How to use DAVID for functional annotation of genes

[Music] in this tutorial I'm going to tell you how to use tools within the David bioinformatics resource to perform gene ontology enrichment analysis and pathway mapping on a list of genes that might have been generated from your experiment the tool we're going to use is the functional annotation tool so if I click on that you then get a chance to upload your a list of genes so you can upload from a file or you can copy paste a list of gene IDs into the box you then need to tell the software the format of your IDE so minor ensembl IDs and then you also need to tell it whether this is the list of your genes you're differentially expressed genes or whether this is a background control list so mine are the list of differentially expressed genes for my experiment then you click Submit list once your data has been uploaded you should see a page that looks a bit like this we've got several different categories and each one has a little cross beside it so we're going to look at the gene ontology first so if we click the cross these are all the different gene ontology searches that have been performed so within gene ontology that abbreviation B P stands for biological process C C stands for cellular compartment and M F is molecular function so these different categories will tell you different things about your genes and then next to BP we have 1 3 5 so 1 refers to the broader categories and as you go through 2 5 it they're getting more and more specific so to look at the data that's been generated we can click on this button chart so we'll start with the BP one biological process one I'll just expand that up so you can see it so here we have a list of the go towns that are significantly enriched in the list of genes that you uploaded you can see that because we're at BP one these are very broad terms so we've got multicellular organism all process developmental process signaling so these are very broad terms which you may find are not specific enough to be helpful for the question that you're asking if we go back and click on for example the most specific BP v so now you can see we've got much more specific categories nervous system development neurogenesis organ development BP all includes all of those tones and you get a mix of the really broad ones and very specific ones and the one that I find gives the most useful data is the BP direct and this contains go terms have been directly annotated by their dspace and it's these direct ones which are ticked by default within the goat within the diva tool so let's click on chart for the BP direct and well how it will look in a bit more detail about what this data is showing us so first of all we'll start at the right hand side so we have some p-value and essentially our adjusted p-value so this is telling you that this gene ontology term is highly enriched in your list of genes and the this list of Gautam's is organized in decreasing level of significance over the time you get down to the bottom these go to terms are less highly enriched if we click on this RT that refers to related terms so it is telling you other sorts of terms and pathways which are related to that go term if we click on the bar called genes this gives us a list of all the genes that were in your list which fall into that goo term category so we can click on the gene name and it tells us a bit about it where it's phoned it tells us what species it's from an RG tells us other genes that are related to that one so as I said this was the biological process gene ontology so you can go and look at the cellular component and see what that's pulled up so this is telling us generally where those genes are found within the cell so here we have plasma membrane highly enriched other terms related to plasma membrane and then we can look at molecular function and this tell us a slightly different things what those genes might be doing inside the cell so they might be binding calcium ions or binding when proteins are binding happen so you can see the different sorts of go tones are telling us different information I'm going to close that out and just show you the other sorts of categories that they've got so we have terms linked disease from the Armen database so we've just got two that seemed to be enriched here we have some functional categories or keywords which upregulated which you might find helpful and then we have other things you might find useful for your question like cytogenetics or relationship to literature poutine demeans but one particular useful one I find is pathways so we have various different pathways in here bébé ID and bio Encarta bio carter seem to be smaller that we have keg and react ohm so react home within the david tool i find less helpful because it doesn't tell you immediately what the pathway is that is being enriched you can click on the link that takes you to the rack tour website and then that will tell you a bit about that pathway but that's a bit more long-winded so I find that the cake pathways are more useful so if you click on the cake pathway so now you can see which pathways are enriched in your list of differentially expressed genes and here it's useful if you click on that name we now have a diagram of that cake pathway and each of the genes in your list has been annotated with a red star now if we scroll down here here we can see all the genes in the pathway that it's drawn and the ones in red are the ones that were in your list of genes this is like wrinkled with these diagrams in that there little red stars are slightly displaced from the genes so you need to imagine the red star is moved down and to the right a little bit so this one belongs with nature in g1 this one belongs with nature and 3 you can download a picture of this pathway by right-clicking and going copy image but that won't include the red stars that will just include the pathway so then you will have to then go and manually annotate your image within PowerPoint or whatever program you like to use to indicate the genes that have been enriched now with any gene ontology you get a lot of information if we go back to for example this one the BP direct wish from my experiment is telling me lots of useful things that I've got things to do with axon guidance and nervous system and went signaling but there's a lot of tones and it can be quite hard to put those together or to come up with a sort of coherent idea of what's happening inside your cells so one really useful thing that David has is down the bottom of this and it's called functional annotation clustering and what that is going to do is going to take all the terms that have been enriched in these blocks above and cluster them now I find it helpful if I only run that tool with the three go term direct categories so I prefer to click off all of the others because I find that that confuses the overall story and makes a bit harder to to see what's going on so I would tend to unclick all of these and so you can see next it no no category selected so I've just got these three and then I click on functional annotation clustering and what that does is it looks at those enriched gold tones and those categories that I clicked and set as well which of those have things in common with each other how can we cluster those go terms to make them a bit more easy to understand and see what's going on and so here we have 49 clusters them and the first cluster contains these four categories of extracellular matrix structural constituent things do is collagen and the ER and what's really nice is the diagram so if you click on this little green and black box sometimes this doesn't work and you have to reload it but essentially it's giving you a cluster diagram and each row is a different gene from your list and each column is one of those four different Google terms and so the green indicates so for example if we look at this bottom one collagen type for alpha 3 chain each of these four go terms is associated with that gene whereas this one at the top to see you on cue and juma necrosis factor related protein two is associated with one of those go terms so you can quickly get an idea of which genes are related to each other because they share go terms if we click on G here that now gives us a list of all the 37 genes that were in that cluster RT is related terms so this is all the other things that are related to those particular terms so extracellular matrix most highly enriched and you see other ones that are related to it and this gives us a like a list of genes that fell into that particular Google category so you can see that this clustering of the annotations can start to give you a much clearer idea of what's going on so here we have things to do with a matrix here we have things to do with wincing lang this looks like it's got links to phosphatidyl inositol signaling for example so you can start to get a better idea of what's happening inside your cell and so construct to think about how to pursue those further I hope you found this tutorial to be useful do us a favor in return and click the like button and leave a comment below to let us know what you liked or what else you would like us to cover in the future we have lots of other tutorials lectures and interesting videos so do browse our channel page and consider subscribing thanks for watching [Music] [Music]

Channel: Genomics Gurus

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Keywords: Genetics, Genes, Transcription, Teaching, University, College, Public Engagement, Science, Lecture, Tutorial, Glasgow, Scotland, UK, University of Glasgow, Glasgow University, Adam West, Katherine West, Transcriptomics, david tutorial, bioinformatics course, bioinformatics, gene ontology analysis david, gene ontology analysis, gene ontology analysis tutorial, gene ontology, functional annotation tool david, functional annotation tool, gene ontology visualization, gene ontology how to use

Id: EuCH5mqRylE

Length: 12min 54sec (774 seconds)

Published: Tue Jul 14 2020