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 <hoan...@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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___________________________________________________________
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