Okay, so welcome everyone to the day three of the workshop. Today we're going to be discussing genome-free transcript reconstruction and the analysis framework we have for studying RNA-seq when you don't have a genome. And the afternoon we'll be focusing on annotating transcripts, so trying to gain functional information about the transcripts that we reconstructed. So a few learning objectives for the modules for this morning. One is just to understand the various challenges involved in reconstructing transcripts from RNA-seq data. We did cover a little bit of that yesterday, but Obi described the string-tie algorithm, and I'll make reference to similar approaches here and how it differs from doing it without a genome. And we're going to become more familiar with the different algorithms and data structures that are involved in doing transcript assembly, particularly genome-free. You'll be able to appreciate the importance of string-specific RNA-seq more so than maybe you had before, and we're going to learn various ways to assess the quality of the assembled transcriptome that we have. So as we know with RNA-seq, especially with the Illumina platform, which is really the dominating technology these days, the reads that you get from the machine are much smaller than the transcripts you're actually starting with. So we need to have a way to be able to take the short reads and reconstruct the full-length transcripts. And there are a couple different approaches for doing this, and it depends on whether you have a reference genome to start with or not. Because if you have a reference genome, you could, of course, just align the reads directly to the genome as you've done in the last couple of days. And then we have tools that can take the alignments and reconstruct transcripts based on those alignments, reconstructing the full-length transcript place-of-form sequences that are best supported by the reads. But of course, there are limitations here because you might not have a beautiful reference genome for the organism that you're working with. Or the genome you have might be a draft genome, or it might be just full of gaps, and it's still useful, but maybe you can't rely on it entirely for the work that you want to do. So we have other options. We can just take the reads and assemble the reads themselves without using a genome. And then if we have our draft genome or we have a genome of a related organism that we want to leverage to gain more insights in our transcripts, we can then align those transcript sequences, those reconstructed transcript sequences to that genome using a splice of liner. And that can give us some more information about where the introns and exons might lie within the structure of the transcript. And there are lots and lots of tools that have been developed for this over the last decade. It's been a very active area of research and development. So you have lots of tools to choose from. Some tools are more popular than others. And you've gotten to learn about certain of these, like Top Hat, Star, High Sat, G-Snap, maybe you haven't heard of, but it's another tool that was developed for doing short read alignment. And then for transcript reconstruction, we had cufflinks, and now we're using string tie, but there are lots of different algorithms for doing that too. And similarly, when it comes to assembling transcripts at DeNovo, genome-free, you now have lots of options. We developed a Trinity software, but of course you have many different tools for doing this sort of thing. So there's some similarities between genome-guided reconstruction and genome-free reconstruction, at least conceptually. The main goal with any kind of transcript reconstruction is to take the RNA-seq reads and to try to encode into some sort of a graph structure. And the graph structure will encode the sequence information, the order of the reads, the orientation of the reads, and indicate overlaps among the reads. And it's just an example of a graph structure that we might have where the nodes represent the sequences that have been already oriented, and the edges indicate the order of those sequences and the orientation and the overlap. And the goal with reconstructing the transcript sequences is basically to find a path from left to right within this order graph that's best supported by the reads or the alignments in case we have alignments of the reads. And given a path through that graph, we can then just reconstruct the transcript as just traversing those nodes of sequence in that order. And different paths through the graph would give you different transcript sequences, so different transcript lines of forms. Now one way we could do this is just to take all the reads and just identify all the overlaps among those reads. And we can just treat each read as a node in our graph and just traverse the overlaps to then reconstruct the final transcript sequences. It's a fine approach, but the problem is that it doesn't scale well. It might work fine when you have thousands of reads, but once you start generating millions and billions of reads, determining all the overlaps among all those reads can be computationally challenging. If you look at the number of alignments that you require for determining overlaps as a function of the number of reads, you can see that it grows exponentially. It's just not something that's feasible with short read data or you have millions and millions of reads that your organ would. Feel free to ask questions. This would be very interactive. So in short, this is really an impractical way of going about short read assembly. So we want to be able to avoid doing all these pairwise alignments or avoid doing all these millions or billions of pairwise alignments to establish relationships between the sequences. And so it's a very popular data structure that's been used to basically encode the information in the reads. And from that, determine which reads overlap each other in the extent of their overlaps. And it's called the de Bruyne graph. And the way you construct the de Bruyne graph is by breaking the reads down into individual camers. The camers are these short strings with a word size. In this case, in this example, we have a word size of five. But in general, it might be 25 or 30. And basically just take all these words, all these overlapping words from the reads. We just start at the one end. We start at the left side and just pull out the first camer. And each camer is going to become a node and a graph. So in this case, we're going to construct this de Bruyne graph by adding this one camer as a single node in this graph. And then what we do is we just move that window over by one base. You can see that this next camer overlaps the first camer by all but one base. It's k minus one length overlap. And we take that camer and then we add that camer as another node in the graph. And then we can just draw an edge between those nodes, indicating that they're adjacent within the read and they overlap by k minus one bases. And we do this for all the reads. If we come across the same camer in different read, we don't add that as a new node in the graph. We basically just reuse the existing node that's already there in the graph. So the number of nodes in the graph is just going to be equal to the number of unique camers that we have in our entire read data set. And once we do that, we can end up with a structure that looks a little bit more complex. But again, every node in the graph is a camer. And every edge indicates a k minus one overlap. And we'll end up with these bubbles and branching patterns in the graph anytime we encounter sequence variation. And that sequence variation could be resulting from, could be a polymorphism, because all we need is a single base change to give you a unique camer sequence. Or it could be a result from alternative splicing. Because alternatively spliced isoforms are going to have some regions that are shared sequences, but they're going to have other regions that are going to be unique to those isoforms. So any of those points will diverge and you'll see these bubbles and branches within the graph. So we built this de Bruijn graph. And one of the first things that we generally do is simplify it, because you'll find that there's these long stretches of unbranched regions. There's just one camer linking to another camer. And we can just collapse those down into single nodes, where that node just has more sequence information attached to it. Or it was basically collapsing down those overlapping cameras into one node with one sequence. So this is a much simpler graph to work with. And then we can basically traverse that graph, just like we did before. From left to right, we can find paths through the graph from left to right. And each path could give us a different transcript sequence, or could reconstruct a different transcript isoform. Now, which path you choose in the graph to traverse is going to depend highly on a number of factors. Earlier on yesterday, when Obi was describing stringtie, there was a network flow algorithm where he was trying to find a maximum flow to define what path was going to be the first path that was going to traverse. So network flow is one way we could try to address this problem and determine which paths we should choose. But there's lots of different ways. There's many different algorithms we're trying to address this. A lot of it boils down to different algorithms in computer science. Some of the more classical algorithms like, what are the minimum paths? The minimum path coverage criterion and determine what's the minimum number of paths you need in order to traverse every node in the graph at least once. There's lots of different ways of doing this. But ideally, the paths that we report are going to be those that are going to be best supported by the underlying RNA-seq data. In this case, we would have four different paths. And if we traverse each of the four paths, we would end up with a different sequence. If we line those sequences, we can see that there's differences. There's a polymorphism in there, and you also have a large deletion. So genome-free transcriptome assembly uses similar algorithms to genome assemblers. They both use this De Bruijn graph structure. But there are some important differences between how a genome assembler uses a De Bruijn graph to reconstruct genomes as compared to a transcript assembler, where it's using a De Bruijn graph to reconstruct transcriptomes. And this is actually one of the key reasons why we built Trinity in the first place, because it was about 10 years ago when Illumina, it was slucks at the time, came on the scene, and we had ways of now generating millions and millions of short reads, and we needed ways to reconstruct transcripts from these data and use them for annotating genomes and doing transcript research. And there were no good tools that were developed at the time to do this, right? There were tools for doing genome assembly. There were tools like Velvet and Abyss where they used De Bruijn graphs and they would reconstruct genomes. But if you took those tools and applied them to transcriptome data or RNA-seq data, you would not get a very good result. We really needed to have tools that were tuned to the differences between trying to assemble RNA-seq data and to get transcripts versus DNA-seq data for genomes. And it's a lot of it boils down to the tools of different expectations and they have different goals. With genome assembly, your expectation is that you're going to have relatively uniform coverage of your reads across the entire genome. There'll be depths in coverage due to GC content and repetitive regions and certain biases, but overall you expect to have relatively uniform coverage across the entire genome. With transcriptome assembly, we have very different expectations here. We have some transcripts that are expressed at very low levels. We have other transcripts that are expressed at extremely high levels. The dynamic range can be 10 to the 5th to 10 to the 7th between them. So we have very different types of transcripts in terms of the read coverage that we expect to see. With a genome assembler, if it comes across a region that has very, very