Question: Differential gene expression analysis using cuffdiff misses key gene
gravatar for rachana.pradhan88
5.5 years ago by
rachana.pradhan880 wrote:

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!






ADD COMMENTlink modified 5.4 years ago by Biostar ♦♦ 20 • written 5.5 years ago by rachana.pradhan880

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.

ADD REPLYlink written 5.4 years ago by Devon Ryan93k

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?

ADD REPLYlink written 5.4 years ago by mikhail.shugay3.4k
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