Thank you Mohammad and Ross for your valuable information. I am re-running Cuffdiff with No effective length correction and also running EdgeR tool to see how the results go. Thanh
On Thu, Jul 18, 2013 at 8:42 PM, Ross <[email protected]> wrote: > Hi, Thanh, > If your primary goal is inference about differential 'gene' expression > taking biological variability into account with biological replicates for > each of two conditions, you might want (eg see Dillies et al., > http://bib.oxfordjournals.org/content/early/2012/09/15/bib.bbs046.longand > http://wiki.galaxyproject.org/Events/GCC2013/Abstracts#Events.2FGCC2013.2FAbstracts.2FPosters.P4:_Comparing_R-based_methods_and_Cuffdiff2_for_analysis_of_RNA-seq_data_in_Galaxy) > to try (and compare!) edgeR (and optionally DESeq and VOOM/limma). A set of > *very much beta* tools is available for admin installation and user testing > from the test toolshed in the statistics section owned by fubar. > > The edgeR tool can optionally run 2 way GLM. It requires raw count > matrices as inputs which can be generated from a GTF/'gene' model of your > choice and any number of mapped SAM/BAM inputs using the htseq based > companion tool in the same tool shed section. Please don't install to a > production machine yet but we're getting good results from it - feedback > and code improvements are welcomed from willing beta testers. > > The R 3.0.x tool shed dependency package in particular is still under > development and is likely to change substantially in the next week or two > as we sort out a sane and generalised Atlas dependency installation. > > > On Fri, Jul 19, 2013 at 2:55 AM, Hoang, Thanh <[email protected]> wrote: > >> Hi all, >> I have been analyzing my RNA-seq data on mouse tissues. My RNA-data is >> single-ended and 51 bp in length. I ran TopHat/Cufflink/Cuffdiff to test to >> differential gene expression >> In the Cuffdiff's output, I got very high RPKM value for some of miRNA >> and some other short genes ( less than 100bp). These genes are in the top >> genes with the highest RPKM. I think the RPKM values of these genes are >> probably too high to be true. >> *test_id* *gene_id* *gene* *locus* *sample_1* *sample_2* *status* * >> value_1* *value_2* *log2(fold_change)* *test_stat* *p_value* *q_value* * >> significant* *ENSMUSG00000093077* *ENSMUSG00000093077* *Mir5105* * >> 5:146231229-146302874* *Epithelium* *Fiber* *OK* *1.53E+06* * 445558* * >> -1.78097* *-355.367* *0.00715* *0.016986* *yes* *ENSMUSG00000093098* * >> ENSMUSG00000093098* *Gm22641* *7:130162450-133124354* *Epithelium* *Fiber >> * *OK* *87894.1* * 36474.7* *-1.26887* *-0.59863* *0.4913* *0.587174* *no >> * *ENSMUSG00000089855* *ENSMUSG00000089855* *Gm15662* * >> 10:105187662-105583874* *Epithelium* *Fiber* *OK* *42868.9* * 21566.5* * >> -0.99114* *-20.7066* *0.0186* *0.039568* *yes* *ENSMUSG00000092984* * >> ENSMUSG00000092984* *Mir5115* *2:73012853-73012927* *Epithelium* *Fiber* >> *OK* *21104.8* * 8317.49* *-1.34335* *-447.314* *0.0001* *0.000354* *yes* >> *ENSMUSG00000086324* *ENSMUSG00000086324* *Gm15564* *16:35926510-36037131 >> * *Epithelium* *Fiber* *OK* *6443.35* * 3664.15* *-0.81433* *-1.52095* * >> 0.2129* *0.301429* *no* *ENSMUSG00000092981* *ENSMUSG00000092981* * >> Mir5125* *17:23803186-23824739* *Epithelium* *Fiber* *OK* *5974.14* * >> 2390.75* *-1.32127* *-0.34111* *0.5746* *0.661937* *no* >> >> I checked some forums and they said that this is the drawback of >> TopHat/Cufflink/Cuffdiff when dealing with short genes. But I am still not >> so clear about this. Anyone got the same problem? What can I do with this >> situation? >> Anyone suggests any other good tools to test for (1) differential gene >> expression OR (2) both differential gene expression and gene discovery? >> >> Thank you >> Thanh >> > >
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