card in, I'm like, Simo is going to know this card. He's going to know where it's from. So I feel like you're going to know this just because of your random knowledge. So let's see if I was right. Card number two, you're up to three points. Not terrible, but you could you have room to improve. The Frost and Flame Dragon is our next you. Oh, let's go. All right. So what's first set and and. Yes, you're in the set of the original printing. Frost and Flame Dragon was from Tactical Evolution. Get easy. And it was when was Tactical Evolution printed? That's the question. OK, let me think about this. 2000 and Tactical Evolution was right before Glidears Assault, which was right before Teladad. So 2007, Tactical Evolution 2007. All right. He had to calibrate all his series together for that answer. Is that your final answer? Final answer. Final answer. Tactical Evolution was released on August 15th, 2007. And so you are correct on both of those two points. So you're up to five points now. Secret rare six points. I think you're going to crush the year in the set. Secret rare next. I knew you're going to crush these. These are the ones I was like, oh, God, she's going to get all these right. OK. Well, you're on fire right now. That's six points in a row. Nice and quick. Mine on Frost and Fire. Frost is a multiple choice. Yeah. Multiple choice here. Number of printings. Now this one might be a little bit more difficult. This is going to be tough. Yeah. And because I did not write it down, let me make sure that I know the answer. I think I do because I did it yesterday, but time out. Wait. While you're looking that up, the reason that I know Frost and Flame Dragon so well is because I actually attended the sneak peek for it. And I remember like people think because everyone loves their chaos monsters, right?  It's like a fire, water, chaos monster, right? Not that it's good, but the artwork was just sick. And so I remember just like a lot of people like the card just because it was, it was just something different at the time. Yeah. I found this card recently in like a collection and I was like, this is one of the coolest looking cards I've ever seen. It's sick. Like the secret rare looks amazing on it. I was like, this is such an unknown card. Like I remember it was like, okay, and Duelings for a little bit. And I had kind of heard of it from there, but I didn't know what set it came from. And then I got it. I was like, this is a secret and tactical evolution. That's crazy. Yeah. All right. The number of printings for Frost and Flame Dragon, here are your answers. All right, what are my multiple choices? Yeah. A, three, B, six, C, one, or D, nine. I like how you put these in like a random order. Like, you know, just go like one, three, six, nine. It's just one, it's anyway. What thought about that? I was like, should I put it in order? I was like, no, it's more mysterious if you do it the other way. It's more sus if you do it this way. Okay. This is going to be tough actually. So I know everything about Frost and Flame Dragon prior to this question. But now, okay. The real question is who would in their mind at Konami would think, you know what, we need to reprint Frost and Flame Dragon. That's what we need to do. I will say, I do think this card has at least more than one printing. I think it has like, I think it has like a super rare or like a common reprint, maybe both. I'm pretty sure it's not just like one of those tactical evolution secrets that has like never been reprinting. It could be, but I swear it has like one extra printing. I'm going to go on a limb and say this card does not have nine printings. That seems insane that this terrible card would be printed nine times. Although- So you don't want to use your lifeline as what you're saying. No, I'm not going to use my lifeline on this. I want to save it for later. Okay. So at this point, I'm between three and six. And this just begs the question, would they have really reprinted Frost and Flame Dragon like three extra times? And like they could have. Like it's just one of those like random bad cards that they could put in one of those. Like I could see them putting it in like a star pack or like just some random set like that. Like no one actually like buys. This does feel like a star pack card.  This is so cool, right? Yeah, this is the coolest card I've ever seen. I bet it's really good. Yeah. I'm just trying to put myself into like the mind of the Frost and Flame Dragon. And like what set would this card be reprinted in? Especially back then, because I feel like there'd be no reason for them to reprint this card now. There's no way this thing has. I'm going to go with three. I'm pretty sure it has at least one extra printing that is not the secret rare. So I'm going to lock in three final answer. Okay. This was a very tricky question. And here's why. Okay. It has the secret rare printing as you listed. Yes. It has the common you mentioned. Okay. And then here's the kicker.  