tophat cannot find bowtie 2 indexes

[mjm...@ucdavis.edu]

Hello,

I'm relatively new to RNAseq and currently having trouble having tophat identify my bowtie 2 indexes. I downloaded my reference sequence from maizecyc, and now I am trying to run tophat. This is what I am getting:

$ tophat /path/to/reference/sequence maize_v3_2014.ebwt PE_read_1 PE_read_2
[2015-10-14 13:51:24] Beginning TopHat run (v2.1.0)
-----------------------------------------------
[2015-10-14 13:51:24] Checking for Bowtie
          Bowtie version:     2.2.6.0
[2015-10-14 13:51:24] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (/path/to/reference/seq/maize_ref_v3_2014.ebwt.*.bt2)

Is the reasoning is that I did not install my indexes properly? I wrote for the bowtie-build command as:

$bowtie2-build maize_v3_2014.fa -o maize_v3_2014.ebwt

note: I am also working on a computer where tophat is in a module format, and so I cannot just simply move the indexes to the bowtie2 index folder on my computer.
Much thanks if you can provide me some sort of solution(s)

[mouton...@gmail.com]

Hello, 

 so this is how I built my bowtie file. As you need to use a gtf later then take the gtf in the same website of your reference genome (like explained in the manual). 

I think that Ensembl is pretty good and has updated genome.

#  ftp://ftp.ensembl.org/pub/release-82/fasta/yourreference

#  Download toplevel.fa.gz These files contains all sequence regions flagged as toplevel in an Ensembl schema. This includes chromsomes, regions not assembled into chromosomes and N padded haplotype/patch regions.

gunzip yourreferencegenome.dna.toplevel.fa.gz

#From the directory containing the genome.fa file, run the "bowtie2-build" command.  

bowtie2-build -f Felis_catus.Felis_catus_6.2.dna.toplevel.fa FelisCatus62

#This command will create 6 files with a *.bt2 file extension.  

Then when you run Tophat2 you don't have to give him the extension of your bowtie file, Tophat will search by himself. Then it is highly recommended but not obligatory to have your referencegenome.fa in the same directory as your bowtie2 indexes. Tophat will be just much faster. Be sure that they have the samename (I rename FelisCatus62  the genome file) then in your command you just have to give the name of the file /yourpath/FelisCatus62 before .bt2 (avoid the "." in your file) and it will work!

Have a good work

Alice