Cufflinks pipeline for splicing differences between animal samples

[seqhorn]

I have been trying to set up a pipeline for identification of differential expression, but in particular differential splicing between samples in a group of animals. A genome sequence exist, but without any gene reference annotation GTF.

So far I have been able to detect plenty of genes that are differentially expressed between the animals, but cuffdiff always comes up with zero (0) splicing tests or significant column values always being 'no' when test is 'OK'.

I can see examples of differential splicing when I manually inspect the data in IGV, so why am I not able to detect these with cuffdiff?

In short my pipeline looks as follows:

tophat genome_ref animal_1.fastq, ..., animal_n.fastq
cufflinks animal_1.hits.bam, ..., animal_n.hits.bam
cuffcompare animal_1.transcripts.gtf, ..., animal_n..transcripts.gtf
cuffdiff cuffcmp.combined.gtf animal_1.hits.bam, ..., animal_n.hits.bam

I have also tried decreasing the minimum count option or increasing the P-value cutoff, but it does not give me differentially spliced genes with significant P-values.

Any thoughts ideas?