It has four duelist league prints. I knew it. I knew it. It was basically these three. But it's actually six because they did four of the same ones. Oh my God. I knew there was some weird thing with this. I knew it.  And it's like, he's right, except they did four at once. Yeah. So it's like, so in the three is really like that could be included into one almost, but it's four different like cards. Yeah. Because it's like the blue rare, the red rare. Yeah. It's all different name colors. So you were right. Both of these, you've been right on. You've just barely missed them. The multiple choice is very difficult. I'm so tilted. Okay. I'm so tilted. You're going to recover right here on the price. You're going to get some points. Am I? I don't know. I'm asking you about the first edition secret from Tactical Evolution. What is the price of the Frost and Flame Dragon near mint condition? I feel like I had a better chance with the sunlight wolf than this. Like this one is hard. This one is really hard. This is one of those cards that's like collectors have this just because it's like, it's a rare first-ed secret rare from like an old set. And it looks sweet. And it looks good. Yeah. Like I would say like, it's just like a neat looking card. I don't know if you know this actually. Like back in the day, this card was money even though it wasn't played. Like people just thought it looked cool. So like it was actually an expensive secret rare. That makes sense. Because this is one of the coolest Yu-Gi-Oh! cards I've ever seen. Yeah. If only it was good. That's the problem. Yeah. That would be nice too. Like now the Chaos monsters, we can't do this again. Sadly. Oh my God. First-ed near mint, Frost and Flame, Tack of... I can also see this like being a card that no one wants and it's like worth nothing. Like I genuinely could see it going both ways. Would I pay more for this than a Ghost Rare sunlight wolf? That's the actual cost. You do have a lifeline. No, we're not using that. It's a pretty hard one. I got an answer. I got an answer. Okay. For the first edition secret rare Frost and Flame Dragon. We're going to go with $125. $125. Is that the final answer? That's the final answer. Yes. Simo. Your streak of not knowing price is continuous. But I will say this is, this is a very hard card. This is one that I wouldn't have known either, to be honest. But when I saw it, I was like, I got to do this one because it's so weird. Okay. It's only $65. Wow. That's what I said. That's what I said. It's less than a sunlight wolf. It's kind of crazy. That's crazy. I thought because it's like an older secret and like especially because those are hard to find like first ed near mint, like a lot of the older cards like are kind of like in like the 80 to $100 range. And so especially because it has like the cool factor, like maybe it'll be a little bit more than that. Like I could see it, but I can also see it being worth $60. Because honestly, that's probably what it should be worth. Honestly, I think your guess is really good because it doesn't have a recent reprint. Like dualist league is, I think, no, I okay. Actually the speed dual GX midterm is where the common came out. Excuse me. That probably hurt the price. That probably hurt the price actually because before then it's it had been 12 years or whatever. Interesting. So yeah, that's still a pretty good guess to be honest. That's one of the harder cards I've asked about the price. So I'd be curious if like we went back prior to the GX box release what like the price of that card was at that point. And maybe it didn't move. Maybe it was still about like 60. I'm clicking on the TCG player graph. The last year, no, it didn't impact at all. Okay. So it's it's actually it's actually gone up. It used to be a $30 card. Wow. This year it was a $30 card. That's insane. That's okay. Wow. That I would not have expected. You know those little secrets are sometimes just insane money, whether they're playable or not. That's just like how it goes. Yeah, they're they're like super rare from that era that are worth that much. I know. So that's pretty weird. All right. Let's go on to the third card. To get every price wrong. That's what we're doing now. Yeah. You're gonna get you're on the price game. You're gonna get everything else right. You're not gonna get the prices right. We're gonna rename the series. The price is wrong. The price is wrong. I think that you're gonna do pretty well. Okay. All