high coverage, it's not going to think this is a highly expressed region of the genome. No, that doesn't make sense, right? It's going to think it's a repeat region. And all the times it'll take those reads and just put them in a separate bucket and maybe deal with it later. So when you apply your genome assembler to transcriptome data, all of your highly expressed genes end up being highly expressed transcripts that are getting put aside into a new bin and maybe not assembled at all. So that's one of the huge problems. With genome assembly, you expect to have a single contig reconstructed per gene. Ideally, you get entire chromosomes reconstructed as single molecules. In the case of transcriptome data, we expect to have multiple contigs reconstructed per gene. If we have evidence for alternative splicing. In the case of genome assembly, we're expecting to assemble large contigs, like entire chromosomes, like I said. In the case of transcriptome assembly, we're expecting not to generate megabase size sequences, but instead smaller sequences. On average, maybe a few kb and two or three kb and many thousands of these. Another key difference has to do with the type of data that you're inputting into the assembler. With genome assembly, the DNA-seq data that you're using is inherently double-stranded. But with RNA-seq data, we can generate strand-specific data. That's really critical, especially if you want to be able to differentiate sense transcription from any sense transcription. This very good strand-specific RNA-seq protocols are very popular right now. If you're generating RNA-seq on your own and you have the option to do strand-specific RNA-seq versus non-strand-specific, always take the strand-specific option if you can. A lot of times they're not perfect, but still 99% of the reads will have the correct strand after sequencing. And again, this is why we developed Trinity about 10 years ago now. It was a tackle, a lot of these problems. And Trinity has come a long way over the last 10 years to improve on this. Another difference between Trinity and other assemblers, just in general, other genome assemblers, other transcriptome assemblers, is how we build our graphs. A lot of other tools will develop one big graph and then just try to tease the transcripts apart from that one large graph structure. In Trinity, we try to partition the data, partition the reads into smaller graphs, where ideally we'll have one graph per gene. And each graph will represent the transcriptional complexity of that gene. And this works out very well for us in terms of being able to perform parallel computing, because each graph basically becomes a separate compute job. Reconstructing the transcripts for that gene represents a separate isolated compute job. And if you have a big compute farm with hundreds or thousands of nodes, we can basically fan off all these jobs onto the compute farm and have them all run in parallel. And then once they're all done, all the results just come back and you have your final assembly. So it's very convenient. So here's a general overview for how Trinity works. We originally called it Trinity because it had three major parts. And there were actually three of us that developed the code. Myself, Moranya Soarer, was a PhD student at the time. She's now a postdoc with Eric Lander. And this is actually her PhD project. And I've been at the brood for 10 years now. And I got involved in the project early on just because I was particularly interested in having good tools for being able to reconstruct transcripts from RASc data to use for genome annotation. And then there's a very talented scientist and engineer, Manfred Greb Herrer, who had a lot of experience in genome assembly. And he's since moved on. And he's now at Uppsala at the SciLife lab as a professor. So I'm the one that remained and I'm keeping the Trinity legacy going to the extent possible. So we have these three different modules. We have inchworm, chrysalis and butterfly. I wrote the inchworm tool, Manfred wrote chrysalis and Moranya Soar wrote butterfly. So inchworm takes the RNA secrets and it builds contigs from those RNA secrets. It does it very fast and it's a very draft sort of context. So it generates context but they're not in any way supposed to represent like a final product of doing transcriptome assembly. And I'll describe how this works. It just takes the unique cameras within the reads and does a greedy extension to build out these contigs. And we'll walk through the details because it's fun as well as interesting. Once we have these linear contigs and chrysalis, these linear contigs basically represent the unique parts of isoforms. So you might have a full length transcript isoform as represented by a few different inchworm contigs. And chrysalis' job is basically to find the different parts, the different inchworm contigs that correspond to, likely correspond to the same gene, just represent different unique parts of an alternative isoform gene, and it clusters them together. And once we have these clusters of contigs that represent the different parts of isoforms, it then constructs this proper de Bruijn graph structure that I described earlier. And once we have this de Bruijn graph, butterfly will come in and it will take the original reads and thread the reads through the graph structure to try to figure out what are the paths that are actually best supported by those reads in the graph. And then it will actually provide you with the final transcript sequences. So I'm going to walk through this, starting with the inchworm algorithm. So what the inchworm first does is it takes a read sequence. And as we're talking about before, we're building a de Bruijn graph. We're going to extract the camer sequences. Now we're not going to actually build a de Bruijn graph. We're just going to do the first part. We're going to just walk through and pull out all the cameras that overlap by K minus one. And that's going to do is instead of building a graph, we're just going to build a table. A table has two columns. One column is going to be the camer sequence. The other column is going to be the number of times we've seen that camera in the entire set of reads. In computer science, we tend to call this a hash table. We basically have a key and a value, key value pairs. So in this case, our key would be a camer sequence. And if we look up that key in our table, it gives the count of how many times we've seen that camera in the data set. And the first thing the inchworm is going to do is it's going to determine a seed camer that it wants to start to use to build out a contig. And the way it defines a seed is it just chooses the camer that has the highest count in the table. So we'll pretend that that's Gattica, the most famous of all camers. So we start with Gattica, and Gattica has found nine times. There's a movie made about it, so it's very famous. So it's found nine times. And we pull that out, and it's basically what we're going to do is we're going to start from Gattica, and we're basically going to build a contig sequence by extending from both ends. We do this in a greedy fashion. And so we start with Gattica, and we look, okay, well, if we're going to extend it by just one base, which base would it be? And since our alphabet for nucleotides only has four characters in it, we only have four possibilities. We can extend with a G and A or T or a C. We just have to figure out which letter are we going to choose to extend our contig. And all we do is just look up each of the four possible overlapping camers in our dataset. So the overlapping camer would start with A, and it would end with one of these four bases. So we just look through and say, okay, look up in our table. How many times do we find Attica ending with a G, and we find four times.  It's only found one time. T, we look up our table. It's not even there. So T is not an option. If we wanted to extend this with a T, we just couldn't because there's actually no camera that ended with T in our entire read set. And then go down to C, and we find that camera is found four times. Ideally, if we found one that was at the highest count, we would just take it and go with it, okay? But in the case where we have ties, it gets a little bit more complicated because then we have to break the tie, all right? So we have a tie. We basically have to look out from the G and the C. We do the same sort of thing. And C, you know, if we just go out one more base, can we break the tie? As we look up all the other camers, and we find, yes, we can break the tie. The A wins. So we're going to go in this direction. Okay. And we basically just keep doing this one base at a time until we get tie cases, and we have tie cases, and we have to go out a little bit further to break the ties. So we just keep extending it one base at a time until you get to a point, and then there's just look up each of the four possible camers, and none of them are on our table. All right? Well, if none are on our table, then we have to stop. All right? So that's how we stop extending at that end. So once we finished extending to the right, we basically do the same thing, but now we extend to the left. Okay? So just basically go in this direction, look up the cameras on our table to figure out what the greedy extension would be. Take that greedy extension and just keep on going until we run out of options, and then we stop. Once we stop, we report that contact sequence. And this would be our first inchworm contig to be reconstructed. And then we go through this contig. Any camer that's in that contig, we remove from our data table. Okay? I remove from our hash table. So any camer that we use can only be used once. Okay? It'll only show up in a single contig. Yeah. So we're going to get to that. That's why this is not the final output from Trinity. So this is just a useful intermediate. This is a useful first phase to basically pull out the unique parts of transcripts that are essentially best supported by the reads, because we're doing this greedy extension. So we have multiple options. You might have a sequencing error and you have the real base, and the real base will oftentimes have a much higher account than the sequencing error would be. Yeah. Do you have a fast way to figure out which is the highest account? Otherwise, it'd be un-squared if you have to go through the whole, right? Yeah, you just sort. You do initial sort step.  Yeah, so you only have to do the sort step once. And is this a hash table? The lookups are quick, so in a constant time. Okay, so we remove the camers from the data table, and we basically just start the process all over again. We choose the next seed that hasn't been used yet, extend it to the left, or extend it to the right, extend it to the left, remove those camers, and keep going. And eventually, your data table becomes completely empty, and that's when inchworm is actually finished. And inchworm reports all of its, the contigs that it was able to reconstruct. Now, but we do have this problem, as Francis pointed out. In the case of alternative splicing, yeah. Yeah, true.  