Hello All,
I am using the tuxedo pipeline to analyse time series based rna-seq data consisting of two replicates per time point. After aligning reads for every replicate using tophat2, I followed the tuxedo pipeline to obtain a merged gtf file using cufflinks and cuffmerge and abundances per replicate using cuffqunat. Finally, I used the following parameters for cuffdiff to obtain differentially expressed genes:
Time series:
cuffdiff -o $outpath -L D0,2h,28h,D2,D4 -p 8 -T --min-reps-for-js-test 2 /merged.gtf $inpath/D0P1.abundances.cxb,$inpath/D0P2.abundances.cxb $inpath/2hP1.abundances.cxb,$inpath/2hP2.abundances.cxb $inpath/28hP1.abundances.cxb,$inpath/28hP2.abundances.cxb $inpath/D2P2.abundances.cxb,$inpath/D2P2d.abundances.cxb $inpath/D4P1.abundances.cxb,$inpath/D4P2.abundances.cxb
Relative to D0:
cuffdiff -o $outpath/D0.2h -L D0,2h -p 8 --min-reps-for-js-test 2 /merged.gtf $inpath/D0P1.abundances.cxb,$inpath/D0P2.abundances.cxb $inpath/2hP1.abundances.cxb,$inpath/2hP2.abundances.cxb
*same command used for D0 relative to other time points
However, in the output file gene_exp.diff, one of the key marker genes is not identified as significantly differentially expressed in the Significant column although the FPKM values for all the time points versus D0 are significantly different (based on the gene_fpkm_tracking file)
The values are:
D0: 0.00954311, 2h:0.80255, D1:0.132429 ,D2:0.0765285, D4:2.01515
Any input on this problem will be greatly appreciated!
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This is 8 weeks late, but the FPKM values you show don't suggest differential expression consistent with a time course. Instead, it looks like there might just be differential expression at D4 (maybe). With a time course experiment, you're asking it to find genes that follow some sort of consistent change between subsequent timepoints, not for there to just be a change at one timepoint.
So you have 2 replicates per time point? Could you provide exact values of count and FPKM for those? Have you tried other options for time course analysis, such as DESeq?