Bowtie-inspec failing me

[ErikFas]

So, I've just gotten past the point where my installation works (thanks for all the help!), and I'm trying to get one of my sets of test data to work.

tophat -r 20 hg19 subset_hct.a_1.fq subset_hct.a_2.fq

It seems to stop at where it wants to make a .fa-file from the .bt2-files:

[2014-06-21 18:35:27] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-06-21 18:35:27] Checking for Bowtie
		  Bowtie version:	 2.2.3.0
[2014-06-21 18:35:27] Checking for Samtools
		Samtools version:	 0.1.19.0
[2014-06-21 18:35:27] Checking for Bowtie index files (genome)..
[2014-06-21 18:35:27] Checking for reference FASTA file
	Warning: Could not find FASTA file /Users/erikfasterius/rna_seq/bowtie_indexes/hg19.fa
[2014-06-21 18:35:27] Reconstituting reference FASTA file from Bowtie index
  Executing: /Users/erikfasterius/bin/bowtie2-inspect hg19 > ./tophat_out/tmp/hg19.fa
	[FAILED]
Error: bowtie-inspect returned an error
Command: bowtie-inspect --wrapper basic-0 hg19

Checking the bowtie_inspect_recons.log yields this:

Error: Encountered exception: 'Cannot open file hg19'
Command: bowtie-inspect --wrapper basic-0 hg19

I'm running the command from the directory where I have my data files, and have my BOWTIE2_INDEXES set to where the .bt2-files are located. They are obviously found (as before I correctly set the BOWTIE2_INDEXES weren't found, since I had tried to set BOWTIE_INDEXES instead due to ignorance), but they somehow can't be opened. I'm still very new to all this, so any help would be greatly appreciated!

[GenoMax]

You could put a copy of hg19.fa (used to make the indexes) in the same directory.

If you are using the stock hg19 genome then you may want to download the genome/indexes/annotation file from iGenomes site. http://support.illumina.com/sequenci...e/igenome.ilmn This bundle contains all the appropriate pieces you would need (with the names of chromosomes in sync) so there would be less problems down the road as you get further into your analysis.

[ErikFas]

I don't have the hg19.fa yet - it wasn't in the file when I downloaded the .bt2-files. Can I get just that from somewhere, or can I manually remake that somehow?

[GenoMax]

You probably want to get that from the same location where you downloaded the indexes from. You can find hg19.fa at many places but there is no guarantee that it will match the indexes you have (in terms of exact build/patch level etc).

Just download the full set for hg19 (sequence, annotation, indexes) from the iGenomes link I posted above.

[ErikFas]

I started with concatenating the assembly sequences (chrom*.fa-files) from the UCSG and using bowtie2-build to get the hg19.fa file, but that still didn't work. Then I just did what you said and downloaded the humongous 27 GB file from iGenomes, which worked!

It took a while to download, though, which was why I didn't do that at first. You're probably entirely correct in saying that the reason it didn't work was that the files were somehow out of sync or with mismatched names somewhere in them.

So, thanks a lot for the help!