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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