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Old 09-11-2014, 02:03 PM   #1
bbm
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Default tophat could not find bowtie index files

I'm trying to align my rna-seq data to ref genome using tophat. I made bowtie2 index files, and it is in the working dir.
The code I used is :
"source /usr/local/apps/tophat/tophat.csh

cd /gpfs_share/hlibyar/tophat

tophat Am_genome ../trim/IAPV_05h_TTAGGC_L003_R1_001.trimmed.fastq
"


This is my error msg.

[2014-09-11 16:36:03] Beginning TopHat run (v2.0.8b)
-----------------------------------------------
[2014-09-11 16:36:03] Checking for Bowtie
Bowtie 2 not found, checking for older version..
Bowtie version: 1.0.0.0
[2014-09-11 16:36:03] Checking for Samtools
Samtools version: 0.1.19.0
[2014-09-11 16:36:03] Checking for Bowtie index files
Error: Could not find Bowtie index files (Am_genome.*.ebwt)

It seems like tophat is looking for ebwt files, but my index are *.bt2
Pls help me here! thanks a lot!
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Old 09-11-2014, 02:09 PM   #2
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The error states that it could not find bowtie2. I assume it needs to be in the path.
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Old 09-11-2014, 03:12 PM   #3
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It found bowtie1 but the right index files (you have version 2 files) are missing. So you can either include path for bowtie2 or create indexes using bowtie1. I assume you want the former.
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Old 09-12-2014, 06:57 AM   #4
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Quote:
Originally Posted by GenoMax View Post
It found bowtie1 but the right index files (you have version 2 files) are missing. So you can either include path for bowtie2 or create indexes using bowtie1. I assume you want the former.
Thanks! But my bowtie2 index are in the current dir?
still need to give the path?
I'll try that.
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Old 09-12-2014, 07:01 AM   #5
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Your default $PATH is pointing to bowtie v.1. executable. Do you have bowtie v.2. installed? That location of v.2 executable should be added to $PATH.

Once tophat finds a version of bowtie (I think it looks for v.2 first then v.1) it will go looking for the indexes. Since it found v.1 it went looking for those indexes (the indexes differ for the two versions).
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Old 03-20-2015, 08:31 AM   #6
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hi
I'm following a tutorial in tophat, but I don't know what is wrong with my code, please help
I have been trying with
tophat -p 2 -G GSM794483_C1_R1.transcripts.gtf -o C1_R1_thout /home/tomas/bin/c1/genome genome GSM794483_C1_R1_1.fq GSM794483_C1_R1_2.fq

tophat -p 2 -G GSM794483_C1_R1.transcripts.gtf -o C1_R1_thout genome.1 GSM794483_C1_R1_1.fq GSM794483_C1_R1_2.fq

or even this
tophat -p 2 -G GSM794483_C1_R1.transcripts.gtf -o C1_R1_thout genome.1 GSM794483_C1_R1_1.fq GSM794483_C1_R1_2.fq
but nothing work...
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Old 03-20-2015, 09:01 AM   #7
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Are you following the tophat tutorial from nature protocols?

Can you post a long listing (ls -l) of files from the directory where you are trying to run tophat from?
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Old 03-20-2015, 09:07 AM   #8
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ls
C1_R1_thout genome.3.bt2 GSM794483_C1_R1_2.fq.gz
genome genome.4.bt2 GSM794483_C1_R1.accepted_hits.bam
genome.1.bt2 GSM794483_C1_R1_1.fq GSM794483_C1_R1.transcripts.gff
genome.2.bt2 GSM794483_C1_R1_2.fq GSM794483_C1_R1.transcripts.gtf
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Old 03-20-2015, 09:23 AM   #9
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Try this:

Code:
$ tophat -p 2 -G ./GSM794483_C1_R1.transcripts.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq
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Old 03-20-2015, 09:54 AM   #10
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tomas@HP-Pavilion:~/bin/c1$ tophat -p 2 -G ./GSM794483_C1_R1.transcripts.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

[2015-03-20 13:53:45] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2015-03-20 13:53:45] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-03-20 13:53:45] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (./genome.*.bt2)
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Old 03-20-2015, 09:57 AM   #11
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i'm working with ubuntu 14, and every file are in the same directory
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Old 03-20-2015, 09:58 AM   #12
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Can you post a long listing of this directory?

Code:
$ ls -lh
Did you get the index files from Illumina igenomes?

Last edited by GenoMax; 03-20-2015 at 10:02 AM.
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Old 03-20-2015, 10:00 AM   #13
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Are not
tomas@HP-Pavilion:~/bin/c1$ ls -lh
total 4,1G
drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
drwxrwxr-x 4 tomas tomas 4,0K mar 20 13:53 C1_R1_thout_new
drwxrwxr-x 2 tomas tomas 4,0K mar 20 12:15 genome
-rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.1.bt2
-rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.2.bt2
-rwxrwxr-x 1 tomas tomas 143 abr 10 2012 genome.3.bt2
-rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.4.bt2
-rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:34 GSM794483_C1_R1_1.fq
-rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:35 GSM794483_C1_R1_2.fq
-rw-rw-r-- 1 tomas tomas 138M sep 13 2011 GSM794483_C1_R1_2.fq.gz
-rw-rw-r-- 1 tomas tomas 325M sep 9 2011 GSM794483_C1_R1.accepted_hits.bam
-rw-rw-r-- 1 tomas tomas 14M mar 20 11:02 GSM794483_C1_R1.transcripts.gff
-rw-rw-r-- 1 tomas tomas 14M mar 20 10:29 GSM794483_C1_R1.transcripts.gtf
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Old 03-20-2015, 10:05 AM   #14
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Those index files do not look right. Where did you get those from? There are two genome.rev.*.bt2 files missing.
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Old 03-20-2015, 10:07 AM   #15
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i get it from http://0-www.ncbi.nlm.nih.gov.elis.t...i?acc=GSE32038
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Old 03-20-2015, 10:14 AM   #16
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Official link for this data at NCBI is: http://www.ncbi.nlm.nih.gov/geo/quer...i?acc=GSE32038 (your link seems to be from Taiwan).

I don't think GEO dataset has fly genome indexes. Those should be downloaded from Illumina igenomes page here: http://support.illumina.com/sequenci...e/igenome.html (See link for BDGP5.25).
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Old 03-20-2015, 10:16 AM   #17
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and of these:
Drosophila melanogaster Ensembl BDGP5 BDGP5.25
NCBI build5.41 build5.3 build5 build4.1

which one should I download?
You have been my informatic super hero

Last edited by lupid; 03-20-2015 at 10:18 AM.
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Old 03-20-2015, 10:24 AM   #18
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Get Ensembl BDGP5.25 since that is used in Nature Protocols. (any of them would work).
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Old 03-20-2015, 10:58 AM   #19
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You are the best, thank, that work
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Old 03-20-2015, 11:35 AM   #20
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Now the error is this
Building Bowtie index from GSM794483_C1_R1.transcripts.fa
[FAILED]
Error: Couldn't build bowtie index with err = 1

I tried to put the fata file in the directory, and not change, what can I do now?
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