So, next we're going to look at how we can use the Integrated Genomics Viewer in order to visualize the results and to them. There are two types of files that we can visualize and that we need to visualize in IGB. The first ones are the top of alignment, and the second ones are the transfer. The GTF files of transcript assemble with. Both types of files will need to be indexed, however, to allow easy access to random locations in the visualization tool. So first I'm going to illustrate how we can sort and then index these files, and then I'm going to show you how you can load them and analyze them in the IGB. For the purposes of illustration, we're going to use one of those examples. As you might recall, The gene TMEM 215, which we identified as being deferentially expressed between control and test samples via. So we're going to remember the name TMEM 215 on chromosome 9. As well as the logos name assigned by S lock 057616 might be needed. We'll start with the transcripts files because that's slightly easier to handle. So, we have the transcripts file in the directory Cufflinks. And you might recall that each sub-directory contained a transcript assembled from that particular sample. So now, let's make a smaller file, let's only expand the transcripts corresponding to chromosome nine. And I'm going to do that using a simple trick. Starting with control, cufflinks, like that. Cufflinks/Ctrl1. The name of the file was transcripts.gtf. I'm going to use here a problem that's called awk. So I'm going to cheat here a little bit. And I'm going to say, I'm only going to repeat those lines that show chromosome nine in column one. Save con 1 yes, sets chromosome nine, I'm going to save that. Usually, you should be able to work with the entire file, but this further narrows down the input to the visualization tool. So let's check that, below there. So, indeed, we're getting only chromosome nine, and I'm going to save that into a file that I'm going to call crtl1.gtf, and I know that those are the transcripts. And I can do that with the second file and so on. And then similarly, we would test. This one. Now, we need these files to be indexed, but in order to be indexed, they need to be sorted. Which they are not. We will use for both sorting and for indexing a tool that's called IGV tools. You can only access them from with in the IGV environment but in this case the actual I shall demonstrate it from the command line. So IGV tools sort. Okay. Oh. Okay. Simply Test1.gtf. Test1.sorted.gtf. So it's IGV tools sort name of the first file, and the input file, and name of the output file. And let's see what we get. And it's very simple, the same thing for the other files. That's three. Then we're going to do that for the controls. And now we can finally indexed them, again using IGV tools, to index Ctrl1.sorted.gtf. And let's take a look. So this created the file Ctrl1.sorted.gtf.idx and we can continue that with the other files. And test three. So this creates the sets of files. Gtf files. As well as the sets of Indexes for these files. That we'll need for the visualization. So here they are. Those are the gtf files. Now we also have to create smaller files, sorted and index files for the alignments. Now the alignment files are all ready sorted and knowing that I was supposed to extract ranges from them, I've all ready indexed them. So if we're looking at Tophat/Test1/. Okay. So we have. In addition to the alignment file accepted we also have the index. So now with the index we can use some tools view, just like I demonstrated in the previous lecture, and extract only those [INAUDIBLE] could belong to chromosome, chromosome nine. So to do that, we would say samtools view, test Tophat/Test1accepted heat. But you understand that I already have the index there. And give me anything that is located on chromosome nine, and I want that as a band file. So I'm going to show that as I'm going to add -b. And save that into, let's say chr9.test1.ban. And I'm going to run this and this is going to take awhile so I will only demonstrate this for Test1 with the understanding that you can repeat this analysis and you can repeat this comment for the other five files So, we can now view the file. Samtools view, chr9.Test1. Just to make sure everything is in order. Okay? And indeed, all of the matches are on chromosome nine. Now we will index the file because it's already sorted. And we'll repeat this analysis, this processing for the other five files. Now we have a set of alignment files and their indices. This is Test1 but we should have for all these examples as well as a set of gtf files that represent annotations and we're ready to view them in the integrated genomic viewer.
