Could not find Bowtie2 index files (genome.*.bt2)

[Juntheboon]

Hi, this is my first time here and I'm currently in the process of self-learning how to use some sequencing software and ubuntu in general. I've been following the steps of this paper:

http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html#an2

I'm working on a server that I currently have no idea what already is installed or not..

I ran this command:

tophat -p 4 -G genes.gtf -o C1_R1_thout genome C1_R1_1.fq C1_R1_2.fq

and this is what showed afterwards:


[2015-03-03 09:44:36] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2015-03-03 09:44:36] Checking for Bowtie
Bowtie version: 2.1.0.0
[2015-03-03 09:44:36] Checking for Samtools
Samtools version: 0.1.19.0
[2015-03-03 09:44:36] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (genome.*.bt2)

How do I resolve this error?

Thank you.

[GenoMax]

Did you download the genome index files from iGenomes: http://support.illumina.com/sequenci...e/igenome.html. Look for the Ensembl drosophila links. This is a big download. The files you need are going to be in this directory hierarchy in a directory called Bowtie2Index. The "genome" part in the command refers to the "basename" (there will be several files that start with genome and then have separate names after .) of the genome index.

It also appears that you are using older version of Tuxedo suite programs. Before you go too far in perhaps you should ask your system administrators if they can update the software for you.

[Juntheboon]

Hi GenoMax,

Yes I already downloaded the iGenome I believe and unpacked it. It was almost 2GB if I remember correctly. It was the BDGP5.25

And yes, I'm using basically a test server to get familiar before getting to use real hardware.. Gotta start washing dishes before getting to work as a chef haha.

[GenoMax]

In that case provide full directory path to /path_to/Bowtie2Index/genome location in your command above.

[Juntheboon]

Do I add this path after the genes.gtf in the command? I'm sorry if this is a dumb question, I'm still very new to all of this.

[GenoMax]

Do I add this path after the genes.gtf in the command? I'm sorry if this is a dumb question, I'm still very new to all of this.

Like this:

$ tophat -p 4 -G /path_to/Annotations/Genes/genes.gtf -o C1_R1_thout /path_to/Bowtie2Index/genome C1_R1_1.fq C1_R1_2.fq

Add the path to genes.gtf file while you are at it.

[Juntheboon]

I got this:

$ tophat -p 4 -G /path_to/Annotations/Genes/genes.gtf -o C1_R1_thout /path_to/Bowtie2Index/genome C1_R1_1.fq C1_R1_2.fq

[2015-03-03 10:21:58] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2015-03-03 10:21:58] Checking for Bowtie
Bowtie version: 2.1.0.0
[2015-03-03 10:21:58] Checking for Samtools
Samtools version: 0.1.19.0
Error: cannot find transcript file /path_to/Annotations/Genes/genes.gtf

I checked through Nautilus whether or not I have the bowtie2 index and I definitely do.. Not sure what the issue is.

[GenoMax]

You need to change the "path_to" part to reflect the actual path you have on your system.

[Juntheboon]

Got it. I think it worked this way.

tophat -p 4 -G /home/jun/Drosophila_melanogaster/Ensembl/BDGP5.25/Annotation/Genes/genes.gtf -o C1_R1_thout /home/jun/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome C1_R1_1.fq C1_R1_2.fq

Now I get another error..


[2015-03-03 10:36:24] Checking for Bowtie index files (genome)..
[2015-03-03 10:36:24] Checking for reference FASTA file
[2015-03-03 10:36:24] Generating SAM header for /home/jun/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome
Traceback (most recent call last):
File "/usr/bin/tophat", line 4072, in <module>
sys.exit(main())
File "/usr/bin/tophat", line 3926, in main
params.read_params = check_reads_format(params, reads_list)
File "/usr/bin/tophat", line 1829, in check_reads_format
zf = ZReader(f_name, params)
File "/usr/bin/tophat", line 1782, in __init__
self.file=open(filename)
IOError: [Errno 2] No such file or directory: 'C1_R1_1.fq'

[GenoMax]

If your sequence files are not in the directory you are running tophat from then provide full path to both of those files.

Start looking at this on the side to understand what we are doing here: http://korflab.ucdavis.edu/Unix_and_...ent.html#part1

[Juntheboon]

Thank you for all of the help. This guide is awesome.