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Old 01-24-2012, 10:16 PM   #1
mrfox
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Default Repost: tophat-fusion outputs empty result

Hi all,
Today I run tophat-fusion-post and got empty result. Then I searched the forum and noticed that this question has been posted:
http://seqanswers.com/forums/showthread.php?t=13096

I wonder if anyone has solved this issue. Based on my test, I think tophat-fusion-post does not read the files in tophat_MCF7 under top_dir (assume our sample is MCF7): I prepared all other required files (ensGenes.txt, mcl, refSeq_sorted.txt, blast_human/) under top_dir but did not provide the tophat_MCF7 folder. Then I run tophat-fusion-post, it returned no error message and gave 0 fusions finally.

My another question is: what is the point of "Making a list of known fusions (fusion_info.txt)" before running tophat-fusion-post? Could anybody answer this?

Thank you all.
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Old 01-27-2012, 08:34 AM   #2
mrfox
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Thanks to Daehwan Kim, one of the Tophatfusion contributors, In my case, the PATH for Tophat aligner was not set properly: it points to the Tophat in the Tophatfusion folder. After I correct that, Tophat-fusion goes well for most of my samples.
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Old 02-03-2012, 11:54 AM   #3
xia687
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Could you tell me how did you correct it?
In my case, I have put the path for TopHat-Fusion in my shell's PATH but still get the empty files. I really appreciate you help!
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Old 02-03-2012, 12:08 PM   #4
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You can run 'which tophat' in your shell to see if the original Tophat's 'tophat' script is the default one or Tophat-fusion's 'tophat' script is. If it still points to the Tophat-fusion installation folder, then you need to modify the PATH in your .bashrc or .bash_profile to put Tophat's folder before Tophat-fusion's.

Hope it helps.
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Old 02-03-2012, 12:22 PM   #5
xia687
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Thanks for your reply. I have tried it but still got empty results. In addition, a note from http://tophat-fusion.sourceforge.net/index.html said that "If you have both TopHat and TopHat-Fusion installed on the same machine, you may need to put the path for TopHat-Fusion in your shell's PATH so that the sub-programs included in the TopHat-Fusion's package are used. ". I wondered when you run the first step of tophat-fusion, which
'tophat' command you used, from Tophat's folder or Tophat-fusion's folder?
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Old 02-03-2012, 12:33 PM   #6
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I do not remember it. My suggestion is that you first set up the PATH correctly and then run the pipeline from the very beginning. If the problem occurs again, it might be wrong with something else. Another case is that in your samples, there are no fusion events at all--it is not necessary that we could observe fusions in each sample.

Good luck!
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Old 02-03-2012, 01:02 PM   #7
xia687
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I am still curious why should we put the path for Tophat before TopHat-Fusion in the shell's PATH. In my previous run, I have added both TopHat-Fusion and tophat directories to PATH and got some problems. Some sub-programs such as "prep_reads" can be found in both Tophat and TopHat-Fusion directories. In this case, TopHat-Fusion calls these sub-program from some other place, but not from where it is supposed to be (e.g. from Tophat, not from TopHat-Fusion's package). As a result, it causes the problem.

Thanks and have a good weekend!
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Old 02-03-2012, 01:06 PM   #8
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It seems your can run it smoothly.
I guess there is some difference between Tophat-fusion's scripts with those of Tophat's, though they have the same file names.

Actually we do not need to go to these details. I believe that their team will improve it and eliminate the confusion.

Have a good weekend.
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Old 02-06-2012, 06:15 AM   #9
wzhang1001
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Default tophat-fusion-post empty results

hello Xia687 & Mrfox,

I have the same problem, and have been puzzled for a while ...

I can run tophat-fusion without any problems. But when I tried tophat-fusion-post, I always got empty results. I have been using MCF7 data that tophat-fusion-post uses as demo so I should be able to repeat their results but I couldn't.

I tried changing the PATH as Mrfox suggested but didn't see anything difference. It looked to me tophat-fusion-post is not using balst and blast_human directory because I renamed that folder and removed blastn and blastall, it did not complain, and the log file always looks like
"
/site/app/x86_64/tophatfusion/v0.1.0/tophat-fusion-post:16: DeprecationWarning: the sets module is deprecated
from sets import Set
[Mon Feb 6 09:46:21 2012] Beginning TopHat-Fusion post-processing run (v0.1.0 (Beta))
-----------------------------------------------
[Mon Feb 6 09:46:21 2012] Extracting 23-mer around fusions and mapping them using Bowtie
[Mon Feb 6 09:46:21 2012] Filtering fusions
0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Mon Feb 6 09:46:25 2012] Blasting 50-mers around fusions
[Mon Feb 6 09:46:25 2012] Generating read distributions around fusions
[Mon Feb 6 09:46:25 2012] Reporting final fusion candidates in html format
num of fusions: 0
-----------------------------------------------
Run complete [00:00:04 elapsed]
ESC[HESC[JESC[HESC[JESC[1;31m... Bye-byeESC[0m