So, but it also, I mean, it depends on the complexity of the data that you're working with, to actually have a billion reads from like a yeast, then it's a huge, yeah. Right, right. So, in the case we have alternative splicing, we have, in this case, isoform A and isoform B. They have some regions that they share, so in this case, they share the orange, they share the green. But isoform A has this yellow piece that is unique to isoform A. Alright, and if we're going to run inchworm on this, you know, inchworm is not going to be able to reconstruct both isoform A and isoform B, because every camer can be used only once. We're going to have to show up in one isoform or the other isoform, assuming that it got one or the other. If we're going to have a graphical representation of these isoform structures, it might look something like this. And if isoform A is lowly expressed and isoform B is highly expressed, we might represent that by weighting the edges. In this case, isoform B is highly expressed. We have this darker edge, let's see if I can actually use mine. There we go, yeah, this works. So we have a thicker edge here indicating that there's more read support, basically, for going this path, because there's more camers for the more highly expressed transcript. Alright, so if inchworm is going to reconstruct a contig from the camers that are represented by these two isoforms, we'll probably just give us this. Maybe start over here somewhere and then take this path and then give us the green segment. So it gives us a nice full-length contig for the more highly expressed isoform. And then later on, it might recede on a unique sequence that's in the yellow region. Alright, when it pulls out that contig, it's going to pull out mostly just the yellow bits, because these two can have no camers shared in common. Alright, every camer that inchworm uses has to be unique. Alright, so these actually have no camers in common, but they can have k-1-mers in common. Okay, so that's sort of the trick here. There's k-1 to help define relationships among these parts of the isoforms. So if two inchworm contigs share k-1, then there's a chance that they might actually be related. And we can confirm that relationship by looking at the reads and saying, do we actually find reads that actually support the link between these two contigs? Alright, that's really the goal of Chrysalis. This comes in, takes the inchworm contigs, looks for k-1 overlaps. If it finds k-1 overlap, then it looks at the reads and says, okay, is there actually reads in the data set that support the junction between these two inchworm contigs? And again, they really do go together. And if that's the case, then it'll cluster them together. There's just a general overview of the Chrysalis framework. So starting with our inchworm contigs, we integrate these isoforms based on the k-1 overlaps. We use the read information as we call it glue. The reads are used as a glue to sort of glue the contigs together. And we also use the read information here too. So if we have two contigs, maybe they don't have a k-1 overlap. In this case, it looks like they actually have overlap. But in any case, we use the paired-end information and paired-end RNA-seq data to also try to associate inchworm contigs with each other as part of the clustering. And then once we have these clusters, then we build the burn graph structure. And this is really how we end up with our many thousands of these clusters or many thousands of graphs. This is how we're sort of partitioning all the data. So then in the next... So in the final step, Butterfly comes in. And Butterfly is actually going to do the transcript reconstruction given the graphs and given the reads. For every de Bruijn graph, it first goes through that stage of compacting the de Bruijn graph. So if we have these long stretches that are unbranched, it'll collapse the nodes down into single nodes. They have more sequence information. And then it takes the reads, and it threads the reads through the graph in order to identify those paths that are best supported by the paths that individual reads take in addition to read pair linking information from paired-end sequencing. The repairs were used during the clustering of the contigs. But other than that now, it's mainly just used for the clustering of the contigs in the crystal stage and then in the final reconstruction stage using from Butterfly. Right, so Butterfly basically just gives us the resulting sequences. We just have a few examples of how this has been useful in mouse. In mouse is a nice test case for us because we actually have a reference genome. So when we reconstruct transcripts to Novo, we can always go back and look at the genome and say, okay, well, how good do we actually do? Here's a very simple case where if we look at the graph structure that we have for one of our graphs, we only have three nodes. This is almost as simple as it gets. And in this case, we have a blue node, a red node, a green node, and we have labels on the edges indicating how many reads actually support linking this node to the next node. There's really only two paths we can take in order to reconstruct transcripts from this. We can go from the blue node to the green node, and we reconstruct one transcript. And if we take the other path through the red node, and then we end up with this other isoform, or this little red piece. Red piece is short. It's only 130 bases here in this red node. And if we didn't have a genome, then all we know is that we have presumably two transcript isoforms. They're different for each other, and that one has the red piece and the other one doesn't. But we don't really know what is that red piece. Is it a skipped axon? Is it an alternative donor or acceptor splice site? An axon has it really correspond to in terms of the overall structure of the transcripts. And you really need a genome in order to be able to infer that. And in this case, if we map these two isoforms back to the genome and look at where do these sequences fall, we can see that the little red bit is actually a skipped axon. Axon that's skipped in one isoform and not the other. So having a genome, even if it's a genome of a related organism, it's really useful for trying to gain more insights into the structure of the transcripts that you've reconstructed to Novo. Can you also let us know how complete rough seek is? How complete rough seek is? For mouse. Yeah, we're going to touch on that. But yeah, so like for any of these data sets. No, no, no, no, no, no. This is an important point. So this goes into the topic of how messy transcriptomics can be. So in general, when we have reference data sets that we're working with for human or mouse, we talked about yesterday, we're talking about I think the human data set which is gen code and there's about 200,000 transcripts in our reference data set. How many genes is that? I think it was like 50,000 genes or something around that. But typically people say like how many genes are there in human and there's a contest and I think they said it was like 30,000 or so. It was the number they settled on. But anyway, we're talking about tens of thousands of genes and maybe a couple hundred thousand total transcripts for mouse or human. And when you do a de novo transcriptome assembly, you end up with a lot more than that. There's a lot of transcription that's happening and there's a lot of transcripts that you'll reconstruct. They're not necessarily included by your reference. And there's a lot of reasons for that. This is going for like hours about this. In general, you'll capture a lot of the known protein coding genes which are pretty well characterized. But you'll end up with tons of non-coding RNA transcripts expressed from different regions of the genome and they won't necessarily be in your reference annotation data set. It might be because the reference data sets are really based on well-studied tissues. Once you start digging into different tissues or even in single cell transcriptomes, you end up finding lots of novel transcription that's just not captured by the reference data set. Again, most protein coding genes are pretty well captured, but there's just tons of non-coding transcription that is presumably biologically relevant. A lot of it has to do with just transcription that really the product of transcription might fall. That's a lot of different ways. Enhancers are transcribed. There's lots of transcription that happens. It's biologically relevant, but the actual product, the actual RNA product itself, whether that actually has a specific function is highly questionable. So how many genes do we have? How many transcripts are there? These are questions that will never be answered in a way that most people will agree on, at least within my lifetime. So just be aware of that. How's that? Is that good enough for us? That's all right. No, it's good. It just doesn't aside. I work on reference data sets. I also work on lots of non-reference data sets. One of the studies that we published almost recently is the salamander transcriptome, the axolotl, the Mexican salamander. It's a model organism for looking at limb regeneration. So it's really fascinating. But the genome is huge. The genome is 30 billion bases, or 10 times bigger than the human genome. We assembled the transcriptome for this. We ended up with over 1.4 million transcripts being assembled. It's got a big genome, but how many genes does it have? It's thought to have around 30,000 genes, like human or mouse. And the genome is just huge because of an ancient repeat expansion due to mobile elements. Wait until you end up with 1.4 million transcripts. We started off with over a billion RNA secretes. The more reads you sequence, the more transcripts you'll end up reconstructing. There's lots of stuff that's very lowly expressed.  There's a lot of that, too. So, yeah, just things to be aware of. But if you start applying things like minimum expression thresholds, let's say one transcript per million, or one FBKM even, is thought to represent around one transcript per cell. So if you want to consider that to be a reasonable threshold, if you filter out all the stuff that's less than, say, one TPM, you'll end up with 100,000 transcripts, and maybe 30,000 genes. But there's just tons of stuff that you can... Once you go deep enough, you have enough material to actually reconstruct transcripts on. And you start seeing lots of regions of the genome lighting up that maybe you hadn't expected. And is it biologically relevant or not? It's maybe. Different cell types, different tissue types. There's lots of really interesting things that are happening that you'll only see once you start digging in very deeply and have a very sharp focus on that cell type. All right, back to business. So another example with butterfly. Here we end up with a case where... We don't actually have two isoforms. We actually have transcripts from two parologous genes. But these genes are highly similar. They're like 95% identical. And they have some regions that are unique and they have some regions that are shared. And we find that they end up getting clustered together into the same graph. So here's a graph and we can see we have regions that are shared here in the middle and we have regions that are unique in each. But because we have good paradigm information, we can use the paradigm linkage information. And if we had longer reads, we could use that too. But in this case, there was short read data. We can primarily use the paradigm read information to tease these apart and basically unzip these structures into their separate parologous transcripts. And this is just to show what we reconstructed and what it looks like when you map it to the reference genome. And you can see they're basically spot on with the reference transcripts. As I mentioned earlier, given the options, strand-specific data is absolutely preferred. Any chance you have of getting strand-specific RNA-seq, I'd be able to capitalize on that. And there's a couple reasons for it. And then one I mentioned earlier is just biologically, you're able to separate a sense transcription from an anti-sense transcription. And I have a nice example. It shows where that's biologically relevant. But another reason is just it simplifies the assembly process because if you have double-stranded data, then you pull out any given camer. That camer is going to be represented in the graph in both its forward orientation and also its reverse complement orientation. They basically mean the same thing. And if we have strand-specific data, then we can actually tell the difference between the forward camer and its reverse complement camer. There are actually two separate nodes in the graph. All right? So the law is to basically separate these things out. And that greatly simplifies our ability to reconstruct the final transcripts. While back, Josh Levin, who's at the Broad Institute, is one of my collaborators. He did a very well-known study looking at different RNA-seq methods that are available for doing strand-specific RNA-seq. And he settled on this DUTP second-strand marking method as being the leading protocol. Now, I'll walk you through what that is briefly. This is basically the standard way that a strand-specific RNA-seq is being done these days and are