Tophat trouble reconstituting index

[Xinlitik]

Hi all,

I'm new to the field so please forgive my ignorance. I setup Tophat, Bowtie, and SamTools and attempted to run tophat on some samples. This is the message it produced:

[2012-11-28 19:56:55] Beginning TopHat run (v2.0.6)
-----------------------------------------------
[2012-11-28 19:56:55] Checking for Bowtie
Bowtie 2 not found, checking for older version..
Bowtie version: 0.12.8.0
[2012-11-28 19:56:55] Checking for Samtools
Samtools version: 0.1.18.0
[2012-11-28 19:56:55] Checking for Bowtie index files
[2012-11-28 19:56:55] Checking for reference FASTA file
[2012-11-28 19:56:55] Generating SAM header for /u/home/mcdb/x/bowtie-0.12.8/indexes/genome
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-11-28 19:58:09] Reading known junctions from GTF file
[2012-11-28 19:58:21] Preparing reads
left reads: min. length=50, max. length=50, 75848803 kept reads (6201 discarded)
[2012-11-28 20:17:00] Creating transcriptome data files..
[FAILED]
Error: gtf_to_fasta returned an error.
open: No such file or directory
[main_samview] fail to open "R1/accepted_hits.bam" for reading.
cp: cannot stat `R1/accepted_hits.bam': No such file or directory

This is a different error than I first posted, sorry. I can't figure out why I am getting the gtf_to_fasta error. I downloaded Ensembl's NCBIM37 for mouse here: http://cufflinks.cbcb.umd.edu/igenomes.html. Then I used the mus_musculus gtf included in it, as well as the genome.ebwt files included in the BowtieIndex folder of the unpacked file. I read in another post that this error could be from the fasta and gtf files having different names, so I renamed them both genome but got the same error.


Any help is appreciated. Thanks!

[Wallysb01]

Can your post your command? It looks like it having trouble just finding the file. You might double check that the paths are correct?

[Xinlitik]

Can your post your command? It looks like it having trouble just finding the file. You might double check that the paths are correct?

Thanks for the reply. I "fixed" the problem by pilfering a labmate's gtf and .ewbt files. The ebwt files had identical sizes so that was just to be safe, so the different GTF must have been the problem. The thing is, I don't know why, hah. Any reason why the GTF included in the link in my first post would not work? The one I used successfully was also ensembl (mm9.ensembl.gtf) so I'm not sure why it failed. Does filename matter? Should I have tried e.g. genome.ensembl.gtf to go with genome.ewbt1234?

Here is the command:

tophat --solexa1.3-quals --no-coverage-search -g 1 -G /u/home/mcdb/dashraf/GTF/mm9.ensembl.gtf -p 8 -o ./$k /u/home/mcdb/x/bowtie-0.12.8/indexes/temp/mm9 $k.fastq

(the /temp/ is because I added the labmate ewbts in a new folder)