"

Any idea? Appreciate your help!
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Old 02-06-2012, 06:19 AM   #10
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Hi wzhang1001,

Did you create the directory structure strictly as instructed in the Tophat-fusion webpage?
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Old 02-06-2012, 06:30 AM   #11
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Yes. I followed the instructions posted on tophat-fusion, and created top_dir and copied ensGene.txt,ensGtp.txt,mcl,refGene_sorted.txt under top_dir, and also created a blast_human directory that conatins human_genomic*(unzipped) and nt* (unzipped) from NCBI.

But looked like tophat-fusion-post didn't look for those blast files which it is supposed to.

Thanks for your quick reply.
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Old 04-17-2012, 07:03 PM   #12
mxqian
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what is the "mcl" for? where to get it?
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Old 04-19-2012, 06:29 PM   #13
kmhwang82
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what is the "mcl" ??
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Old 04-24-2012, 10:54 AM   #14
xia687
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mcl (known gene fusions) can be obtained from previous tophatfusion version (in annotation directory), http://tophat-fusion.sourceforge.net...n-0.1.0.tar.gz
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Old 06-17-2012, 01:48 PM   #15
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Default an observation

not sure if you've all got this to work, but my version of tophat-fusion-post (from tophat2) points to:

blast/

for the blast database, not blast_human/ as listed in the tutorial webpage.

you can change the code, or just put the blast db's into top_dir/blast

good luck,

sf
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Old 06-27-2012, 06:54 PM   #16
fjrossello
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Quote:
Originally Posted by seqfast View Post
not sure if you've all got this to work, but my version of tophat-fusion-post (from tophat2) points to:

blast/

for the blast database, not blast_human/ as listed in the tutorial webpage.

you can change the code, or just put the blast db's into top_dir/blast

good luck,

sf
Hi Guys,

Seqfast is right. The folder name should be blast and NOT blast_human as described in the manual. For your information, using the described top_dir structure with the proposed amendment I was able to replicate the example results.

Cheers,

Fernando
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Old 10-02-2012, 10:32 AM   #17
tankman
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Default tophat-fusion-post

hi fjrosello,

I've posted all over the place about this but maybe you can help me also try to replicate the tophat-fusion-post example they have. I've tried everything and still get empty results at the filtration stage (it doesn't even BLAST anything), and I'm completely baffled why.

Here is some output if you wouldn't mind taking a look, or if easier just send me a copy of your directory structure and command history to see where I diverged. Thanks a lot.

tm

[tankmanb01@node7-3 copy_files]$ ls -lrt
total 251520
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:29 tophat_BT474_mixF.fastq
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:29 tophat_KPL_final
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:29 tophat_MCF7_final2
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:30 tophat_SKBR_final2
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 65 Oct 2 13:54 blast -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/blast
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 71 Oct 2 13:54 refGene.txt -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/refGene.txt
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 71 Oct 2 13:54 ensGene.txt -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/ensGene.txt
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 63 Oct 2 13:54 mcl -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/mcl
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 65 Oct 2 13:54 index -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/index
drwxrwxr-x. 7 tankmanb01 tankmanb01a 32768 Oct 2 13:56 tophatfusion_out
-rw-rw-r--. 1 tankmanb01 tankmanb01a 255743631 Oct 2 14:01 combofusion
-rw-rw-r--. 1 tankmanb01 tankmanb01a 1334123 Oct 2 14:04 fusions_example.out
-rwxr-xr-x. 1 tankmanb01 tankmanb01a 79424 Oct 2 14:05 tophat-fusion-post_ALT
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 5 Oct 2 14:25 blast_human -> blast
[tankmanb01@node7-3 copy_files]$ find tophat_* -name "run.log" -exec head -1 {} \;
/packages/tophat/2.0.4/bin/tophat -p 64 -o tophat/tophat_BT474_mixF.fastq --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 50 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19
BT474_mix_1.fastq BT474_mix_2.fastq
/packages/tophat/2.0.4/bin/tophat -p 64 -o tophat/tophat_KPL_final --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19 SRR064287_1.fastq
SRR064287_2.fastq
/packages/tophat/2.0.4/bin/tophat -p 64 -o tophat/tophat_MCF7_final2 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19 SRR064286_1.fastq
SRR064286_2.fastq
/packages/tophat/2.0.4/bin/tophat -p 45 -o tophat/tophat_SKBR_final2 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 50 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19 SKBR3_mix_1.fastq
SKBR3_mix_2.fastq
[tankmanb01@node7-3 copy_files]$ find . -name "fusions.out" -print
./tophat_BT474_mixF.fastq/fusions.out
./tophat_KPL_final/fusions.out
./tophat_MCF7_final2/fusions.out
./tophat_SKBR_final2/fusions.out
[tankmanb01@node7-3 copy_files]$ tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 index/hg19
[Tue Oct 2 14:29:37 2012] Beginning TopHat-Fusion post-processing run (v2.0.4)
-----------------------------------------------
[Tue Oct 2 14:29:37 2012] Extracting 23-mer around fusions and mapping them using Bowtie
[Tue Oct 2 14:29:43 2012] Filtering fusions
Processing: tophat_BT474_mixF.fastq/fusions.out
Processing: tophat_KPL_final/fusions.out
Processing: tophat_MCF7_final2/fusions.out
Processing: tophat_SKBR_final2/fusions.out
0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Tue Oct 2 14:30:00 2012] Blasting 50-mers around fusions
[Tue Oct 2 14:30:00 2012] Generating read distributions around fusions
[Tue Oct 2 14:30:00 2012] Reporting final fusion candidates in html format
num of fusions: 0
-----------------------------------------------
[Tue Oct 2 14:30:00 2012] Run complete [00:00:22 elapsed]
[tankmanb01@node7-3 copy_files]$
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Old 10-02-2012, 08:46 PM   #18
fjrossello
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Quote:
Originally Posted by tankman View Post
hi fjrosello,