really good kits for it. So we start with our RNA sequence template. The first thing we're going to do, we're going to do a first-strand synthesis with just normal DNTPs. I generate a first-strand CDNA. But then when we do the second-strand synthesis, instead of using thymine, we throw in uracil. And we know uracil is not a standard DNA base. It's an RNA base. And if this ever ever... You don't actually find DTTPs as a naturally occurring base as far as I know. That would be a dangerous thing to have happen in the cell. But there are cases where DNA can get damaged and cytosines end up being deaminated to uracil. And that's a problem. And it's not repair able to lead to a mutation. But we have really good DNA repair enzymes that will recognize, hey, if I see a uracil and DNA, I know it doesn't belong. It'll pop it out. And then the rest of the DNA repair machine will come in and actually replace it and restore it to its original base. But in molecular biology, we can actually leverage these enzymes. And we can put them in a tube and we can sell them as a kit. And any place we have this uracil and our second-strand synthesis, we can basically remove using these DNA repair enzymes. So this is a bit of a trick. Let's take a step back here. When we do the second-strand synthesis and we include the use in the second-strand, we then do adapter ligation. You can see we have the yellow. We have the red. Y adapters. Very NHN. And if we didn't remove the use and we're going to do sequencing, let's say we started the sequencing at the yellow primer. Then our sequencing would go into the blue for some sequencing products. And we start over here in the yellow. It would lead into the pink for other sequencing products. We'd effectively be sequencing both strands. But if we use this fancy DNA repair enzyme to remove the use, this pink strand cannot be used as a template in PCR. We're visually destroying the pink strand. So now, all the sequencing that we do from the yellow has to go into the blue. We'll never see any yellow going into pink. That's really the key point here, is how we get our strand-specific sequencing. Because only one of the strands can actually be used as a template for sequencing downstream. It's really just very, very clever. And then why is this important? I have a couple of examples here. Here's an example. It's important for a lot of things, sense and any sense, of course. But if you're working with compact genomes, where the genes are very closely spaced next to each other, there's a lot of transcription, where the transcription from one gene can run off into the other gene. Then having the strand-specific RNA-C can really help tease them apart. So this is just a case. One of our favorite cases we do a lot of work with, Espombe. And here we have strand-specific RNA-C. And these are just the reference genes down here. So here we have one reference gene that's going to the right. We have another gene that's on the bottom strand going to the left. And since we have strand-specific RNA-C, we can actually separate out the coverage and look at the coverage on the top strand versus the bottom strand separately. So here we can see the top strand coverage and we can see that this transcription overlaps in the regions between these two genes. But because we have strand-specific RNA-C, we can actually tease that apart. And when we do the actual reconstruction using Trinity, we actually do reconstruct these transcripts as separate transcripts, each in their proper orientation, and they're allowed to overlap here in the middle. It's not a problem. But if we didn't have strand-specific RNA-C, what would we end up with? Yeah, one big transcript. If we look at the coding regions, we see, oh, it was the huge coding region on the upper left and there's a huge coding region on the bottom right. But other than that, we do just reconstructing transcripts. It's like one big transcript. And that's really not ideal. Here's another example. And again, in our S-Pombe vision used. In this case, we have three genes. They're all on the bottom strand. And we have our strand-specific RNA-C coverage profiles in the middle. And we can see that, at least for the genes that are on the ends here, the RNA-C coverage that we have is consistent with the transcribed, you know, the coding region orientation for that reference gene. But in the middle here, we can see for this myosas-specific protein kinase, the transcription that we have is actually on the other strand. If we didn't have strand-specific data, then we wouldn't recognize that basically every bit of transcription that we have here is antisense transcription. And it actually turns out to be important for this organism that many of these myosas-specific genes are down-regulated by antisense transcription. So it's important biology that we can capture here if we have access to the strand-specific methods. Now, when we run Trinity, and you'll have some experience doing that shortly, if you haven't used it before, the main output here is just a multifastifile. Okay, so we have nothing super fancy to look at. It's just a fastifile, like any other fastifile. There's a couple of things that are useful to note here, though. So we have the accessions for the transcripts, and I've got to update this with our current representation, but basically you have an accession value for the transcript, and the prefix for that accession value is meant to represent a gene identifier, and it's a gene in error quotes, because it's not like a real gene in a lot of cases. These are just transcripts that are clustered together, and presumably they come from the same gene, but we're still not good at teasing apart parologous transcripts from differential isoforms of the same gene, but they're related in some way. They have some sequence that's shared in common, and they'll have different isoform numbers. So in this case we have a prefix, here we have a sec one and we have a sec two, but they share the same prefix. So we know that basically from the same gene, but they're different isoforms of that gene, and they have some sequences that are shared, and they have some sequences that are unique. And we can look at the path representation here. This is basically indicating the node in the graph, just a node number, and the region in the sequence that corresponds to that node in the graph. So if we compare these two paths, we see the paths are the same, but this one here has this blue bit here in the middle, that's not in the other one. This is the sequence that's really unique to this isoform. In this example, this blue piece is actually the red piece from the mouse isoform I showed you earlier. Skip that's on and one isoform is not found in the other. So this can be somewhat useful. And there are other tools, this is a tool called Bandage that we're not going to have time to play with, but as a nice gooey interface, you basically upload your trinity facile file into Bandage, and it'll give you a graphical view of pieces that are unique, pieces that are shared, and it'll give you a pretty structure. So just Google Bandage, trinity, assembly. Yeah, so this is a pretty cool software, and they have different versions, windows, Linux, Mac, but you can basically upload your trinity assembly into this. We could try it later depending upon if we have time for it or not. And if you have, yeah, this is actually, this is a genome, this is not a trinity transcript. This would be a crazy trinity transcript if that's what it was. But basically, you'll end up with a whole bunch, like all of your genes will be shown down here, and if you have alternative splicing isoforms, then you click on one that has interesting structure, or zoom in on it, and gain some more insights into that. All right, any questions? How often do you have cameras that are completely, that are really different than just by random chance, right? So you have like a connection. All right, yeah, it doesn't happen that often, unless you have large gene families. What is K? K is 25 for what we use in trinity, and 25 is relatively unique. Once you start getting below like 19, I think you start running into trouble. But yeah, 25 is a relatively unique camera size. But you still have issues. You have olfactory receptors and mice, you got like a thousand of these things. Some are more closely related than others. Other genes like kinases, they have shared kinase domains. There will be certain domains that just show up at high frequency and occasionally have a camera that will show up. So it happens, but in all of the experiments that we've done for trying to figure out what's the optimal K value for trinity, 25 seemed to be the optimal. Yeah, yeah, sure. So alumina is pretty good. There's not too many mysteries, but we are trying to combine packed bio-data for the length of the error in the creature. Right.  Right, right. So eventually there will be a version of trinity that can handle a wide variety of different data types. But we're talking at least like a couple years away. The latest version of trinity is actually capable of incorporating packed bio-reads along with aluminum data. But these are packed bio-reads that have been carefully error-corrected. So you have to go through the whole ISOC pipeline or use some other error correction strategy. Because trinity is not going to be able to handle long reads that have lots of, you know, like 85% identical to reference. 15% error is another way of saying that. But yeah, ideally, you know, in terms of like the longer roadmap for where trinity is headed, nanopore, dirty or packed bio, and other retypes.


Okay, welcome back. We'll be moving on to our next lesson and in the just for y'all to follow through. We're going to be looking at the second chapter that is understanding human needs in your textbook. And I'm at page nine. We're going to be looking at understanding human needs through a biblical perspective. But before we go there, I just would like to take on Kennedy's question. Kennedy asked the question that Kennedy asked is please talk about palliative care when you are to terminate. What are the best steps to take? Okay. So in palliative care, that there may be times and conditions in which you may you may find yourself not really being able to completely terminate a contact, especially when it comes to issues with palliative care, terminal illness, that it may be essential and needed that you have regular follow ups with your councillors also for those who are going through, you know, challenging situations, maybe dealing with a child who is differentially abled, or maybe someone who's going through a physical disability. There may be certain situations where termination may not really take place, but then there is a follow up that happens and this follow up generally takes place, maybe once in a couple of months, where there is a quick recap of where they have been. So you wouldn't completely terminate in some of these cases. You can sometimes leave it open for a re-contact and a regrouping of sessions, maybe for a short period of time, and then there may be a lull for some point of time and then it comes back.  