Hi Ying. Some comments below.
On 2010-10-18, at 10:22 PM, Ying Ye wrote: > Dear edgeR users and developers， > > I have few questions about edgeR when recently I use it for 454 > pyrosequencing data: > > 1. prior.n > According to users' manual, we may not use too low prior.n in > moderated tagwise dispersion approach. But in my dataset, there are > more than 15 samples in each comparison group and the freedom is > larger than 30. prior.n <- estimateSmoothing(d) gives 0.0005329. So I > am wondering if I could use 0.0005329 since I have rather big number > of samples in each group. Or I should adjust prior.n into 10 according > to the manual's suggestion. Well, its hard to give a prescription for prior.n for all datasets. Since you have so many degrees of freedom, you shouldn't need prior.n as high as 10. You might try something lower, say 1-3. > 2. TMM > I am not sure if this is also applicable to 454 microbiota data. > I suppose I should do TMM normalization as well since the > normalization factors from my samples have a big variation (f is from > 0.41 to 4.58). Is that right? I must admit that I'm not intimately aware of all the nuances of microbiota data, but I will say that those factors you mention above are generally lower/higher than we see in RNA-seq data. I'd say its probably best to look at some "smear" plots -- through maPlot() for example -- to assess whether the TMM normalization is appropriately capturing shifts due to composition or the like. As always for exploratory analysis, it would be good to look multidimension scaling plots -- see plotMDS.dge(). There is no substitute for looking at your data. > 3. p_value > According to your experience, is it reasonable and reliable to > use p_value < 0.05 as significance criteria? or only <0.01 can be > reliable. First off, you'll probably want to do some multiple testing correction, which can be done through the topTags() function. As to where to set the threshold on significance, that is a matter of your false discovery tolerance ... the status quo is 5%, but you may want to be more or less stringent. Hope that helps. Mark > I am a new users in this package and hope you may give some > suggestions. Many thanks! > > Ying Ye > > > > > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioc-sig-sequencing mailing list > Bioc-sig-sequencing@r-project.org > https://stat.ethz.ch/mailman/listinfo/bioc-sig-sequencing ------------------------------ Mark Robinson, PhD (Melb) Epigenetics Laboratory, Garvan Bioinformatics Division, WEHI e: m.robin...@garvan.org.au e: mrobin...@wehi.edu.au p: +61 (0)3 9345 2628 f: +61 (0)3 9347 0852 ------------------------------ ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:6}} _______________________________________________ Bioc-sig-sequencing mailing list Bioc-sig-sequencing@r-project.org https://stat.ethz.ch/mailman/listinfo/bioc-sig-sequencing