I've posted all over the place about this but maybe you can help me also try to replicate the tophat-fusion-post example they have. I've tried everything and still get empty results at the filtration stage (it doesn't even BLAST anything), and I'm completely baffled why.

Here is some output if you wouldn't mind taking a look, or if easier just send me a copy of your directory structure and command history to see where I diverged. Thanks a lot.

tm

[tankmanb01@node7-3 copy_files]$ ls -lrt
total 251520
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:29 tophat_BT474_mixF.fastq
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:29 tophat_KPL_final
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:29 tophat_MCF7_final2
drwxrwxr-x. 3 tankmanb01 tankmanb01a 32768 Oct 2 13:30 tophat_SKBR_final2
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 65 Oct 2 13:54 blast -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/blast
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 71 Oct 2 13:54 refGene.txt -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/refGene.txt
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 71 Oct 2 13:54 ensGene.txt -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/ensGene.txt
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 63 Oct 2 13:54 mcl -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/mcl
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 65 Oct 2 13:54 index -> /scratch/tankmanb01/projects/RNA_Cholangiocarcinoma/AN/tophat/index
drwxrwxr-x. 7 tankmanb01 tankmanb01a 32768 Oct 2 13:56 tophatfusion_out
-rw-rw-r--. 1 tankmanb01 tankmanb01a 255743631 Oct 2 14:01 combofusion
-rw-rw-r--. 1 tankmanb01 tankmanb01a 1334123 Oct 2 14:04 fusions_example.out
-rwxr-xr-x. 1 tankmanb01 tankmanb01a 79424 Oct 2 14:05 tophat-fusion-post_ALT
lrwxrwxrwx. 1 tankmanb01 tankmanb01a 5 Oct 2 14:25 blast_human -> blast
[tankmanb01@node7-3 copy_files]$ find tophat_* -name "run.log" -exec head -1 {} \;
/packages/tophat/2.0.4/bin/tophat -p 64 -o tophat/tophat_BT474_mixF.fastq --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 50 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19
BT474_mix_1.fastq BT474_mix_2.fastq
/packages/tophat/2.0.4/bin/tophat -p 64 -o tophat/tophat_KPL_final --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19 SRR064287_1.fastq
SRR064287_2.fastq
/packages/tophat/2.0.4/bin/tophat -p 64 -o tophat/tophat_MCF7_final2 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19 SRR064286_1.fastq
SRR064286_2.fastq
/packages/tophat/2.0.4/bin/tophat -p 45 -o tophat/tophat_SKBR_final2 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 50 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM index/hg19 SKBR3_mix_1.fastq
SKBR3_mix_2.fastq
[tankmanb01@node7-3 copy_files]$ find . -name "fusions.out" -print
./tophat_BT474_mixF.fastq/fusions.out
./tophat_KPL_final/fusions.out
./tophat_MCF7_final2/fusions.out
./tophat_SKBR_final2/fusions.out
[tankmanb01@node7-3 copy_files]$ tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 index/hg19
[Tue Oct 2 14:29:37 2012] Beginning TopHat-Fusion post-processing run (v2.0.4)
-----------------------------------------------
[Tue Oct 2 14:29:37 2012] Extracting 23-mer around fusions and mapping them using Bowtie
[Tue Oct 2 14:29:43 2012] Filtering fusions
Processing: tophat_BT474_mixF.fastq/fusions.out
Processing: tophat_KPL_final/fusions.out
Processing: tophat_MCF7_final2/fusions.out
Processing: tophat_SKBR_final2/fusions.out
0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Tue Oct 2 14:30:00 2012] Blasting 50-mers around fusions
[Tue Oct 2 14:30:00 2012] Generating read distributions around fusions
[Tue Oct 2 14:30:00 2012] Reporting final fusion candidates in html format
num of fusions: 0
-----------------------------------------------
[Tue Oct 2 14:30:00 2012] Run complete [00:00:22 elapsed]
[tankmanb01@node7-3 copy_files]$
Hi Tankman,