Some of them just leave, some of them, you know, say they don't want to terminate, but they will come back after six months. So it, like I said, there aren't any watertight compartments on how this needs to be done. Remember, this is a relationship and a lot can be accomplished when you begin to relate to people in their time of need. So even when, for example, if there are some clients who've actually terminated or who've said that they want to take a break of six months, and in between, you know, when I'm probably looking up a list of people to pray for, they kind of pop in my head and I just, you know, quickly send them a message and find out how they're doing. We may have a one-off session, just like a follow-up and then, and keep it at that. So I wouldn't say there are specific pointer steps, but you can keep it open, especially when there are long-term needs that may be there, especially for elderly, you know, elderly senior citizens who are left home alone without real support. So a counselor who's actually just helping to build contact once in a while really helps them to deal with whatever they may be going through, right? Okay. All right, we're going to get on to our fresh chapter, and this, I think, is one of the most exciting chapters in this book. And I'd like you all to see this, not from a counselor's perspective, but I'd like you all to see it from a perspective of your own understanding and understanding your own human needs and how you can grow through this. So I'm just requesting if we can keep questions to the end so that I can, you know, finish this at one floor because I don't want to break it up and take it on to the next class because the entire essence of this is lost. So if you can keep your questions to the end, we'll probably have around five, ten minutes at the end to work through some of these questions. Just give me a minute as I just share the slides. Okay. So the next chapter that we are looking at is understanding human needs. Okay, so we've kind of established what counseling is about. We know that it is a process. A counseling is based on a relationship that has empathy, that has acceptance, that has trust. And within this relationship, the counselor focuses on the client's feelings, the thoughts, the actions, and also helps guides, empowers them to cope with what they may be going through to explore options to make their own decisions, to make them responsible for those decisions. And one of the goals of Christian counseling is to bring every person to a maturity in Christ. So we've established the beginnings of this. Okay. And what we need to look at is, so maybe let me give you an example. Let's say when you buy a phone or buy some kind of appliance or a gadget, what you need to do is you really need to look back and find out how this entire thing works. Okay.  But maybe for me, on an older generation, require that manual or maybe let's say something that's new you haven't used before, you go back to a manual to understand how it works. And even in counseling, something that, you know, we need to understand is that when you are learning, or when you're learning how to deal with man, you also need to understand this man or what his personality or what these what goes on inside of the person. Okay, now that's that's a very crucial thing for us as for for a counselor to do that. So even, you know, psychologists and those who are in the secular field place a big emphasis on this thing on the fact that that one of the thing the most important things one must do before they counsel is to understand a personality or to find out a theory of personality. As Christian counselors, we know that understanding who God is, his nature, who his purposes, what he has for us is the foundation of understanding even man. Okay, and that becomes foundational in our relationship with the counseling.  Okay, so to get to get to an understanding what are we, what are we going to be looking at when we look at personality, we need, we understand that we need like I've saying we need to understand the nature of God, and we need to understand the nature of man. So when we're looking at the nature of God, what are we doing is to finding out an understanding who is God, what is our image of God, what to, where do we get our image of God, how do we understand who God is, and the image of picture we have of God is influenced by many factors. Okay, we, we understand it by the study of scriptures, we understand it through our own prayer life to our personal prayer life, through the teaching of the word that we have received. Some of some of some of things that we understand about God is what has been modeled by our own parents, or what has been revealed by the Holy Spirit. Okay, so when we look at at what is our image of God, and when we look at all that we said collectively, we see that God is eternal. He is powerful. He's perfect. He's personal. He is relational. So we see these different aspects of God. Okay, so he's eternal that he, there is no beginning and end to him. He's powerful. He has his sovereign. He has everything in his power and control and authority. He's perfect. There is no blemish in God, no lie, nothing in God that is imperfect. He's personal. That is he relates. He's a relation. He's a personal God that he, he has, you see in the Bible of how there are emotions that God displays what his thoughts are towards us.  We see that the relationship that he shares in the Trinity, the son, the father, the son and the Holy Spirit. He's a relational there and his desire to be relational to his creation also. So these are some of the attributes that we see. Okay. So we, we agree that, you know, a theory of personality is important and we understand as Christians that we base our foundation on what the Bible says about the nature of God. Now what does the Bible say about the nature of man? What does the Bible say about the nature of man? We understand that man is made in God's image. And if we really need to understand people and all their problems, we need to understand how God made man and how he, how, how he created us to be. So scripture shows us that we were made in the likeness of God. And thus you and I are seen as image bearers, Genesis 126 to 27, very familiar verse. It says, then God said, let us make human beings in our image to be like us. So God created human beings in his own image, in the image of God he created them, male and female he created them. So he created us in his image, in the same nature and image that he is, he created us. So he created us in the image of God. So what does it mean to be an image bearer, to be an image bearer God and to reflect God. So it means to resemble God when I'm, so when you look at yourself in the mirror, it is an exact replica of, of who you are. That's what you're seeing in the mirror.  Now we need to understand this because if we don't, if we don't understand this, we are greatly, you know, limited in our approach, in the way that we approach people. For it's only when we understand the design that has gone into how we are made that anything significant can be done. Like, you know, only if you know what, what buttons lead you to do something on the phone, can you actually work out a problem. So similarly, only until you know your design, would you be able to know what can significantly work out of that. So why is it we need to understand man? It is, if we need to repair something, an understanding is needed of how it is made. So the more complex the problem, the more greater or detailed should our understanding be.  But let's say with the fever, there are some hundred other things, there is a lot more of detailed tests that he needs to do to really understand what's going on. So the greater the understanding, the easier it is for us to work alongside with God to seek how we can bring back and restore the image God has for fallen man. Okay, so we understand the nature of God, we understand the nature of man that we are his image bearers, and we are created to be, we were created to be like him. So when we look at this of how God made man, we see these three specific points, okay, that people are God made man as a free moral agent. Which means he gave each one of us the will and the right to make a choice. He's given us the ability and the decisions and the ability to make those decisions. However, we don't have the right to choose our consequence. We have the right to choose what we want to do, but there may be a consequence that will follow both in this life and for eternity. So that's how we see or how we see man has been made. Secondly, man is been made as an eternal being. If you look at Matthew chapter 25 verses 41 and 46, it says then the king will turn to those in the left and say away with you you cursed once into the eternal fire prepared for the devil and his demons. So man is an eternal being and God has placed eternity in our hearts.  So God made man as a free moral being, a free moral agent where we use our own wills and the right to make choices but not to choose the consequence we were made as an eternal being. And thirdly, God has given the right for man to choose his eternal destiny. He has given us the right to choose that as it says in John 5 24. It says, I tell you the truth, those who listen to my message and believe in God who sent me have eternal life. They will never be condemned for their sins, but they have already passed from death to life. And that choice is something that God has given each one of us. So to understand this is important for us as we move ahead in knowing what are the needs that we have. So as we were explaining this, what does it mean to be an image bearer of God or bearer of God's image or bearer of divine image? Now if we look at this, we try to represent it as best as possible. To be an image bearer like we figured out and we understood is to resemble God and reflect his nature. Now God, before the fall, prior to the fall, Adam and Eve or man was perfectly valued, perfectly loved and was perfectly significant. This were inherent attributes in man because he bore the image of God. I know sometimes it gets very difficult for us to understand this because right now in the fallen state that we may be in, we always need to be loved, to be accepted, to be significant. But because of what Jesus did for us on the cross, we experience this perfectly loved, being perfectly valued, being perfectly significant. Yet there are glitches in that, right? So prior to the fall, Adam and Eve never experienced this need of wanting to be loved or wanting to be valued or wanting to be significant. These three things was already inherently there in them. But this changed when fall happened, when sin took place. But they still do have the capacity for security or significance and worth, but they have no way of actually satisfying it. So as the bearers of divine God's image, we have these inherent attributes that were perfectly loved, valued and having significance or having meaning and purpose. But it all changed when sin came into this entire equation. Now quickly, just to help us understand, and I know some of this we dealt with in emotional wholeness, but just to quickly bring this together. There are passages in the Bible that clearly establish the fact that man is a triune being that is composed of the spirit, the soul and the body. And some of those that we spoke about was 1 Thessalonians 523. It says, I pray God, your whole spirit and soul and body be preserved blameless unto the coming of our Lord Jesus Christ. So we are made in his image in this way. Now, just to quickly bring about and establish some difference between these three is, let's look at the spirit first. When before sin or before the fall, the spirit of man was one with God. The spirit of man was one with God. And that's where I spoke about, we have those inherent attributes of just being loved and valued and having that purpose. There was never a question about it because the spirit of man has was joined with the spirit of God. But when the human race fell in Adam, sin closed that window of the spirit. And that part of the spirit became like a death chamber. And it remains so in every unregenerated heart until the light and the life giving power of the Holy Spirit floods this dead chamber with the life and light giving power of the new life we have in Christ Jesus, right? So we were one with God in spirit where we had value and love and acceptance. When the fall happened, it became like a death chamber until a point of time we face regeneration by the power of the Holy Spirit because of our new life in Christ Jesus. So it is then that the spirit of man becomes the sphere of God consciousness. It's that inner or private place of man where the work of regeneration takes place. So that's the spirit, okay? Now when we look at the soul, remember man not only has a living soul, but he is a living soul. So the Bible says in Genesis 2.7, and the Lord God formed man of the dust of the ground and breathed into his nostrils the breath of life. So he became a living soul. So we must be careful not to confound that which is spiritual and that which is soulish. We have seen that the spirit of man is the sphere of activity where the Holy Spirit operates. Just so is the soul, the sphere of activity where Satan