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**GenoMax** · 03-20-2015, 09:14 AM

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).

**lupid** · 03-20-2015, 09:16 AM

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

**GenoMax** · 03-20-2015, 09:24 AM

Get Ensembl BDGP5.25 since that is used in Nature Protocols. (any of them would work).

**lupid** · 03-20-2015, 09:58 AM

You are the best, thank, that work

**lupid** · 03-20-2015, 10:35 AM

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?

**GenoMax** · 03-20-2015, 10:49 AM

Use this file instead of the GTF file you are using: /path_to/Drosophila_melanogaster/Ensembl/BDGP5.25/Annotation/Genes/genes.gtf

**lupid** · 03-20-2015, 11:48 AM

don't work u.u
tophat -p 2 -G ./genes.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

[2015-03-20 16:13:45] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2015-03-20 16:13:45] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-03-20 16:13:45] Checking for Bowtie index files (genome)..
[2015-03-20 16:13:45] Checking for reference FASTA file
Warning: Could not find FASTA file ./genome.fa
[2015-03-20 16:13:45] Reconstituting reference FASTA file from Bowtie index
Executing: /home/tomas/bin/bowtie2-inspect ./genome > ./C1_R1_thout_new/tmp/genome.fa
[2015-03-20 16:13:54] Generating SAM header for ./genome
[2015-03-20 16:13:55] Reading known junctions from GTF file
[2015-03-20 16:13:59] Preparing reads
left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
[2015-03-20 16:18:58] Building transcriptome data files ./C1_R1_thout_new/tmp/genes
[2015-03-20 16:19:07] Building Bowtie index from genes.fa
[FAILED]
Error: Couldn't build bowtie index with err = 1
tomas@HP-Pavilion:~/bin/c1$

**GenoMax** · 03-20-2015, 12:03 PM

Can you show a current long listing of the files in directory?

Code:

$ ls -lh

As shown on page 570 of Nature protocols article it may be best to create at least this softlink in your current directory. I am assuming you have copied other files locally (will know from the listing above).

Code:

$ ln -s /path_to/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .

**lupid** · 03-20-2015, 12:10 PM

tomas@HP-Pavilion:~/bin/c1$ ls -lh
total 4,4G
drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
-rwxrwxr-x 1 tomas tomas 117M mar 15 2012 genes.fa
-rwxrwxr-x 1 tomas tomas 54M may 23 2014 genes.gtf
-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
lrwxrwxrwx 1 tomas tomas 29 mar 20 16:51 genome.fa -> ../WholeGenomeFasta/genome.fa
-rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
-rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.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
tomas@HP-Pavilion:~/bin/c1$

I don't know if I did what you propouse, but when I try, this happend
tomas@HP-Pavilion:~/bin/c1$ ln -s /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .
ln: fallo al crear el enlace simbólico «./genome.fa»: El archivo ya existe
tomas@HP-Pavilion:~/bin/c1$

**GenoMax** · 03-20-2015, 12:15 PM

This link is not pointing to the right directory:

lrwxrwxrwx 1 tomas tomas 29 mar 20 16:51 genome.fa -> ../WholeGenomeFasta/genome.fa

So do this

Code:

$ unlink genome.fa

ONLY if above does not work then

Code:

$ rm -f genome.fa

Then create the soft link again

Code:

$ ln -s /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .

Try tophat.

**lupid** · 03-20-2015, 12:31 PM

after try both options still didn't work
here my console
tomas@HP-Pavilion:~/bin/c1$ rm -f genome.fa
tomas@HP-Pavilion:~/bin/c1$ ln -s /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .
tomas@HP-Pavilion:~/bin/c1$ ls -lh
total 4,4G
drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
-rwxrwxr-x 1 tomas tomas 117M mar 15 2012 genes.fa
-rwxrwxr-x 1 tomas tomas 54M may 23 2014 genes.gtf
-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
lrwxrwxrwx 1 tomas tomas 91 mar 20 17:23 genome.fa -> /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa
-rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
-rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.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
tomas@HP-Pavilion:~/bin/c1$ tophat -p 2 -G ./genes.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