right. The next card is Yazi Evil of the Yang Zing. Okay. So first question is year and the set this card was released in. Well, this helps that we just did this in frog. That does help. Yeah. Set was the Oh God. The new challengers new challengers. I always want to call it next challengers, but it's new challengers. So it's new challengers and that was released in 2014-2015. It's one of those two. Duelist Alliance was right before that. That was August. So new challengers was the false set. So that would have been November. You do not have a lifeline. So it's all up to you. I don't need a lifeline. I'm going into the encyclopedia one moment. Try to like use another set as like a as a basis year wise. Necroz is 2015. Yeah. Yeah. So 2014, 2014 new challengers, 2014. Final answer. Yes. The new challenge technically has a the at the beginning, but I'm not going to dock you for that. Excuse me. The new challengers. I just let you know for future CMO encyclopedia moments was released on November 7th, 2014. There we go. Just like you said. So two points. So you are, you have six points out of these questions. You're crushing these. Yeah. Now, do you know the rarity of the original printing? Secret. So the way I do these is they have two printings and either one is correct. So this had both a secret and an ulti. So you get that That's true. I had the ulti printing as well. Yeah. And also had an ulti. But yeah, but you got the secret right. So that's three points. It also helped that I pulled this in progs. It's very silly. That'll help. Yeah. Good memories. Let's see if you can get a multiple choice question. Correct. How many printings does the Yazi have? First answer. Okay. A. Five. B. Three. C. Six. Or D. Eight. I want to say when this got reprinted in the mega tin, I think they kept it secret. I don't think they downgraded it to ultra in the mega tin. They may have, but I swear they kept it secret because they may not have been to the point with mega tins where they actually swapped rarities around yet. So it's minimum three. I know that. Now the question becomes where the hole is in the encyclopedia of CMO of what printings did it get after those initial three? I feel like it had an ultra print in like a some reprint set at one point. So that would be four, assuming that's even correct. Don't forget about your lifeline. I have the lifelines, I'm aware. The fact that sword soul was such a popular deck, it could have been that like Yazi got a reprint in some set as well at that point. Like I could see there being like a super Yazi. Maybe it was like, was there like an OTS pack printing of Yazi maybe? I might be just making that up. I feel like it's between five and six. I know there's more than three. I'm pretty sure it has at least four. I know. I think I'm pretty sure. Yeah. Logic. You're nailing it. I'm going to rule out eight. I don't think it has eight printings. Okay. That seems like officially rolling it out. I like it. I think it's five or six. I think it's five or six. Okay. You've used your own lifeline here. Yeah. I use my own lifeline. If you're right. If I'm right. Yeah. I also could have just removed the correct answer. But I feel like the fact you specifically gave me five and six is to mess with me. Or is it? Or is it? Yeah. I could have just been three the whole time and I'm just making stuff up now. I'm going to say I'm going to say five. Final answer. Final answer. Five printings of the Yazi. Simo. You've gotten one right. Five printings. Let's go. Okay. So what were the other two? You nailed it. So the ultra printing was from Ghost from the Past. Okay. And then you mentioned an OTS pack print. They got a common in there in OTS 17. It's funny. I'm like I felt like it may have not even been a super. I almost thought it was a common. But Yazi seems like too good of a card to print a common. So I thought it may have just been in the super slot. But okay. All right. It was a common in OTS 17. So you basically nailed all of them. So the Yazi's been your best one. Not bad. It's been your best one. And now the question is do you know the price of the new challenger's first edition ultimate rare near mint? No. Oh my god. Can you get within 30% to at least get one point? I don't know. I've only been within 50% of my best so far. Yeah. You haven't been close yet. But now's the time. All right. Are any like actual good decks playing Yazi right now? I don't think so. I'm like I'm so out of the loop with like just random stuff. But it's also like an ulti like Yazi.  