operates making sure that the affections and emotions of man are targeted. So Satan knows full well that he dominates the soulish part or the psychical part or the part of our minds. He knows that we as his victims are people of emotions and for him it doesn't matter whether you cry or you are sentimental. As long as your spirit does not come in contact with God's spirit, he continues to attack the soulish part of it. So the soul is the seat of our passions, our feelings, our desires and Satan is satisfied that he can master these in this area. So it is also the seat of affections, the right or wrong of love, hate, lust, the appetites of our body. Like you know it says in Peter, I think it's 2 Peter 211, it says, abstain from fleshly lusts which war against the soul. So the soul of man has his affections and desires which are not directed to God until after the spirit has become regenerated. So man cannot love God nor the things of God until he is born from above. A man may have a troubled conscience or be stirred emotionally but still can remain dead in trespass and in sins. So man's desires and affections are turned towards God only when he realizes his sinful condition and how God's grace is there in salvation. So when the spirit of God illuminates the spirit of a man with light and life, the man begins to yield his affections of his soul, desires, affections of his soul to God. So it is important to understand this and of course man being a triune being, he also has a body in which we have the interaction with the outside. And we will talk about this a little more in length. So when we're looking at us being the bearers of God's image as image bearers, how do we resemble and reflect God? And there are five areas of functioning that earlier, this is what I was, Sam I was talking about these five areas of functioning. So as a bearer of God's image, we have these five areas of functioning. The first one is our spiritual being. So as God is spirit, this finds expression in man as the image of God. So at the core of our personality lies an area of being that can function effectively only when the spirit is in contact with God. There is that God shaped vacuum and we have that deep yearning for a close and that intimate relationship with God. So that's the spiritual being and this is there even in everyone, in every person God has created. There is a place of vacuum that only God can fill and there is that deep yearning for that close relationship with God so that they can bear that image. So that's the first one, that's a spiritual being. The second is the rational being. The rational being is man having the ability to reason, the ability to understand, the ability to comprehend and scripture places a big deal or a great deal on the emphasis on man's ability to think. So we are spiritual beings, we are rational beings, we are volitional beings. What is volitional beings? The volitional being is the part of you or the faculty where you have which gives you the ability to make a choice or faculty of choosing the will. So man like God has the power to set his will to choose and even to move in different directions. So man's not a puppet, God didn't make man as a puppet but was given a free will and many elements or things that will influence our decisions. But the Bible is extremely clear that we are responsible for those choices. You are given the free will but we are responsible for those choices. So God has given man a will to choose and to decide. The next part is man, God has made man as an emotional being. Now we see that even God is an emotional being and some of the examples that I can bring up is in Hebrews 4.15 it says the high priest understands our weakness for he faced the same things that we did and yet he did not sin. So Jesus he faced the same emotions that we are facing and he went through those temptations and tests of time but yet he did not sin. These emotions play a big role in determining whether our life is meaningful or miserable. So we experience emotions which we were originally designed to experience. God designed us to experience those emotions just like how he has those emotions. And lastly is the physical being. Now God does not have a body as he is spirit but in his creation of us as human beings the image of God is designed to function in a frame. It needs a frame to function through which our spirits and our soul can be effectively expressed. So when we say we are bearers of God's image there are these specific five areas that we function from. Now I will get into detail as to why that is important. Now when we look at being bearers of God's image what we are also seeing is that we carry, when we say we are image bearers, we carry in our nature the image of God our Creator and this reality is the foundation of all Christian counseling. That God in making us like himself what did he do? He bestowed in us something called as dignity. He bestowed dignity upon us. But because of the fall people are not only dignified they are also depraved, they are wicked. So when man sinned the image of God was damaged. The image was never lost but it was marred and it was corrupted by sin. Which means the design, this image or this design that we have been given has been violated by sin and we become self-centered. There is a lot of self-concern and there is an attempt to make our lives work by our own effort rather than dependency on God. I am just going to bring this up once again. We are God's image bearers and we carry the true nature of God, of our Creator God and that's where we see that yes I am an image bearer of God. But because of the fall, so when we are his image bearers we are given that dignity. But because of the fall we are not just have dignity but we also are depraved. And when we sinned that image of God became damaged or became marred or became collared, became blurred. It did not go away, the image was never lost but it became marred and was corrupted by sin. Which means the design has been violated by sin and that's why there are times of being self-centered and feeling that sense of self-preservation. Which is an attempt to ensure that we can work out things on our own life and don't require the dependency that we need from God. So what happened? When we look at, if you remember the earlier slide, as a result remember before the fall we had those innate inherent attributes of being loved, of being valued or belonged or secure and significant or having a meaning and purpose. But as a result of sin these attributes have become needs. So people still have the capacity for security, significance and worth but they have, because they have gone away from God they have no way of satisfying it. So what they do is there is that urge or the drive to meet that need of security, to meet that need of significance, to meet that need of love. And it is looked all over the place but not to God. And that's the fallen nature of man, that we depend on everything outside in the sin-fallen world to make us feel those needs, those attributes again. But we find that we fall into further sin. And these unmet needs, you know, that need when, so let me give you an example. So for people who don't feel loved, look at multiple relations for where they can feel loved. People don't find value in themselves, they hope their accomplishments will give them value, their money will give them value, maybe a relationship will give them value, maybe a name will give them value. Or at a place where there is no significance, they're looking at different things in life to give them that place of significance and purpose. They may be going in lengths to find this in order to find that sense of significance and security. So these, and it becomes needs and man continues to look for it all over and these needs become powerful motivators. So remember that the needs we are talking about becomes very vital to the effective functioning of a personality. So unless these needs are met, you don't function in yourself as a person or as a personality. So to quickly help us understand how needs become strong motivators, so let's look at a very common example.  So there is a need, you feel thirsty and the desire becomes the desire to quench that thirst becomes a motivation. And that leads to a certain behavior where you probably go and get yourself something to drink and the goal is you don't you don't thirst anymore. Okay, so this is a very common example. So similarly, if you were to look at it at some of the needs that man has we were talking about the need to be loved. The need to feel secure, the need to be significant. So let's say here there's a need to be loved. The motivation is, you know, the desire or the, the, the, the, the, the pool to have that that sense of emotion to be fulfilled. And maybe the, and then that leads to a certain behavior. So there's probably, you know, maybe in, in different relationships, they are, they're hoping to feel that sense of need till they reach the goal of having fulfilled that specific need within them. Okay, so when we look at needs, we need to understand why do people need to live effectively is because they, because it's important for them to fulfill their needs. It's important for them to fulfill these personal needs that are there for them. So, and I'd like to categorize these needs into three kinds or bucket it into three kinds. So the first one are those casual needs or those casual longings. Often when these are not met, you know, life can just go on the same. It doesn't, it doesn't really matter. And I will bring about a certain example, certain examples to help us understand. The second is those critical needs that if these are not met, life can be a little difficult and, and a bit strenuous. And the last is those crucial, most important needs that if these are not met, the person is God just not able to function. Okay, let's, let's look at these one by one casual needs, the casual longings of a heart, what are those casual longings of a heart. So, for example, you know, these are these are some those needs that ranges from being very trivial to significant like for example, you know, I wish I can have chicken kebab today. Or I wish, you know, this restaurant is open for us to eat today. I hope it doesn't rain today. You know, I hope my people all like my cake, right. Life is really not affected by these, if these needs are not met, if they're not met, you experience some kind of discomfort, but you're able to go along. Or let's say, you know, I wish I go to the shop and get that bright color top I want. It doesn't affect you as much. So these are those casual needs that one may have. Let me give you an example. For example, you know, you booked a holiday in the summer for a break to a resort on the day of your travel. Let's say one of your family members come down with high fever and is unwell to travel. You may be disappointed, right? Or you may be sad that you couldn't do it, but it's not going to impact you as much. So these are casual needs. All of man, we all have these casual needs. Let's look at the next one, which is the critical needs. What comes in these critical needs? Critical needs are those that are often related to relationships, to money, to career, to shelter. Like critical longings are those that are met in your relationship with maybe your spouse, with your children, with your friends, all of which are important and legitimate and their important desires for quality of life and for the enjoyment of life.  