[2015-03-20 17:24:20] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2015-03-20 17:24:20] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-03-20 17:24:20] Checking for Bowtie index files (genome)..
[2015-03-20 17:24:20] Checking for reference FASTA file
[2015-03-20 17:24:20] Generating SAM header for ./genome
[2015-03-20 17:24:21] Reading known junctions from GTF file
[2015-03-20 17:24:24] Preparing reads
left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
[2015-03-20 17:29:19] Building transcriptome data files ./C1_R1_thout_new/tmp/genes
[2015-03-20 17:29:26] Building Bowtie index from genes.fa
[FAILED]
Error: Couldn't build bowtie index with err = 1
tomas@HP-Pavilion:~/bin/c1$

**GenoMax** · 03-20-2015, 01:43 PM

Do this

Code:

$ head -5 genes.fa

and then move the genes.fa file to something else? I am not sure where that file came from

Code:

$ mv genes.fa genes.fa.BKP

**lupid** · 03-20-2015, 02:48 PM

Don't u.u
I changed the name of the gtf file, because I read that maybe has to have the same as the rest of the files, but don't work either, here my console
/bin/c1$ tophat -p 2 -G ./genome.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

[2015-03-20 19:36:20] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2015-03-20 19:36:20] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-03-20 19:36:20] Checking for Bowtie index files (genome)..
[2015-03-20 19:36:20] Checking for reference FASTA file
[2015-03-20 19:36:20] Generating SAM header for ./genome
[2015-03-20 19:36:20] Reading known junctions from GTF file
[2015-03-20 19:36:24] Preparing reads
left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
[2015-03-20 19:41:34] Building transcriptome data files ./C1_R1_thout_new/tmp/genome
[2015-03-20 19:41:42] Building Bowtie index from genome.fa
[FAILED]
Error: Couldn't build bowtie index with err = 1
tomas@HP-Pavilion:~/bin/c1$ ls -lh
total 4,3G
-rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15
-rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15.txt
drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
-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
lrwxrwxrwx 1 tomas tomas 91 mar 20 17:23 genome.fa -> /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa
-rwxrwxr-x 1 tomas tomas 54M may 23 2014 genome.gtf
-rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
-rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.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

**GenoMax** · 03-20-2015, 04:07 PM

Did you download bowtie2 and tophat executables or compiled them yourself? Are you running 32-bit/64-bit Ubuntu?

Since you have copied all other files to the current directory why don't we copy the genome file too

Code:

$ unlink genome.fa
$ cp /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/WholeGenomeFasta/genome.fa .

**lupid** · 03-21-2015, 05:27 AM

I'm using Ubuntu 64 and download the executables
I try to put the file in the same directory as you suggest and nothing
/bin/c1$ ls -lh
total 4,4G
-rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15
-rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15.txt
drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
-rwxrwxr-x 1 tomas tomas 54M mar 20 20:02 genes.gtf
-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
-rwxrwxr-x 1 tomas tomas 117M mar 21 10:16 genome.fa
-rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
-rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.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
tomas@HP-Pavilion:~/bin/c1$ tophat -p 2 -G ./genes.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

[2015-03-21 10:16:47] Beginning TopHat run (v2.0.13)
-----------------------------------------------
[2015-03-21 10:16:47] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-03-21 10:16:48] Checking for Bowtie index files (genome)..
[2015-03-21 10:16:48] Checking for reference FASTA file
[2015-03-21 10:16:48] Generating SAM header for ./genome
[2015-03-21 10:16:50] Reading known junctions from GTF file
[2015-03-21 10:16:54] Preparing reads
left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
[2015-03-21 10:22:22] Building transcriptome data files ./C1_R1_thout_new/tmp/genes
[2015-03-21 10:22:29] Building Bowtie index from genes.fa
[FAILED]
Error: Couldn't build bowtie index with err = 1
tomas@HP-Pavilion:~/bin/c1$

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