13 months ago by
RNA-seq tutorials can be found here for example protocols using the tool as wrapped for Galaxy: https://galaxyproject.org/learn/
More is at the tool author's website. There is also a google group for the tool suite. See Manual + Help here: http://cole-trapnell-lab.github.io/cufflinks/
Low coverage of splice junctions could cause these to be not considered when transcripts are created by Cufflinks. This is the simplest reason why splices detected by Tophat were not promoted to multiple alternative transcripts assembly by Cufflinks.
Are you using a reference annotation input at this step (optional with all tools)? As a guide (identifies and includes novel transcripts) or as truth (only uses/reports known transcripts)? What was the source? Illumina sources are linked here: http://cole-trapnell-lab.github.io/cufflinks/getting_started/#using-pre-built-annotation-packages
If not from Illumina, does the reference annotation contain the attributes for p_id and tss_id (and optionally gene_name if you want gene labels included in the output)? Are the chromosome identifiers for the reference annotation an exact match for the chromosome identifiers on the reference genome used?
Was a custom reference genome used? Did you run NormalizeFasta on it first?
Support hub: https://galaxyproject.org/support/
Let us know if the above does not help. Sharing some details of what you are doing will aid understanding the data and what (if any) the usage problem is.