Hi Jen, Thanks for the reply. I am using the Galaxy Main instance, and simply chose the mm10 reference genome from the pull-down option list. The BAM index statistics on one of my Tophat files reports that it has a large number of reads that are aligned to to chrMŠ so at least the alignments are there. I used the same mm10 reference genome with Cufflinks. Since I didn't use a custom genome, it seems as though there should not be any identifier mismatchesŠ(?) Tim On 2/19/13 1:54 PM, "Jennifer Jackson" <jen@bx.psu.edu> wrote:
Hello Tim,
The problem is either that the reference genome build used does not contain the mitochondrial genome sequence, that the reference annotation file that you are using with the Cufflinks does not include mitochondrial annotation, or that the identifiers between the reference genome and the reference annotation are a mismatch (one is labeled "M" and the other "chrM" or possibly "MT").
Some links to the build: http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/mouse/ http://www.ncbi.nlm.nih.gov/genome?term=mus%20musculus (open "Representative" to see the Mito as "MT", it is not listed on either link under chromosomes.)
If you pulled out the fasta sequence yourself and are using it as a custom reference genome on the public Main Galaxy instance at https://main.g2.bx.psu.edu/ (usegalaxy.org) or installed it locally, then it could be missing the mito chromosome/genome.
If you are using another public or private/local Galaxy (not Galaxy Main), you can contact them to find out how the genome was created (if it included the mito).
For either of the two above - you could also run a tool like "NGS: Picard (beta -> BAM Index Statistics" to see if the mito chromosome is present in the Tophat output and what it is named. If included, compare the chromosome identifier/name to the reference annotation file your are using. The identifiers must be exact or tools will not function properly. More help, please see: http://wiki.galaxyproject.org/Support#Tools_on_the_Main_server
An alternative is to use either the mm9 or mm10 genome at the public Galaxy Main Galaxy instance. The variants, described on the "NGS: Mapping -> Bowtie" tool form, contain the mito chromosome named as "chrM". These are sourced from UCSC: http://genome.ucsc.edu/goldenPath/credits.html#mouse_credits also navigate to "Genomes -> mammal -> mouse -> mm10" to see more details
Hopefully this helps,
Jen Galaxy team
On 2/19/13 9:43 AM, Brown, Tim wrote:
I have RNA-seq data that I have run through Tophat using the mouse mm10 grc38 reference genome. When I use Cufflinks on these data, the mitochondrial genome transcripts are excluded from the analysis. I would like to see that data. Is there an option within the toolbox that can remedy this? I'm a newb here.
Thanks for any help, Tim
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