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Old 03-24-2011, 04:33 AM   #1
cedance
Senior Member
 
Location: Germany

Join Date: Feb 2011
Posts: 108
Default Tophat -j option error

Hi,

I ran tophat with the command shown below on a pair of paired-end read and obtained the bam file including the insertions, deletions, and junctions .bed file.

Code:
 tophat -o <o/p dir> -r 73 --mate-std-dev 19 -p 6 -F 0 --solexa1.3-quals <path-to-index> <pe_read1.fastq> <pe_read2.fastq>
Then, as specified in the documentation,
Quote:
NOTE: Tophat can align reads that are up to 1024 bp, and it handles paired end reads, but we do not recommend mixing several "types" of reads in the same TopHat run. For example, mixing up 100bp single end reads and 2x27bp paired ends into the same TopHat run will give bad results. If you'd like to combine results from several "flavors" of RNA-Seq reads, you can run first with one of your sets, and feed the junctions produced by that run into future TopHat runs as externally supplied junctions with the -j option
I used the junctions file obtained in this run to run tophat again with the -j option on the single end read. This single end read also has similar bp length but I just thought its better to run this way.

Code:
tophat -o <o/p dir> -p 6 -F 0 --solexa1.3-quals -j <path-to-junction-file . bed> <path-to-index> <se_read.fastq>
And I get this error:

Quote:
Mapping reads against SLY_2.40 with Bowtie
Joining segment hits
Mapping reads against SLY_2.40 with Bowtie(1/3)
Mapping reads against SLY_2.40 with Bowtie(2/3)
Mapping reads against SLY_2.40 with Bowtie(3/3)
Searching for junctions via segment mapping
Retrieving sequences for splices
[FAILED]
Error: Splice sequence retrieval failed with err = 1
Does anyone have a clue what the error is? I have invested quite sometime to fix this error...
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Old 03-25-2011, 05:05 AM   #2
cedance
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Location: Germany

Join Date: Feb 2011
Posts: 108
Default

Okay, I managed to get it to run. I did not know about the bed_to_junc converter (the file to use is after converting junctions.bed to say, junctions.junc file).
This post helped me resolve in case anyone needs it: http://seqanswers.com/forums/showthread.php?t=3381
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-j junction, bowtie, fastq bam sam, single paired end, tophat error

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