Hi!
I'm new to RNAseq, and I'm trying to find genes/isoforms that are differentially expressed in two samples (one from wild type mouse and one from knock out). I have no replicates.
To do this I used SpliceMap, then Cufflinks/Cuffcompare/Cuffdiff.
The problem is that about 40% of the genes, 30% of the isoforms and practically all of the entries in 0_1_tss_group_exp.diff are considered significantly differentially expressed. This is obviously not logical, so I have concluded that something must be wrong. I just don't know what.
Is there anyone out there who can help me shed some ligth on where my mistake is?
These are the options used:
cufflinks -o data/KO_cufflinks -m 78 -s 35 -p 8 good_hits_KO.sam
cufflinks -o data/WT_cufflinks -m 78 -s 35 -p 8 good_hits_WT.sam
cuffcompare -o ccKOogWT -r mm9ch.gtf -R data/transcriptsKO.gtf data/transcriptsWT.gtf
cuffdiff -m 80 -p 8 -o data/cuffdiff ccKOogWT.combined.gtf good_hits_KO.sam good_hits_WT.sam
I'm new to RNAseq, and I'm trying to find genes/isoforms that are differentially expressed in two samples (one from wild type mouse and one from knock out). I have no replicates.
To do this I used SpliceMap, then Cufflinks/Cuffcompare/Cuffdiff.
The problem is that about 40% of the genes, 30% of the isoforms and practically all of the entries in 0_1_tss_group_exp.diff are considered significantly differentially expressed. This is obviously not logical, so I have concluded that something must be wrong. I just don't know what.
Is there anyone out there who can help me shed some ligth on where my mistake is?
These are the options used:
cufflinks -o data/KO_cufflinks -m 78 -s 35 -p 8 good_hits_KO.sam
cufflinks -o data/WT_cufflinks -m 78 -s 35 -p 8 good_hits_WT.sam
cuffcompare -o ccKOogWT -r mm9ch.gtf -R data/transcriptsKO.gtf data/transcriptsWT.gtf
cuffdiff -m 80 -p 8 -o data/cuffdiff ccKOogWT.combined.gtf good_hits_KO.sam good_hits_WT.sam
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