cuffdiff problem--Performed 0 isoform-level transcription difference tests

如何解决cuffdiff运行结果中表达量为0的情况？

cuffdiff -o cdiffout -b ref.fasta -u -p 15 --library-type fr-firststrand 
-L H_3,O_3,F_3 cuffmergeout/merged.gtf  
tophatout/H-1-3/accepted_hits.bam,tophatout/H-2-3/accepted_hits.bam,tophatout/H-3-3/accepted_hits.bam 
tophatout/O-1-3/accepted_hits.bam,tophatout/O-2-3/accepted_h its.bam,tophatout/O-3-3/accepted_hits.bam 
tophatout/F-1-3/accepted_hits.bam,tophatout/F-2-3/accepted_hits.bam,tophatout/F-3-3/accepted
_hits.bam

Processed 33185 loci. [***************] 100%
Performed 0 isoform-level transcription difference tests
Performed 0 tss-level transcription difference tests
Performed 0 gene-level transcription difference tests
Performed 0 CDS-level transcription difference tests
Performed 0 splicing tests
Performed 0 promoter preference tests
Performing 0 relative CDS output tests
Writing isoform-level FPKM tracking
Writing TSS group-level FPKM tracking
Writing gene-level FPKM tracking
Writing CDS-level FPKM tracking
Writing isoform-level count tracking
Writing TSS group-level count tracking
Writing gene-level count tracking
Writing CDS-level count tracking
Writing isoform-level read group tracking
Writing TSS group-level read group tracking
Writing gene-level read group tracking
Writing CDS-level read group tracking
Writing read group info
Writing run info

解决办法：先使用 cuffquant 对每个样本估计 gene 和 transcript 的表达量，然后再用 cuffdiff 进行差异表达的分析。

原理是：distribute your computational load over a cluster；save your time.