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Old 09-24-2012, 05:00 AM   #1
frymor
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Unhappy igv error with thophat2

Hi everybody,


I am running the tophat2 command like that:
Code:
tophat2  -p 4 -r 500 -o OUT_DIR chromosome.MT.fa R1.fastq R2.fastq
After indexing the fastA file with bowtie2.
It all runs perfectly well and I have now a bam file from the calculations as well as the junctions and indel files.

short background information:
To imitate a circular genome of the mitochondria, I added 500 bases at the end.
With that I can try to identify PE reads which mapped at the "end" of the circular genome, where the second of the pair is mapped at the "beginning" of the chromosome.

I wanted to upload the bam file onto igv, but each time I get an error message.

Code:
INFO [2012-09-24 13:48:04,624]  [DirectoryManager.java:149] [main]  IGV Directory: /home/USER/igv
INFO [2012-09-24 13:48:04,630]  [Main.java:77] [main]  Startup  IGV Version 2.1.24 (2563)09/13/2012 09:23 AM
Fetching user directory... INFO [2012-09-24 13:48:04,798]  [Main.java:78] [main]  Default User Directory: /home/USER
INFO [2012-09-24 13:48:07,106]  [GenomeManager.java:101] [main]  Loading genome: genomes/Mus_musculus.MT.500add.fa
INFO [2012-09-24 13:48:07,116]  [GenomeManager.java:181] [main]  Genome loaded.  id= Mus_musculus.MT.500add.fa
INFO [2012-09-24 13:48:07,311]  [CommandListener.java:78] [Thread-6]  Listening on port 60151
INFO [2012-09-24 13:48:28,107]  [IGV.java:1455] [pool-1-thread-1]  Loading1 resources.
INFO [2012-09-24 13:49:05,210]  [IGV.java:1455] [pool-1-thread-3]  Loading1 resources.
ERROR [2012-09-24 13:49:05,747]  [CachingQueryReader.java:392] [pool-1-thread-4]  Error loading alignment data
java.lang.ArrayIndexOutOfBoundsException
        at org.broad.igv.feature.genome.SequenceHelper.getSequence(SequenceHelper.java:164)
        at org.broad.igv.feature.genome.GenomeImpl.getSequence(GenomeImpl.java:356)
        at org.broad.igv.sam.BisulfiteCounts.incrementCounts(BisulfiteCounts.java:51)
        at org.broad.igv.sam.BaseAlignmentCounts.incCounts(BaseAlignmentCounts.java:74)
        at org.broad.igv.sam.CachingQueryReader$AlignmentTile.addRecord(CachingQueryReader.java:602)
        at org.broad.igv.sam.CachingQueryReader.loadTiles(CachingQueryReader.java:327)
        at org.broad.igv.sam.CachingQueryReader.getTiles(CachingQueryReader.java:218)
        at org.broad.igv.sam.CachingQueryReader.query(CachingQueryReader.java:140)
        at org.broad.igv.sam.AlignmentDataManager$2.run(AlignmentDataManager.java:319)
        at org.broad.igv.util.LongRunningTask.call(LongRunningTask.java:54)
        at java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:334)
        at java.util.concurrent.FutureTask.run(FutureTask.java:166)
        at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1110)
        at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:603)
        at java.lang.Thread.run(Thread.java:679)
Has anyone encountered this problem too?

What is the meaning of it?

I have the feeling, that this has something to do with the additional 500 bases at the end, so that some of the reads map as an overhang of the circular genome.
Is there a way to find this kind of mapping?

I would appreciate ant help or ideas.

Assa

Last edited by frymor; 09-24-2012 at 06:08 AM. Reason: new information. I ran the same tophat command with the MT genome w.o. the additional 500 bases and it works fine on igv
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Old 09-24-2012, 06:22 AM   #2
NicoBxl
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did you indexed your genome before

samtools index accepted_hits.bam
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Old 09-24-2012, 06:28 AM   #3
frymor
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yes I did.
I even ran samtools flagstat on the bam file to see if the bam file is corrupt.
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Old 09-24-2012, 06:30 AM   #4
NicoBxl
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For the reference in IGV you have to use the same sequence you used for the alignment (so the sequence with the additionnal 500 bases)
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Old 09-25-2012, 02:12 AM   #5
frymor
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Quote:
Originally Posted by NicoBxl View Post
For the reference in IGV you have to use the same sequence you used for the alignment (so the sequence with the additionnal 500 bases)
That I did as well.
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Old 09-25-2012, 03:54 AM   #6
Jim Robinson
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Hi, this looks like an IGV bug (obviously). I am trying to reproduce it, in the meantime could you try the "early access" version to see if the bug is fixed there, if not please send or post the stack-trace from that version. You can download this version from:

http://www.broadinstitute.org/software/igv/download_dev

Sorry for the difficulties.

-- Jim
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Old 09-26-2012, 07:19 AM   #7
frymor
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unfortunately it didn't help.

I think it has something to do with the reference genome I am using. I have a lot (and I mean A LOT) of mismatches, almost every third base is a mismatch.

If I upload the 'real' mitochondrial genome it doesn't trouble me. I can upload the files and it also looks normal.
So there is some kind of a problem with the addition of the 500 bases from the beginning of the mitochondrial sequence to the end of this same one. Even though it was this genome modified genome I applied tophat2 to.

The new version of igv didn't help much. It shows the same error massage.
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Old 09-26-2012, 07:29 AM   #8
Jim Robinson
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Hi,

Could you contact us at igv-team@broadinstitute.org? We can arrange to maybe transfer your reference genome and a test file.

Also, the new early access version of IGV should not return the same stack trace, some of the classes referenced in the previous stack trace don't even exist. Seeing the stacktrace from the "early access" version might be enough to find the problem.

Jim
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