And these become critical. So if this becomes unmet, one will definitely experience some discomfort. Sometimes the sorrow lasts like, for example, when you lose a job or when you lose a loved one, the sorrow lasts for a while, but recovery can happen over some time. They are critical. It does matter to you. It makes a difference if it isn't there, but it doesn't throw you completely off balance, right? And maybe let's look at an example of that for us to understand that. So the company that you work in has decided to retrench and you're sure that your job is at stake. You've always managed with a single income, but now with two kids at school and a huge loan to pay off your concern. So you do experience that sense of worry and concern and difficulty as to how things are going to manage. So these are what are called as, sorry, the critical needs that are there which needs to be fulfilled. The next one we're going to look at, which is the most important part of us as human beings is those crucial longings, those important needs that require, that needs to be fulfilled in us for us to keep going on. So these are the most basic and profound longings of the heart. And these desires or these needs must be met if life has to be worth living. So nothing can fill that core except what we are designed to experience and that's what we were designed to experience. Nothing can satisfy these crucial needs that comes from a kind of relationship which only God offers. When these needs go unmet, people experience that spiritual, emotional and psychological problems.  But because of sin, this becomes needs and this becomes our crucial need and the need to feel secure, the need to have that sense of worth, the need to have that significance. And that is something that if goes unmet gets people into having spiritual, emotional and psychological problems. So these crucial needs is the security, the self-worth and the significance. And as human beings, as we grow up, we find this in our relationship with our significant loved ones, maybe our parents or people who we grow up. And that is what becomes a window of God's love to us. However, we realize that the relationship that we have with our earthly people can never be perfect as our relationship with our heavenly Father. So what happens is these crucial personal needs, these needs of love, acceptance, self-worth, security, significance becomes needs that become strong motivators. Because every one of us longs for something. Every one of us longs to have these things met in us. What does the Bible say about longings or needs? God never condemns us for those needs. God doesn't condemn you for being thirsty. He says, is anyone thirsty? Come and drink. Or the scripture that shows as the dear pants for brooks or water brooks. So my soul pants for the living God. So we all long for something. We all long to have these needs met within us. So all of us long for something. All of us, everyone is controlled by what they long for. When there is a need to be loved, we go searching after it. And any of these needs that are not satisfied, like we said, leads to those problems, spiritual, psychological, emotional problems that come about. So to understand how do we see these needs? How do we see these needs even in our councillors? Where are they at? What is the needs that actually motivate them? What are those crucial needs that they may need to have? So just quickly, just to help us to understand through a case example, so that we can... This is packed in an actual case so that we understand what are the needs you feel this person has, Miss S. So Miss S comes to the councillor for help to be more confident in her job. Her mental state reveals that she has feelings of sadness and a discontentment to life. Her past history reveals two broken relationships. Her childhood had been difficult with her trying to cope with a father who was addicted to alcohol, which led to severe discord between her parents. Her work was being affected due to her mental state. She is due for a new job interview and wants to give it her best shot. So what needs do you assess this young lady to have? So I'm just going to help us understand this so that, you know, we'd be able to pick this easier. So what is her emotional state? She's sad. She's discontented. She's dissatisfied. There's anxiety. There is fear of not being able to reach her goal. So there is a sense of a lack of self-worth. And we see that there have been problems in the way that she is related to her alcoholic father. There's been discord there. So she's grown up not being loved, not being secure in a home. And that becomes a need. And she's looking for that significance in different things. She comes to help find confidence in her job because in some scheme of understanding, she probably feels that the job is what is going to give her value, is what's going to give her significance, is what's going to bring her about self-worth. So her lack of self-worth, her lack of security and significance, she's hoping to find it all in this new job which will give her this. Yet the goal of meeting that need is uncertain. She's not sure if that's going to go. So we assess and see that her crucial needs is what she's gone through. As a result of the different things she's gone through, she's in a place where she has none of those crucial needs met. And she's looking and hoping to find it. So this need, it becomes a motivation to find a better job so that she can have a sense of worth, have a sense of value, a sense of security, a sense of significance in that. So that's just to give you an example of what that is. So like we said, the crucial personal needs that God has put into us is threefold. The sense have to have security, to have self-worth and to have significance. It's on that that we stand and we get that in our relationship with God. So as we said, the fall breaks all of that and these things becomes our needs, becomes our motivators to keep moving forward. Okay, I'm going to just move fast. Okay, let me go through this. So I want to explain these three crucial needs. So here's an example.  So what need does this evoke? So what need does this evoke? There is a sense of insecurity. Security is a place where you sense being unconditionally loved and having a sense of belonging. So for a person who's lost everything, everything, there is a place where there comes that place of insecurity that the person experiences. And we see that even in certain, you know, when we look at scripture, it shows, you know, where Jesus promises living water and he gives us an invitation. He says, anyone who is thirsty, come to me. Anyone who needs out of that living water to come to him. Or again, in Psalm 103 verse 4, it says, he redeems me from death and crowns me with love and tender mercy. So he's the one who brings about that security. Even, you know, there are times where you hear David saying that in Psalms. It says, I thirst for you as a parched land thirst for water. So, you know, if you look at that line that we keep discussing as we've spoken about our identity, it says, you don't know who you are till you know who's you are.  So for this person that we spoke about, you know, one lost in the earthquake, the sense of security has become the people, the things, the values around. But when we begin to know that we are gods and our identity in him is always what carries us through, we become strong in our spiritual and in our emotional selves. Okay, so that's the sense of security, the need to be unconditionally loved, the need to belong. The next need that we are looking at is the need for self-worth. So again, an example, imagine a scenario where you were an indispensable part of the organization you worked for. Soon, one came smarter and better than you. Your organization says they are in need of you no more. And this is, you will have a lot of people come to you like this. What have they lost? They have lost their sense of self-worth, then sense of value. Okay, the sense of value that helps them think that they are of some importance. And you see that often people base their value on the wrong things. Like this quote, it says, I am not what I think I am. I am not what you think I am. I am what I think you think I am. So a lot of times people base their self-worth on what others think of them. So if others don't think well of them, then they don't think highly of themselves too. And then they begin to look for people or places where they can fulfill this sense of self-worth and value. But the truth is our self-worth comes only from our identity in Christ. And we understand that as believers we understand that our self-worth comes only from our identity, from what we have in Christ. Okay, the last need that we have is the sense of significance. Now imagine a scenario where you as an aged parent have been asked by your child to leave the home and live in an old age home. Since you are too old to be of any use to your grandkids, what do you think you have lost? You have lost a sense of significance or a sense of purpose. What is significance? Significance is a sense of meaning and purpose that we have for our lives. What am I here for? What is the reason that I am on this earth? And this is something you would see across people.  I did all that I wanted. I splurged. I had pleasure. I did all that I wanted. But I've come to a place on not understanding what the meaning and purpose of my life is. Now God has designed us in a way that we can function effectively only if we stand on the purpose that he gives us. Without that, there is nothing that we have for ourselves. So we see that the sense of significance, our real worth comes from the fact that God has something for us to do. Ephesians 2.10 says that we are God's masterpiece created in Christ Jesus to do good works and something that he has planned for us to do long ago. So that significance comes only when we know that God has a specific purpose for us. So when we are looking at people, when you're looking at your counselling, what you're looking for is to understand where they are in these crucial needs. It is either that they are looking for a sense of being loved, sense of being significant or a sense of value. So quickly, I will be done in five minutes, just the last part of it. Now, in order for us to understand why problems begin in the person's life, it is, like I said, it is necessary to examine these five areas of functioning. So our physical being, we need to remember that the body is prone to dysfunction and disease. So anything that disturbs the thought or the soul can have an effect on the body. So we cannot, as a counsellor, we should not be ignoring the physical aspect. Because if you ignore that aspect, you may be missing out a very vital component which can be contributing to a person's problem. So the physical being is the area of functioning that we need to look at as we work in through counselling. Then comes, yes, the emotional being. The emotional being, as we said, is, like we said, it plays a major role in determining whether our life is meaningful or miserable. And we need to come to help to see, help the individual see that emotions are experienced because we were designed to experience it in the first place. But what we need to understand is to look to take care of those signal emotions. What do we mean by signal emotions? Sometimes there are, these signal emotions arise within us indicating that there can be a malfunction. And they are often compounded by a refusal for us to face it and feel what is going on as part of us. For example, let's take, sorry, let's take anger and resentment. You know, we believe that we must reach a certain goal in order to feel good about ourselves because something has been blocked by someone or something outside of us. And the usual reaction we have is anger and resentment. Something is blocked and we feel someone is responsible and we react with anger and resentment. And that is a signal emotion. And that helps us to see that something is going wrong. Like for example, when the wife thinks that her worth is only in having a career or earning an income. And when that becomes denied, she becomes very angry and resentful because there is an undermined goal. Or let's look at guilt or shame or embarrassment or self-pity or contempt. When the goal we are pursuing is not attainable or the failure to reach it, if we fail to reach it, it produces that sense of guilt and shame. This is because we believe that life is found by our own efforts. And when we fail to reach that specific goal, we face guilt. We face shame for not reaching that goal that we have wrongly set over us. It becomes an unreachable goal. Like for example, parents putting pressure on their children to perform leads them to a place of guilt and shame because they feel it is unreachable. And that becomes a signal emotion within them that needs to be dealt with. Or the last one of anxiety and fear that the