It does those back during like sword soul era. This would be expensive because I'm pretty sure this card spiked up in price during then just because like it's Yazi. You are perfect on the Yazi so far. Do not blow it. I know I can't ruin it. Oh my god. Do I use the lifeline here to get the perfect Yazi but there's still two more cards. Two more cards but you've only used what? One lifeline? I've only used one lifeline foolishly. Okay. You've only used one and you haven't used the multiple choice one or the guess guessing the price one. Those are the hardest questions. So this is a tough one. I feel like this one is so dependent on if it's playable or not. What's your ballpark right now? Like what are you thinking? I'm in like the I'm in a low range. I'm in like I'm in like the 40 to 50 range because it's like the ulti is probably the most valuable one of the ball because between that and the secret people would prefer that but like it's not being played in anything and if it is being played in anything it's like one deck at most. I'm kind of just tempted to say like 40 but that even seems kind of high honestly. I'll say 40 bucks. Why not? 40 dollars. You locking that one in? Sure. All right. 40 bucks for the ultimate rare seems kind of high. It seems high. It seems high. He said it seems high. It is indeed high. This card is 15 dollars. I'm like this is this is way too expensive for this. Yeah. I had a feeling. This is not the perfect Yazi but you did make one more step toward perfect so this next card you're going to get them all right. Okay. All right. All right. Question number four or the card number four I should say lightning storm. Okay. Which year and which set was this card originally printed in? Okay. Lightning storm is ignition assault. When ignition assault was released is a better question. I'm pretty sure it was like in or around during the pandemic. I think it was like before which would be like I think it's like end of 2019 beginning of 2020. Time for the encyclopedia. Well this is where the whole when the encyclopedia starts to form. Yeah. Just because I wasn't playing like as hardcore during that. This is when Prague started actually. Oh yeah. It took you a while to get to the later set so. What was the green set that was rise of the duelist? That introduced starlight rares. So rise of the duelist was first. So then there's a yellow set. The yellow sets typically the fall set. So then that means ignition assault was the red set which means it was probably the winter set. So I'm going to go with ignition assault 2020 final answer. Okay. Ignition assault is correct and it was released on January 31st 2020. So you nailed it. Rise of the duelist did not introduce starlight rares though. Oh no. What's it called? Rising rampage. Rising rampage. Sorry. Yes. You were close. You were close. I thought you were about to go way off when you said that. I was like where is he going with this? No, no, no. I was going just based by the color initially. Yeah. Okay. Okay. Well you still nailed it. You got it right. Now do you know the rarity of the original lightning storm? So the original lightning storm was a secret rare but it was also a starlight rare as well. You are correct on both of those. So that might help you with your multiple choice coming up. So that is another point. You've nailed all of those so far onto the multiple choice which has been pretty tough. You got the last one though. How many printings does lightning storm have? Here are your answers. I'm ready. Option A, 15. Option B, 5. Option C, 10. Or option D, 11. Okay. So secret rare starlight rare. This is where things go off the rails because I'm pretty sure this has a rarity collection print which would add seven to that. So then that would put us to nine. I believe prior to that though, it also had a ultra rare reprint because I remember everyone was like clamoring that lightning storm is so expensive and then they printed like the ultra in some side set or something and then that like cut the price in half effectively which was good. Then it went down from like $100 to like $40 or $50. So that would put us to 10. The question is where does it keep going from there? That is the question that you have to answer. Lucky me, lucky me. I'm going to go with 11. I don't know why my guts telling me 11. Not that my guts been doing me a good job in this episode but I'm going to go with 11. Final answer. The correct answer is A, 15. Printing. Wow. That's insane. You got to remember this is Konami. Staples get printed into the ground. That's true. That is true. All right. What are the other four? Okay. Let's see. 2022, 10 of Pharaoh God's prismatic secret. Oh, the prismatic secret and stuff too. I think the ultra you were thinking of was Kingscorp which also had a collector rare in there. Then Magnificent Maven's had an ultra rare. Oh, did Maven's have two? Maven's had the ultra Pharaoh's rare. The ultra Pharaoh