Cufffdiff returns 0 conf_low fpkm and high conf_hi fpkm for EVERY gene

[fattest0828]

Hi all,

I am using tophat 2.0.13,cufflinks 2.2.1 to do tophat-cufflinks-cuffmerge-cuffdiff pipeline.

Here is the command I typed:

tophat -p 40 -G genes.gtf -o Testes_thout genome Testes_1.fq Testes_2.fq ;
tophat -p 40 -G genes.gtf -o Ovary_thout genome Ovary_1.fq Ovary_2.fq 

cufflinks -p 40 -o Testes_clout Testes_thout/accepted_hits.bam 
cufflinks -p 40 -o Ovary_clout Ovary_thout/accepted_hits.bam

cuffmerge -g genes.gtf -s genome.fa -p 40 assemblies.txt

cuffdiff -o diff_outTestesOvary -b genome.fa -p 40 -L Testes,Ovary -u merged_asm/merged.gtf ./Testes_thout/accepted_hits.bam ./Ovary_thout/accepted_hits.bam

Here are the first few lines from genes.fpkm_tracking in Ovary_clout:

tracking_id	class_code	nearest_ref_id	gene_id	gene_short_name	tss_id	locus	length	coverage	FPKM	FPKM_conf_lo	FPKM_conf_hi	FPKM_status
CUFF.2	-	-	CUFF.2	-	-	1:250485-252044	-	-	184.641	160.348	208.935	OK
CUFF.1	-	-	CUFF.1	-	-	1:179054-185161	-	-	7.27643	6.33654	8.21633	OK
CUFF.3	-	-	CUFF.3	-	-	1:185295-185655	-	-	8.2666	5.1426	11.3906	OK
CUFF.4	-	-	CUFF.4	-	-	1:373845-374208	-	-	2.84731	1.00884	4.68578	OK
CUFF.5	-	-	CUFF.5	-	-	1:372769-373658	-	-	1.61943	0.953847	2.28502	OK
CUFF.6	-	-	CUFF.6	-	-	1:374673-375367	-	-	1.59958	0.823667	2.37548	OK
CUFF.7	-	-	CUFF.7	-	-	1:241807-246436	-	-	26.1398	24.2019	28.0776	OK

It seems fine after cufflinks since all gene have a relatively normal conf_low fpkm and conf_hi fpkm.

However, after cuffdiff and cummeRbund ,when I plot the bar chart of my genes of interest, the error bar is so high that I have to check what is wrong.

Then I find the problem:

All genes in cuffdiff output file have a fpkm_conf_low of 0 and very high fpkm_conf_hi(about 2.5 fold of the fpkm, some are even higher).


Here are the first few lines from genes.fpkm_tracking in diff_outTestesOvary:

tracking_id	class_code	nearest_ref_id	gene_id	gene_short_name	tss_id	locus	length	coverage	Testes_FPKM	Testes_conf_lo	Testes_conf_hi	Testes_status	Ovary_FPKM	Ovary_conf_lo	Ovary_conf_hi	Ovary_status
XLOC_000001	-	-	XLOC_000001	ENSORLG00000000022	TSS1,TSS2	1:305571-308712	-	-	0.131856	0	0.503528	OK	0	0	0	OK
XLOC_000002	-	-	XLOC_000002	-	TSS3	1:369365-372285	-	-	5.3763	0	21.747	OK	49.8755	0	196.266	OK
XLOC_000003	-	-	XLOC_000003	-	TSS4	1:376708-384133	-	-	17.121	0	48.4596	OK	16.607	0	46.3991	OK
XLOC_000004	-	-	XLOC_000004	ENTPD7	TSS5,TSS6	1:384471-401100	-	-	5.3448	0	17.0895	OK	7.11206	0	19.6361	OK
XLOC_000005	-	-	XLOC_000005	-	TSS7	1:402148-407074	-	-	8.27844	0	23.0242	OK	21.7837	0	62.2343	OK

Do you know what happened?

Thanks.

Best,
Feng