Hi Ryan, Thanks for the explanation. Best. Atul On 12/19/2013 02:01 PM, Ryan wrote: > Hi Atul, > > This has been asked before in a slightly different context, but in > general it is acceptable to further subdivide your groups after > estimating dispersions. At worst, you will end up with an overly > conservative test because your dispersion estimation will overestimate > the dispersion for your final set of groups. In your case, you are > estimating dispersions with one group and then splitting that group > into two. > > -Ryan > > On 12/19/13, 10:03 AM, Atul Kakrana wrote: >> Hi Ryan, >> >> Many thanks for your reply. Do you think it's theoretically correct to >> change the grouping before exact test for norm.tags2? I have no idea >> about how estimateDisp() works due to my non-statistics background and >> just want to make sure that I am not making any mistake. >> >> Best >> >> Atul >> >> >> On 12/18/2013 05:55 PM, Ryan wrote: >>> Hi Atul, >>> >>> I believe that all you need to do is to restore the correct grouping >>> before calling exactTest, i.e. something like: >>> >>> norm.tags2$samples$groups = c(1,2) >>> >>> >>> -Ryan >>> On Wed Dec 18 13:08:12 2013, Atul Kakrana wrote: >>>> Hi All, >>>> >>>> I am working on sRNA-seq data and with no replicates and using >>>> edgeR to >>>> identify differentially expressed tags and using an arbitrary BCV >>>> value >>>> (0.3) based on suggestion in edgeR manual: >>>> >>>> "Simply pick a reasonable dispersion value, based on your experience >>>> with similar data, and use that for exactTest or glmFit. Typical >>>> values >>>> for the common BCV (square- root-dispersion) for datasets arising from >>>> well-controlled experiments are 0.4 for human data, 0.1 for data on >>>> genetically identical model organisms or 0.01 for technical >>>> replicates. " >>>> >>>> But I came across this post which mentions a conservative way to >>>> calculate BCV: >>>> [BioC] EdgeR: general advice on using edgeR for sRNA analysis: >>>> https://stat.ethz.ch/pipermail/bioconductor/2013-August/054239.html >>>> >>>> I am getting an error calculating BCV using the approach discussed in >>>> post. Here is my code >>>> >>>> norm.tags2 <- norm.tags >>>> norm.tags2$samples$groups = c(1,1) >>>> norm.tags2 <- >>>> estimateDisp(norm.tags2,robust=TRUE,winsor.tail=c(0.05,0.2)) >>>> results = >>>> exactTest(norm.tags,dispersion=norm.tags2$trended.dispersion) >>>> >>>> Error in exactTest(norm.tags, dispersion = >>>> norm.tags2$trended.dispersion) : >>>> At least one element of given pair is not a group. >>>> Groups are: >>>> >>>> I believe the error is because I have grouped samples as replicates >>>> for >>>> calculating BCV while they are being compared against each other in >>>> exactTest. Can I calculate the dispersion values from samples? rather >>>> than setting arbitrarily? >>>> >>>> I would really appreciate any help. Awaiting reply. >>>> >>>> Atul >>>> >>>> >>>> >>>> >>>> >>>> [[alternative HTML version deleted]] >>>> >>>> _______________________________________________ >>>> 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 >>> > > >