Hi Galaxy Support,
I want to use cufflinks to quantify my own bam file, which is not from galaxy tophat. I tried several times but failed to get id. I can get fpkm. I import hg19 gtf file from ucsc. This time I got gene-id but all fpkm is 0. I am really frustrated with this. Please give me some instruction about setting parameters.
Hello,
There is probably a mismatch between the chromosome identifiers in the inputs. These Galaxy wiki sections can help:
GalaxyNGS101#Reference-based_RNA-seq
Hope this helps, Jen, Galaxy team
Here are the settings below. The fpkm has value but gene-id shows CUFF_id.
Do you know how to map this? What else do I need to set up?
| Input Parameter | Value | Note for rerun |
|---|---|---|
| SAM or BAM file of aligned RNA-Seq reads | myown.bam | |
| Max Intron Length | 300000 | |
| Min Isoform Fraction | 0.1 | |
| Pre MRNA Fraction | 0.15 | |
| Use Reference Annotation | Use reference annotation guide | |
| Reference Annotation | 19: UCSC Main on Human: refGene (genome) | |
| 3prime overhang tolerance | 600 | |
| Intronic overhang tolerance | 50 | |
| Disable tiling of reference transcripts | No | |
| Perform Bias Correction | No | |
| Use multi-read correct | Yes | |
| Apply length correction | Cufflinks Effective Length Correction | |
| Global model (for use in Trackster) | No dataset | |
| Set advanced Cufflinks options | No | |
| Job Resource Parameters | no |
See the comment above. The settings look fine - the incomplete results are almost certainly linked to the contents of the GTF input.
But note - the chromosome identifiers still must be a match between the BAM and the GTF. The GTF file is uncompressed, so these can be just visualized. For the BAM, you can convert to SAM and take a look at the header. When these are a match between the two files, and the GTF has the key attributes, then Cuffdiff functions optimally. When there is a mismatch or missing attributes, odd errors or incomplete calculations can result.
This is another wiki section with more about this tool, including additional troubleshooting help and links into the manual and supporting publications. That said, what I have shared as fixes address the most common issues users encounter. These are requirements not just when using this tool suite in Galaxy, but when using the tools line-command as well.
Support#Tools_on_the_Main_server:_RNA-seq
This is the latest setting. I still got CUFF.id instead of refseq id or gene id. Anyone can help me?
Inheritance Chain
The reference GTF was from UCSC? Then it does not contain the attributes that Cuffdiff requires for certain key functions.
The most direct way to obtain a GTF reference annotation file that contains the key attributes is to use the version from iGenomes. Download the tar file locally, unpack it, and then upload the genes.gtf dataset to Galaxy for use with these tools.
A description of these attributes and how they are used by Cuffdiff is in the manual here: http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/index.html#cuffdiff-input-files