visualizing RNAseq read coverage in Ensembl genome browser

Question

[tospo]

Apologies if this is somewhat basic - I usually work with smaller genomes and use local custom genome data viewers but now I have to work with some mammalian genomes and this is all new to me.

I simply want to visualize RNA-seq read coverage of my samples on the Ensembl genome browser. I understand that I can't upload a huge bam file but actually all I need is coverage plots for several samples, not the full alignments. I also don't need the whole genome - I mainly need to check a few gene regions.

To my surprise I so far failed to find out how others do this. What I have done now is to use samtools "depth" to get the coverage data for a region of interest from my bam file and then to convert that into WIG format using a custom Perl script. This works for a single sample but so far I couldn't get it to work if I want to combine coverage data for several samples into one track.

Here is a little toy example that does add two tracks and they are both plotted in the Ensembl mouse genome browser when I upload them as WIG but they are on top of each other although two separate y axis are drawn (but only one is used). Here is the wig file:

track type=wiggle_0 name="track-1" description="track 1" visibility=full autoScale=off viewLimits=0:25 color=50,150,255 priority=1 graphType="bar"
variableStep chrom=X span=100
74007187 10
74007287 20
74007487 5
74007687 13
variableStep chrom=X span=10
74007787 20
74007797 25
74007807 10
74007827 20
track type=wiggle_0 name="track-2" description="track 2" visibility=full autoScale=off viewLimits=0:25 color=0,200,100 priority=2 graphType="bar"
fixedStep chrom=chr19 start=49307401 step=300 span=200
variableStep chrom=X span=100
74007187 3
74007287 15
74007387 24

I guess I have two questions:

1. How can I get this to display so that both tracks are visible (preferably with a single wig file like I have tried here) - is there a way to make them overlay transparently or to force them being plotted on the two different y axes?
2. Am I doing something silly to being with? How do others visualise RNAseq data on Ensembl if you don't have a server to host your bam files? That must be a common problem, right?

Thanks for your help!

[mxs]

Hm... Excellent point! Let me start with the second question. In my own personal experience usually research groups have some local machine available and they often higher freelancers (or sometimes have their own (bio)ioinformaticians ) to setup a local mirror of the browser containing only relevant data for their work (Ex. mouse , human, fly etc.) . There are several reasons for that. If you have a lot of data (which you usually do) then passing them over the network takes time and if you need few seconds to move from one track position onto the next, it piles up and a lot of time is wasted. (In my opinion this is a valid concern) In several other cases where I've been setting up such local mirrors the primary concern of the group was security. People did not want to just upload data "somewhere" on the net where they had no clue why and who might be looking at it (While this might be a serious problem for the industry, I don't see it as a threat in academia - research areas are just too diverse). Back to your question, if you don't have server, then you do it exactly as you are doing it right now, because this is the way the system is designed. And if you have one, then I would recommend a local mirror.

As far as the first question goes. there is nothing wrong with the way you are doing it right now. If you try uploading it to for example UCSC GB (change chrom=X to chrom=chrX) it displays tracks correctly in two lines. But when I tried it on ensemble it always forces everything into same line. So I guess its a small bug on their account. As far as transparency issue goes that is way over my head (I don't do esthetics)

cheers

mxs

Answer 1

[Emily]

If you can put your BAM file onto a URL, you can attach it and view it directly in Ensembl. Our software will generate a read depth for you, and allow you to view the reads themselves as well. You can put the file in something like a Dropbox or Google Drive. There's a help video here on doing this.

[tospo]

I know I can put bam files on a server to link them to Ensembl but I didn't consider options like Google Docs or Dropbox because I thought the size limits these services impose would be a problem but I just realised that Google Docs actually allows 15GB which will be enough for now for me. Might be worth pointing out these options explicitly in the help video

[Emily]

Help videos are not easy to change, but I could edit the description text to include it.