rare. Yeah. And wait, and the secret Pharaoh rare too. So there was four or three in there. Three in the Magnificent Maven. So you have three in there. You had two in Kingscorp. Yeah. I definitely forgot about like the special alt rarities. I think that's right. Yeah. The high rarities were made insane right there because they did two in Kingscorp and then they did three later and then they did nine in another one. So they've been going crazy on Lighting Storm. So that one was pretty tough. You were almost there. If you thought of one more, you might have bumped it out to 15. Yeah. Okay. But that's fine. You're at what? I think 13 points now. So you're doing okay. Final card. Let's see if you know this one. Wait, wait, the price. Oh, you know the price? You forgot the price. Oh, wait, no. Oh, yeah. That's the whole show. Don't forget that. Don't forget that. First episode. We're trying to figure it out. Okay. The price of the card. Let's not forget that. First edition Starlight Rare Lighting Storm. Near bit. I'm going to actually just do myself right now by saying this. I think I'm going to be the most accurate with this compared to everything else I've said thus far. And I actually- That's how I was trying to skip it. I didn't want you to get this. I have a reasoning for it. So the reason is I know that with this set of Starlight specifically an ignition assault, it got like over printed or like there was some issue with like the production of them and there's like way more copies of these Starlight Rares in circulation than there should be. So all of the ignition assault Starlights are like half the price of like regular Starlight Rares even if they're playable. God, how much did they get tanked those the question? I guess I could have maintained some value though because they're still like the highest version. I don't know. 15 printings. That's a lot. Okay. I'm going to I'm going to preface this. I feel like prior to the 10 billion reprints, I think like Lightning Storm Starlights were around like 200, 250 something like that because they were kind of like half of what like a normal Starlight was, which was like four or 500. With all the reprints, I feel like they could have gotten chopped down by like 10, 20, 30 percent. So going based off that, just because there's so many in circulation but people still like their high rarity stuff, I'm going to go with 175 and that still seems high to me but I'm going to go 175 final answer. Oh, you do have a lifeline, by the way. Don't worry about that. I'm saving that for last. I'm saving that for last. You're saving it for last. We already got this far. We got this far. You're sure you're going to save it? Yes. You're sure you're going to save it and you're walking this in? Yes. Final answer. All right. 175. You were correct. This price has been chopped down by rarity collection but it's been chopped all the way down to $97. Oh my God. Wow. Yeah. Tell me about it. I invested in this car in 2022. I bought it for my $1,000 thing for $300. That's, oh my, that's grotesque. That's, that's, oh my God.  It is very cheap now, $97. It makes sense. When you flood the market, like what do you expect? Right? Yeah. And there's like, now there's a 25th anniversary printing. There's collector air ulter. There's everything. Yeah. I think the collector, not the collector, but the 25th anniversary, like all the versions of it specifically, there's a lot of other nice versions of a lightning storm that aren't Starlight. Right. You don't need to buy the Starlight anymore, which now you can buy for $97. So maybe you do want to buy it. I guess. Yeah. That's true. Yeah. So. Wow. I thought you were talking yourself down there but that's fine. That's fine. I still thought it was just like, it's an old Starlight for a playable card. I thought max rarity, people still want it, but I think the rarity collection just affected it way more than I realized. So I mean, I can't be upset. I figured this one would be a little tricky because I just checked it the other day for my two year check-in and how I've lost $500 of my thousand dollars already just by holding the cards. Okay. You're at 13 points. Pretty good. But this is going to be your best one yet. The final card is Enemy Controller. Oh. Oh, let's go. Let's go. Year in set. All right. So Enemy Controller original printing is Ancient Sanctuary. Ancient Sanctuary came after Invasion of Chaos, which was 2004. So it's 2004. Final answer. Final answer. Ancient Sanctuary, correct, was released on June 1st, 2004 for two points. You got all eight points out of there. You nailed all of those. All eight points. You crushed it. Do you know the original rarity though? Ultra rare because I have a play say. So you're