emotions often begin as feelings of doubt or apprehension or uncertainty. And these arise out of some failure that we may not be able to reach. That failure that we won't be able to reach that goal, which makes us feel a sense of anxiety and fear. So though the goal is reachable, we are plagued with uncertainty. And there are those uncertain goals. For example, you're not willing to do an exam because you don't trust that you're going to do well. And as a result, you don't take it up. So a counselor who pays little or no attention to this emotional part of it does not regard the person as an entire being because that is a very important part of their personhood. Next one we said was the rational part, the rational being of a person. The rational, you know, scripture says as a man think it in his heart, so is he. So it is to know that the person has the capacity to think the counselor needs to help the person to think and search for solutions. The often the wrong thinking of the human mind goes unchallenged. What have been their process of thoughts, you know, thinking that they may need a job to make them feel good about themselves. They may need to pursue fame to make them feel significant. These are wrong patterns of thinking. And by overlooking that, we can be, you know, we're not really helping the person holistically. So to deal with them at their rational parts of it. The next one is the volitional being. The volitional being, like we said, you know, we have wrong beliefs. When we have wrong beliefs, there are wrong goals. And behind most of the problems, there is a wrong goal. If there is no understanding of the fact that the person can choose a counselor will fail to help the person to responsibly make a right choice to change what is happening in his life. So just like God, you know, just like God has the power to set his will, he gave us the place to choose and to move things in a certain direction. So we need to help the counseling to make those right choices because when when we think properly or when we think right in alignment with God, we are also able to make those right choices. And the last one is being a spiritual being. Now being a spiritual being only when it becomes clear why people act and behave the way they do because of these strong spiritual needs of security. Of self-worth and significance. When we begin to understand that this is their core spiritual crucial need. And then when the counselor pays attention to these deep longings, you know, we begin to help them at the core of their personality. We begin to help them see that this is what is unmet and this is what is taking you to this place. You know, maybe there's a sense of feeling unloved and that's why you're trying out multiple relationships. There is a need to feel significant and that's why you work 24-7 at your job losing everything else to it. So helping them to come to a place of understanding that. So the three central convictions that we have when we look at the personality is that people are made in the image of God with these five clear areas of functioning. That sin has broken and distorted this image. And what does counseling do is to work with God to restore them back to that image. So that's what we are aiming at as we do that. Okay. I will move away from the slide just get on to the last. So what are we doing to restoring a person back to their image? We're looking at these five areas by physically. You are looking at what is a vital component that contributes to their problem. Emotionally, you're acknowledging their feelings. Rationally, you're challenging the wrong thoughts or the wrong patterns of thinking and thereby helping them to make a good decision where they take responsibility towards that change. And help them in their spiritual place of the core of their personality. So when you're looking at counseling, you're basically looking at following this specific path. Okay. Now what is there are some often there are two parts that we generally follow. The first one is the path one is one holds that God fills the circle from outside in. First, he makes us comfortable. We trust him for health and wealth. Then as that develops, maybe our relationships improve. Our marriages are strengthened. We learn to enjoy our money or sexuality or personality as we relate to others. Then together as a community, we praise God for his goodness. His blessings leads us to a presence. But this is not a path to maturity. When we think that if everything our casual needs are met, then our crew critical needs are met and then there is satisfaction. This is not the path to maturity. What is the path to maturity? The path to maturity is when the deepest fullness comes from our knowledge of God. And that we find our significance, our worth, our security by pursuing God. When we have a fellowship with God, our fellowship with others and things around us becomes better. The latter becomes perfected and the total our total satisfaction does not work on until we reach heaven. But then it comes on forever. So if we have our crucial, our most inner needs met, the other things fall in place and our satisfaction comes by knowing that he is the one who fulfills us. Like in scripture it says, seek the kingdom of God above all else and he will give everything you need. So what is that place of maturity that we are hoping our clients to bring in is to restore them back to that image of God. To reconnect their spirits back to God. Wherever that insecurity or inferiority or insignificance comes about is to bring them back by restoring and reconnecting their spirits back to God. So that their problems or their psychological problems come to a place of null and void because they have seen every one of their needs being met in the presence and in the power of God. Yes, I know I've done actually a really quick marathon but it really helped to work these out together. I know we are off time, we are five minutes into time but is there any specific questions that y'all would want to bring up? Any specific questions? Okay, Elisha said, counseling in effect can be an effective evangelism too. I think when God when done with Christ focus, absolutely. You know, that's why when we were speaking about difference between Christian and secular counseling, Christian counseling deals with the heart of the matter with deals with these crucial needs that the only thing that can meet us is God and our identity in God. Everything else becomes a chasing after the wind, right? So that's the huge and biggest difference in this that when we begin to see that we don't have to earn our security, our acceptance, our insignificance, we don't have to earn it. And that's what people don't see, those who do not belong to Christ don't see. They attempt to earn it on their own efforts, maybe in work, in relationships, in sex, in money, in looks, they earn it. But for those who believe we are, it is there inherently given to us and we live a life out of fulfillment out of that, even though there may be issues with relationships, even though there may be issues with other things around, maybe not a good job or physical disability, whatever. But because of what comes from within, we live fulfilling lives and that's what we are hoping to focus on, right? Any questions? It gives me goosebumps when I keep doing this lesson because it's so beautiful that we can experience this. Yes, I will, I will ensure that you can have the presentation. Yes, Sam, you'll go ahead. I think you've lifted a hand. Thank you. Thank you, Prasad. Prasad, I think like you mentioned, one is it's such a crucial concept to understand and you had just this one hour, so I don't think it's fair to... I'm thinking if it's possible at all to just spend some more time on this a little bit next week, if at all it's possible. Sure. Yeah, I think I'm dealing a little bit with needs, you know, so like the thoughts around needs, I mean one perspective is we have these needs and hence we act out of these three nature of needs. Yeah. A lot of things that are acting out of these needs and I'm thinking, so there are these other school of thoughts, especially the secular school of thoughts and borderlining to Buddhism as well, which talks about all of these needs are actually illusion and the one solution is to kind of strip oneself out of these needs. And so that's one. But even the other thing is like, so as, you know, so I think as Christian counselors, if our goal is to help our counselors to rightly adjust their displaced needs to the source, who is God. So in that, so there's the danger of, you know, displacing it versus, you know, I'm imagining a person who might say like, I don't have the need to be loved. Like, you know, like, I just don't feel that need to be loved. I can love people and I can serve. So maybe a person like so how do I identify like I don't have a need of self worth. Like, could they be people who select, you know, I don't have a need for self worth and I don't need to be recognized. I just, I'm happy with who I am. So I'm having like, does this do do these needs. Like one blanket rule is to say that yes, everyone has these, which may be true. So exploring that like, is it an absolute truth or could there be people who may have the needs but they might on the surface might might be claiming like, you know, I don't need a self worth. Yes. It's a lot of thoughts around that. Yeah, so we can I think we will probably spend maybe half an hour next week, a one clarifying doubts and maybe just going through this quickly again. So if you all have questions and I'm sure a lot of you have them as you're thinking about it, please read through the second chapter, I will add in the notes as well for you. I mean the presentation as well for you so read in, put down your thoughts and your questions and we will handle it to the first first couple of minutes in the next class so we'll do that. Just to Samuel just to add to what you said is that these are attributes and needs that universally man experiences, even look for those, you know, like many of when you're looking at Buddhism for those who you're saying that they're they're called to strip themselves of their needs. Now that that seems a lot intellectual in its in its way. But emotionally is it something that is that is practical because we were all wired with that that need to be loved need to be maybe in it's in different degrees maybe some requires more significance but then these are those three crucial ones. I'd say, and I think if let's say a person who does not have the need to be loved, there seems to be a significant issue in their personality, or as a result of their upbringing or a result of their experiences in their lives they've just shut that need because it's never been fulfilled for them in their growing up so it becomes like an absolute dead chamber for them that they don't explore it because it's never been. It's not something that they've ever experienced, and they've come to lie and deceive themselves that that they don't need to be loved, or it isn't something that is an important component I would see that there definitely has a history to that. And so if you I'm going to use loosely use this word called sociopath that people generally use. If you look at those who have a personality issue like that definitely have difficult childhood experiences. They have unemotional experiences in their childhood that makes them to understand that love is not an emotion that should be felt. So it's a deception it's a lie that people stick on to when they may come to a place of understanding that they don't need it and so they are free from it. And that makes them even more vulnerable to a place of of of raw of sin or wickedness because nothing comes out of love or there isn't there isn't any anything that drives their actions there isn't even a conscience that that's actually working which means it's completely dead and in spirit, but this is something. So that's how I'd see it. Maybe I'll also mull over that question and see if there's, you know, in any other way that that that can be answered as well. Okay. Yeah, thank you so much for for staying in this long and for your patience. We will meet again next week. God bless. Thank you.