Cuffdiff Results ZERO / 0 FPKM for all genes

[gopal_botany]

Dear All,

I am new to this community and please accept my greetings....

I have performed denovo assembly of plant transcriptome by using SOAP-denovo and I have used Tophat ---> Cufflink -----> Cuffdiff pipeline for downstream analysis.

Respective commands are given below -


tophat --coverage-search --microexon-search -r 200 -o /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/Flower /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/SAF_TOPHAT_REF /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Flower_adapter_trimmed_R1.fastq /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Flower_adapter_trimmed_R2.fastq

tophat --coverage-search --microexon-search -r 200 -o /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/Root /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/SAF_TOPHAT_REF /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Root_adapter_trimmed_R1.fastq /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Root_adapter_trimmed_R2.fastq

tophat --coverage-search --microexon-search -r 200 -o /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/Shoot /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/SAF_TOPHAT_REF /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/shoot_adapter_trimmed_R1.fastq /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/shoot_adapter_trimmed_R2.fastq

tophat --coverage-search --microexon-search -r 200 -o /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/Leaf /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/SAF_TOPHAT_REF /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Leaf_adapter_trimmed_R1.fastq /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Leaf_adapter_trimmed_R2.fastq

tophat --coverage-search --microexon-search -r 200 -o /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/Leaf /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Tophat/SAF_TOPHAT_REF /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Leaf_fastx_trimmer_R1_adapter_trimmed.fq /home/gopal/SAF/Transcriptome/New_Analysis_24_Dec_2013/Leaf_fastx_trimmer_R2_adapter_trimmed.fq

cufflinks accepted_hits.bam

cuffmerge path.txt


cuffdiff -N -p 40 -o ../cuffdiff/new/ merged.gtf accepted_hits_FL.bam accepted_hits_LF.bam accepted_hits_RT.bam accepted_hits_ST.bam

My query is why cuffdiff results zero FPKM in all genes and each sample, while FPKM values were there in clufflink results.

Please help me.....

Thanking you.

[TiborNagy]

The parameters looks OK. Maybe you mixed accepted_hits_*.bam in the command line. It is hard to tell, because we do not know how do you created accepted_hits_FL.bam, accepted_hits_LF.bam, accepted_hits_RT.bam and accepted_hits_ST.bam

[gopal_botany]

Thanks TiborNagy,

As I written above the step wise step execution of pipeline... it will help you to understand how these files generated.

Thanks