locking that in. Locking that in. That is correct. I think you got all those points too. I got all those points. I did not miss a single point on any of those. You crushed all those. So you're up to 16 points. Okay. This is where things are going to go off the rails again. You are one for four so far. Okay. The number of printings, your options are. By the way, you have a lifeline. So no reason not to use it, unless you just want to not. A, 10, B, 24, C, 18, or D, 31. Okay. I'm going to use my lifeline. So go ahead and make it easy for me. Okay. We are going to remove 10 and 18. Oh, how courteous of you. You picked the two I knew it wasn't going to be. Thanks. All right. It'll be a little too easy if I gave you the right answer. Yeah. Thanks. So for people who aren't aware, Enemy Controller has like 9,000 common printings because they printed it in like eight million starter or structured X just because it's like an easy card to reprint. I'm not even going to begin to try to list all the different variables of Enemy Controller because we would literally be here for like an eternity if I try. It's either 21 or 31. It's not that many. You mean 24 or 31? Yeah, 24. Sorry. I can't read. Okay. Honestly, I'm going to say 31 because I think there are just 31 printings of this card just straight up. Final answer. Yes. 31 printings is correct. Oh my God. Okay. First, I'm sorry. That's insane that there's 31 printings of a single card in this game. 31. I cannot believe this. When I got on the page, I was like, oh my, I had to go to the second page on TCG Player to see all of them.  All right. I'm going to, I'm going to speed run this. Battle Pack 2. Battle Pack 3. Even Arsenal Chapter 1. Battle Pack 1. Champion Pack 1. Dual Pack Battle City. Dark Revolution Volume 2. Two. And those are the 31 printings. High rarities, low rarities on some of those repeats, but wow, that is a lot of cards. You got that one right. A lot of unique rarities too because it has like the black rare. It has like the beard. Yeah. The black rare. It has a lot of like the weird rarities that only ever came out in like one particular set. So just kind of, it's one of those cards where it's like, oh, what card has the most different rarities? Like this is probably, this probably has a printing and everything except ghost in all honesty. Yeah. Ghost, Starlight, all those like really high end ones. Yeah. But on the lower end, it's got almost everything covered. Yeah. All right. You are perfect on the enemy controller. Let's see. You still have your lifeline for this final guess, which is the price, which we are not going to skip. What is the price of the first edition ancient sanctuary ultra rare? Oh, okay. It's, I thought you're going to give me the ultimate rare, funny enough, which those look gorgeous by the way. I might have to swap them out. No, I'm going classic. I do like the classic. Also, anyone who's a fan of the, was it the dark revelation super or dark beginning super? It has a really nice super print from back in the day. Yeah. Yeah. The dark beginning stuff. Those are clean. Those are clean. I just read them all, but that was a lot. I don't remember any of them. Okay. So original, original AST econ. So I get to, so my lifeline is, I get to ask if it's higher or lower than like some amount, right? Yeah, whatever amount you want to pick and I'll say higher or lower. This one's weird. Enemy controller is a card that like goes in and out of modern Yu-Gi-Oh a lot of the times, just because it's like just generically good. You are within striking distance of 20 points, by the way. If you get within 10%, you're going to get 20. Oh, okay. And I have a lifeline to help me get there. Yeah. The question is how much do people really value the ancient sanctuary Ultras? Because I feel like if you're going by max rarity, obviously now the Ultis exist, which sort of like blew all the rest of them out of the water. But I actually think a lot of people thought the dark beginning or dark revelation super was technically a higher rarity than the Ultra. So I think those actually may be a bit more expensive than the Ultra one or they're like close. So, but I don't think they're that expensive. I think they're definitely on the lower end, even if they're like seeing some play. There is one way to find out. There is, and that's with the lifeline. I am going to ask, is it higher or lower than $50? This card is higher than $50. Okay. Honestly, I'm kind of surprised it's that much, but okay. I was hoping you would say lower, and then I'd be able to get a lot more accurate, but now this sort of- Now there's a whole world of possibilities for you. Okay. Is it more than the sunlight? That's the real benchmark. I don't think they're