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Old 08-12-2014, 07:55 PM   #1
thickrick99
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Location: Washington

Join Date: Jul 2014
Posts: 21
Post Tophat Can't Find Bowtie2 Index!?!?!

Hi everyone,

So I am familiar with using tophat for alignment and I used it successfully a few weeks ago with the following command:

tophat -p 4 –G ./Homo_sapiens.knownGene.hg19.sorted.withGenes.gtf --transcriptome-index=transcript_data/known hg19 ERR188021_1.fastq ERR188021_2.fastq &

When I previously ran this, it worked, however I ran it today with different fastq files and it gave me this error:

Error: Could not find Bowtie 2 index files (–G.*.bt2)

It seems like for some reason tophat thinks that -G is the base name for the index files instead of hg19. I have tried adding the full path for the hg19 but that gave the same error, I also tried getting rid of the --transcriptome-index part and that didn't change anything either. I need to have the -G reference transcript when running tophat but I don't know why this error is occuring. I tried to recreate the index files from bowtie2-build and put all 6 files into a folder titled Index which was located on my desktop and then in the above command, changed hg19 to /Path/Desktop/Index/hg19. This also failed as well.

This is really frustrating because the same exact command was working just a few weeks ago. I have top hat version 2.0.11 and bowtie2 version 2.2.3

I really need some help on this since I spent many hours trying to solve this problem. Any help would GREATLY be appreciated.

THANK YOU!!!

Last edited by thickrick99; 08-12-2014 at 07:58 PM.
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Old 08-12-2014, 11:41 PM   #2
WhatsOEver
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Location: Germany

Join Date: Apr 2012
Posts: 215
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as far as I remember, tophat is quite sensitive to the correct order of parameters. The error, however, looks more like you forgot a space somewhere - put the full path to the gtf and and the index in a variable and refer to that, this makes it easier to see errors like that. I do have the same versions of tophat and bowtie2 installed and just tried the following (which worked fine)

Code:
b2Index=/path/to/index/files
gffFile=/path/to/gtf/file
tophatDir=/path/to/tophatOutputDir

tophat -o $tophatDir -p 8 -i 20 -G $gffFile $b2Index /path/to/your/fastq/files
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bowtie 2 indexes, rna-seq, tophat 2.0.11

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