Hi Jim, Thanks so much for clearing that up for me! Joseph On Tue, Feb 11, 2014 at 9:04 PM, James W. MacDonald <jmacdon at="" uw.edu=""> wrote: > Hi Joseph, > > The flaw in your reasoning is here: > > > "Let's assume that the process represented by GO_A is such that it > cannot be simultaneously upregulated and downregulated;" > > You aren't measuring a process. You are measuring gene expression. And the > up-regulation and down-regulation of genes can have inhibitory or excitatory > effects on a particular process. > > In addition, GO terms aren't even necessarily related to a single process. > Instead, we use them as a stand-in for the underlying pathways that we hope > to measure (but don't really know much about). If we had better pathway > information we wouldn't even be bothering with GO terms at all. > > So you can certainly contrive a situation where you would only want to > consider up-regulated genes for a particular GO term, but that situation is > unlikely to hold in general. And when you are doing a multiple > hypergeometric tests, using all the GO terms in your universe, it is not IMO > a good idea to make very strong assumptions, especially if you don't need to > do so. > > Best, > > Jim > > > > > On Tuesday, February 11, 2014 3:36:13 PM, Joseph Shaw wrote: >> >> Hi Jim, >> >> Thanks for your reply. >> >> My worry, originally, was that that failure to differentiate between >> upregulated and downregulated processes would lead to spurious >> results. >> >> Let's create another scenario. Assume we have a group of genes >> identified as upregulated and another group of genes identified as >> downregulated. Furthermore, assume two subsets: one belonging to the >> upregulated group and one belonging to the downregulated group. Each >> subset is associated with several GO terms including one GO term which >> is common to both subsets - let's call this common term GO_A. >> Now, it may be the case that, individually, when tested against a >> defined gene universe, neither subset yields statistically significant >> results for GO_A, but combining the aforementioned subsets and testing >> against a gene universe does, in fact, yield a statistically >> significant result for GO_1. >> Let's assume that the process represented by GO_A is such that it >> cannot be simultaneously upregulated and downregulated; if this is the >> case, wouldn't it be incorrect to combine the upregulated and >> downregulated gene lists? >> >> Let's return to the example provided in your previous mail. >> My understanding of the GO DAG is far from exhaustive, so it's very >> possible that I'm wrong, but, given that the GO terms become more >> specific as we move towards leaf nodes, would we eventually arrive at >> a terms representative of negative regulation of programmed cell death >> and positive regulation of programmed cell death? >> If this is the case, assuming there was a sufficient amount of genes >> identified as differentially expressed for both enhancer (identified >> as upregulated in our experiment) and preventer (identified as >> downregulated in our experiment) genes so as to yield statistically >> significant results for separate tests. Would it be incorrect to >> conclude that negative regulation of preventers of programmed cell >> death and positive regulation of enhancers of programmed cell death >> have both been shown to be statistically significant significant? It >> seems to me that both these results are compatible. >> >> Joseph >> >> On Tue, Feb 11, 2014 at 2:00 PM, James W. MacDonald <jmacdon at="" uw.edu=""> >> wrote: >>> >>> Hi Joseph, >>> >>> I think you are making a simplifying assumption that isn't helpful. In >>> other >>> words, you are assuming that up-regulation of a set of genes means >>> something >>> different than down-regulation, or a mixture thereof. But this flies in >>> the >>> face of much that we know about biological processes. >>> >>> As an example, say we have a set of genes with 'programmed cell death' as >>> their GO term. And further assume that some of these genes enhance this >>> process, and some prevent the process. Now if most of the enhancers are >>> up-regulated, and most of the 'preventers' are down-regulated, are you >>> prepared to say these genes should be tested separately because the >>> up-regulated genes are involved with a different process than the >>> down-regulated genes? >>> >>> Best, >>> >>> Jim >>> >>> >>> >>> >>> On Monday, February 10, 2014 6:43:52 PM, Joseph Shaw wrote: >>>> >>>> >>>> Hi all, >>>> >>>> I am in the process of performing some ontological analysis with >>>> GOstats. Given that GOstats doesn't require any information on >>>> relative increases or decreases in expression for its hypergeometric >>>> testing procedure, am I correct in assuming that it does not >>>> differentiate between upregulated and downregulated genes? >>>> >>>> If this is the case then providing a list of differentially expressed >>>> genes (both upregulated and downregulated) to the testing procedure >>>> will result in ontology results where upregulation and downregulation >>>> may be confounded. >>>> In other words, combining upregulated and downregulated genes and >>>> comparing the resulting list to the gene universe will enable the >>>> testing procedure to identify regulated ontological processes, but it >>>> won't be able to identify whether the processes are upregulated or >>>> downregulated. In fact, given that there is no distinction provided as >>>> input, it may even be both. >>>> >>>> To me, it seems that in order to prevent this from happening two >>>> separate testing procedures should be performed: one comparing >>>> upregulated genes to the gene universe and one comparing downregulated >>>> genes to the gene universe. Is this approach advisable? Is there a >>>> correct protocol which addresses the above issue? >>>> >>>> Joseph >>>> >>>> _______________________________________________ >>>> Bioconductor mailing list >>>> Bioconductor at r-project.org >>>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>>> Search the archives: >>>> http://news.gmane.org/gmane.science.biology.informatics.conductor >>> >>> >>> >>> -- >>> James W. MacDonald, M.S. >>> Biostatistician >>> University of Washington >>> Environmental and Occupational Health Sciences >>> 4225 Roosevelt Way NE, # 100 >>> Seattle WA 98105-6099 > > > -- > James W. MacDonald, M.S. > Biostatistician > University of Washington > Environmental and Occupational Health Sciences > 4225 Roosevelt Way NE, # 100 > Seattle WA 98105-6099