Hi Thanh, This is due to Cuffdiff correcting for the size of smaller transcripts, the authors call it the "effective length correction". It is supposed to correct the loss of shorter transcripts upon size selection in creating your RNA-seq library. The default setting on Galaxy is to use the "effective length correction". Cole Trapnell, the creator of the "Cuff-suite tools", discusses this length correction here: http://seqanswers.com/forums/showpost.php?p=76430&postcount=32 Some library preparation protocols don't include a size selection. The one we favor, and Illumina recommends, ScriptSeq v2 from Epicentre (owned by Illumina), does not include a size selection step. It would be great if there was an option in the Cuffdiff wrapper in Galaxy to turn off the effective length correction. Cheers, Mo Heydarian PhD candidate The Johns Hopkins School of Medicine Department of Biological Chemistry 725 Wolfe Street 402 Biophysics Baltimore, MD 21205 On Thu, Jul 18, 2013 at 12:55 PM, Hoang, Thanh <hoangtv@miamioh.edu> 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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