over 100. I think that's a bit extreme for this card, especially now that the Ulti exists. I feel like the Ulti would have really knocked the price of this down. So you're between 50 and 100. I'm between 50 and 100. And keep in mind, your higher or lower is just telling you it's higher. It doesn't necessarily, it's means, you know, it doesn't mean it's $5,000. It's just higher than 50. I can also see them being worth like $52. That would be quite interesting. Yeah. I feel like a good price for this card is like 60, 65. That's like sort of where I'm at with it. Like maybe 70. Where is it? Where is the price? That's the question. All right. Will you get any points on the- Well, I get any points on the final, on the final question to be continued for next episode. If you get one point, it's a win. If you get one point, it's a win. That's how we're reviewing this. I'm going to go with- I'm trying to hedge my bets in case I'm like two- If it's lower or like higher than like what I'm thinking. Why are you hedging your bets at this point? You haven't gotten any right. I just go for it. Because I'm trying to get at least one point, Rukson. I'm trying to get at least one point. That's fair. That's fair. But if you get three, you get 20. Okay. I'm going to be incredibly specific. $63.50. Final answer. It has to be on the dollar. Oh, okay. $63.50. Okay. 63. Okay. Locking it in? Locking it in. Oh, man. I wish you would guessed closer to your over-higher or your 52 randomness. Because this card is $53. Oh my gosh. But you do get points. That's within 20%, I think. Right? So, yeah, that should be within 20%. So, I think you get two points. All right. Yeah. So, you get two points. You get 19. You were so close. We're so close to 20. So close to 20. All right, Simo. You finished with 19 points, which overall is a pretty decent performance. But according to the price scale, you weren't quite there. You're one point away from getting that $50 prize. You do get that $25 prize. So, you got something. The viewers can win $12.50. Thanks to you. Shout out to you, Simo. You're also going to win $12.50 as well. So, I tried, Chat. I'm sorry. Look, it's the first episode, so it could only go up from here. And honestly, you did well on the stuff I thought you would do well on. So, you didn't let me down. So, I'm really proud of you. It'll be interesting to watch other contestants to see maybe they're really good with the pricing, but maybe the inverse of me, where they don't know the years of the set or maybe the exact set. So, it'll be interesting to see playing to people's strengths, how it ends up working out at the end. But at the end of the day, if you don't know your prices, you're not going to win the money. So, that's definitely my downfall. Right. So, I mean, the most points come from the prices because it is guessed that price season two. So, maybe if somebody knows prices and doesn't know the original releases as well, they could still probably outscore you just because they'll get like three or four points on the price guessing, where you were getting three every time, but you knew two of the questions, but they were worth less. Exactly. If this was checking your Yu-Gi-Oh! knowledge for cards and set releases, then I would get that $1,000 easy, but... That's why I kind of want to add those in, just so like if someone like you, even if you didn't know all these prices, which I gave you some pretty difficult questions, I think in terms of prices, even if you don't know them, you could get some points based on some other knowledge. And it's really fun to go back and I don't know all this printing stuff. So, seeing like 31 prints of econ was something I've never thought about before. Insane. So, I thought it'd be really interesting for people to see. So, Seymour, thank you for coming on. It's been super fun and I'm really excited to do more stuff with you in the future, which may or may not happen. Who knows? We might be recording something after this. I appreciate it. Thanks again, Seymour. And guys, make sure you guys go follow Seymour on his channel.  If you guys enjoyed this episode of Guess That Price, make sure to click the playlist to see other videos from Guess That Price Season 1 or Guess That Price Season 2. It's only episode one, so next time we'll have, you know, an updated thing. But there will be playlists available for you guys to go check out the old episodes and the newer ones. Shout-out to Total Info Show Daxter, Puffins of Doom, Renesto Dina, America Doidster, Leo Guine, 62, Brad KK Beatz, and under-tied show Ian Musa, Junior Marding, Robert F. Thomas McLean, Cheng Leng, and Joey Castle. Thank you guys for supporting the channel. I'll see you guys in the next one. Peace.