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-   -   CuffDiff 0 FPKM Output/Incorrectly Identified Differential Expression? (http://seqanswers.com/forums/showthread.php?t=15473)

cw11 11-14-2011 07:25 AM

CuffDiff 0 FPKM Output/Incorrectly Identified Differential Expression?
 
I’m having some problems with CuffDiff – or rather, the output I am getting is completely baffling to me. I believe the program is incorrectly identifying genes as differentially expressed due to an error.

I have three conditions that I am testing – we’ll call them GG, AG, and AA. For Condition GG, I have 2 samples, for Condition AG, I have 3 samples, and for Condition AA, I have 5 samples. When I run CuffDiff, I get a list of differentially expressed genes with q-values less than 0.05. However, when I actually examine the FPKMs for my genes, I see results like this:

GG: 0 (OK)
AG: 11.6888 (OK)
AA: 10.7249 (OK)

In this instance, CuffDiff reported GG as being differentially expressed relative to AG, and GG as being differentially expressed relative to AA.

Viewing my reads in IGV, however, I see MANY reads for this gene in ALL conditions and samples. I cannot understand why my FPKMs are 0. I have also calculated the RPKM values for this gene’s transcripts using SeqGene, as a “quality control” of sorts. When I do this, all RPKMs for this gene are > 1; a representative transcript has RPKM values like this:

GG: 2.31, 2.04
AG: 2.63, 2.59, 2.5
AA: 2.32, 2.15, 2.42, 2.33, 3.02

For my pipeline, I am usingTophat to map reads and find splice junctions, followed by Cufflinks to find novel transcripts, CuffMerge to merge these novel transcripts together with a reference genome as guide, and CuffDiff to identify differences in transcript expression. For reference, here is a summary of the calls I’ve made (from most to least recent).

CuffDiff:
Code:

bsub -P Lithium -q iweek -R rusage[mem=8] -R span[hosts=1] -n 4 -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/07_CuffDiff_10.19.2011_01/output.txt cuffdiff -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/07_CuffDiff_10.19.2011_01 -p 12 -L AG,GG,AA /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/06_CuffMerge_10.19.2011_02/merged.gtf /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_TGTGTG_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_GAGAGA_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_ACACAC_1/accepted_hits.bam /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_CTCTCT_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_TGTGGA_1/accepted_hits.bam /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_GAGAAC_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_ACACCT_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_CTCTTG_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_CTTGTG_1/accepted_hits.bam,/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_TGGAGA_1/accepted_hits.bam
CuffMerge:
Code:

cuffmerge -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/06_CuffMerge_10.19.2011_02 -g /psych/neuro/people/wright/Project_3_RNASeq/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf -s /psych/neuro/people/wright/Project_3_RNASeq/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/02_DeMultiplexingCode/F_02_FileForTestMerging.txt
Where FileForTestMerging looked like this:
Code:

/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_ACACAC_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_ACACCT_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_CTCTCT_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_CTCTTG_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_CTTGTG_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_GAGAAC_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_GAGAGA_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_TGGAGA_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_TGTGGA_1/transcripts.gtf
/psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_TGTGTG_1/transcripts.gtf

Cufflinks:
Code:

bsub -P Lithium -q iweek -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_ACACAC_1/output.txt cufflinks -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/04_Cufflinks_10.18.2011_09/FastQDemultiplexed_ACACAC_1 -g /psych/neuro/people/wright/Project_3_RNASeq/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_ACACAC_1/accepted_hits.bam
Tophat (one of 10 calls):
Code:

bsub -P Lithium -q iweek -R rusage[mem=8] -R span[hosts=1] -n 4 -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_CTTGTG_1.txt tophat -o /psych/neuro/people/wright/Project_3_RNASeq/01_RNASeq_Analysis/03_TophatTest_10.12.2011_11/FastQDemultiplexed_CTTGTG_1 -p 4 -a 5 -F 0.0 -r 100 --segment-length 20 /psych/neuro/people/wright/Project_3_RNASeq/hg19-male /psych/inf/cwright/05_DemultiplexedFastQFiles/FastQDemultiplexed_CTTGTG_1.fq /psych/inf/cwright/05_DemultiplexedFastQFiles/FastQDemultiplexed_CTTGTG_2.fq
Any thoughts, help, comments on the CuffDiff output (or suggestions on the pipeline, for that matter) would be greatly appreciated!

gringer 11-14-2011 12:08 PM

Does your gene have another isoform that could be taking all the reads? Cufflinks considers isoform abundance in its FPKM calculations, so that might have something to do about it.

lmf_bill 11-14-2011 01:17 PM

I got the similar problems. For some genes without isoforms, cufflinks or cuffdiff estimate 0 FPKM, but the real case is they are expressed high.

cw11 11-15-2011 05:54 AM

Thanks! What has everyone doing to deal with these false positives? Just filtering out genes which have FPKMs of 0 for one of the conditions?

(I also understand from a different thread - http://seqanswers.com/forums/showthread.php?t=14864 - that the tuxedo group is planning to address some of these issues in their upcoming version 1.2.0... anyone know when this is expected to come out?)

pengchy 04-27-2015 03:59 AM

The problem still exist for cufflinks version 2.2.1. I checked One example that one gene without any reads hit, be reported FPKM 0. However, not all genes without any reads hit were reported. How to explain?


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