Re: [Useq-users] mRNA-seq with USEQ

[David N. <Dav...@hc...>]

Hello Xiao-yu,

Let's see....


 1.  Using the Tophat SAM file shouldn't be a problem.  I assume it maps junction reads to a particular location in genomic coordinates.  None the less I haven't tried it so to be safe realign your data using Bowtie and a Bowtie index that contains a chromosome of known junctions.  The usage guide describes this in some detail, http://useq.sourceforge.net/usage.html .
 2.  Looks like DESeq didn't complete, it didn't produce an output file that USeq could read in.  There should be several temporary files that were left in your save directory.  These contain a big matrix files of raw data, the R shell script for executing R and an R output error file.  Open the script and error log in a text editor and take a look at the messages.  Also, try executing the shell script by copying and pasting each line into an R command line terminal.
 3.  If you like, de-identify your data and post it and I can give it a quick debugging.

Check out the http://useq.sourceforge.net/outputFileTypeDescriptions.html link, most of the results folders are named after the application that generated them so check out the usage docs manual processing sections for what is to be expected.  http://useq.sourceforge.net/usage.html

-cheers, D


--
David Austin Nix, PhD | Bioinformatics Shared Resource | Huntsman Cancer Institute | 2000 Circle of Hope | SLC, UT 84112 | Rm: 3165 | Vc: 801.587.4611 | Fx: 801.585.6458 | dav...@hc... | Skype/iChat: LiveNix | WebSite: http://bioserver.hci.utah.edu | DAS/2: http://bioserver.hci.utah.edu:8080/DAS2DB/genome



On 7/27/10 8:14 AM, "Liu, Xiaoyu (Xiao-Yu)" <Liu...@ma...> wrote:

Hello David,

This is Xiao-yu Liu from the Mayo Clinic. I am starting to experiment
with mRNA-seq tools and got interested in Useq based on my colleagues
recommandation. I run into a few questions and wondering if you could
help me with.

1. when providing raw alignment to Useq, is there any drawbacks if I use
the SAM file made by Tophat? ( I belive it contains the alignment at
junctions)

2. I had an exception when calculating negative binomial p-values:

..........................................................
Calculating negative binomial p-values and FDRs in R using DESeq
(http://www-huber.embl.de/users/anders/DESeq/)...
java.io.IOException:
R results file doesn't exist. Check temp files in save directory for
error.

        at
edu.utah.seq.analysis.MultipleReplicaDefinedRegionScanSeqs.executeDESeq(
MultipleReplicaDefinedRegionScanSeqs.java:318)
        at
edu.utah.seq.analysis.MultipleReplicaDefinedRegionScanSeqs.calculateDESe
qPValues(MultipleReplicaDefinedRegionScanSeqs.java:434)
        at
edu.utah.seq.analysis.MultipleReplicaDefinedRegionScanSeqs.run(MultipleR
eplicaDefinedRegionScanSeqs.java:124)
        at
edu.utah.seq.analysis.MultipleReplicaDefinedRegionScanSeqs.<init>(Multip
leReplicaDefinedRegionScanSeqs.java:85)
        at
edu.utah.seq.analysis.RNASeq.definedRegionScanSeqs(RNASeq.java:152)
        at edu.utah.seq.analysis.RNASeq.<init>(RNASeq.java:90)
        at edu.utah.seq.analysis.RNASeq.main(RNASeq.java:482)

Do you know what's the possible causes? Could you help me to find a way
to avoid it?

3. I still got some results from the run. There are quite a few
folders/files in the output directory. Do you have a documentation
explain how to read/understand them. The doc probably is somewhere
online, but I have not find it yet.

Thanks!

Xiao-yu Liu, Ph.D.
Informatic Specialist
Mayo Clinic
507 284-8728