Find below my directory structure:

Total 48K
32K drwxr-xr-x 2 fernandr hpcmimr 16K Jun 22 12:33 blast
0 -rw-r--r-- 1 fernandr hpcmimr 38M Jun 22 12:33 ensGene.txt
0 -rw-r--r-- 1 fernandr hpcmimr 7.2M Jun 22 12:33 ensGtp.txt
0 -rw-r--r-- 1 fernandr hpcmimr 398K Jun 22 12:33 mcl
0 drwxr-xr-x 3 fernandr hpcmimr 161 Jun 22 15:18 old.tophat_MCF7
0 -rw-r--r-- 1 fernandr hpcmimr 11M Jun 22 12:33 refGene_sorted.txt
0 -rw-r--r-- 1 fernandr hpcmimr 12M Jun 22 12:33 refGene.txt
0 -rw-r--r-- 1 fernandr hpcmimr 1.7G May 7 2011 SRR064286_1.fastq
0 -rw-r--r-- 1 fernandr hpcmimr 1.7G Mar 15 2011 SRR064286_2.fastq
4.0K -rw-r--r-- 1 fernandr hpcmimr 3.5K Jun 30 19:25 tophat2_fusion_post.e3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.o3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.pe3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.po3330717
4.0K -rw-r--r-- 1 fernandr hpcmimr 2.0K Jun 22 15:18 tophat2_test.e3327346
4.0K -rw-r--r-- 1 fernandr hpcmimr 854 Jun 22 12:06 tophat2_test.job
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.o3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.pe3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.po3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Oct 3 14:34 tophat_fusion.out
0 drwxr-xr-x 7 fernandr hpcmimr 223 Jun 30 19:25 tophatfusion_out
4.0K -rw-r--r-- 1 fernandr hpcmimr 763 Jun 30 18:07 tophat_fusion_post.job

As you can see, I have not used symlinks at all. I know it may sound counter intuitive but you should give it a go when all the necessary files are in the directory where the program will be run.

Find below the commands used to generated the output - similar, if not identical - to yours:

/nfs/home/fernandr/biotools/tophat-2.0.3.Linux_x86_64/tophat -o tophat_MCF7 -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /nfs/home/fernandr/biotools/references/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome SRR064286_1.fastq SRR064286_2.fastq

/nfs/home/fernandr/biotools/bin/tophat-fusion-post -p 24 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 /nfs/home/fernandr/biotools/references/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome

I hope it helps.

Cheers,

Fernando
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Old 10-03-2012, 03:09 AM   #19
tankman
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Hi Fernando,

Thanks a lot for your help. 2 questions:

1) Did you run only align the (SRR064286) MCF7 reads using tophat-fusion and then use tophat-fusion-post on just that lone fusions.out file (as opposed to all of KPL, SKPR, BT474, and MCF7)?

2) Did you also use tophat 2.0.3 to align your reads?

I still can't get the damn thing to work, even after removing the sym links (which really shouldn't matter!) and trying tophat-fusion-post version 2.0.3

ahhhhh!

Would it be trouble to pass me the exact output of tophat-fusion-post?

thanks,
tm

tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 index/hg19
[Wed Oct 3 07:06:25 2012] Beginning TopHat-Fusion post-processing run (v2.0.3)
-----------------------------------------------
[Wed Oct 3 07:06:25 2012] Extracting 23-mer around fusions and mapping them using Bowtie
samples updated
[Wed Oct 3 07:06:48 2012] Filtering fusions
Processing: tophat_MCF7_final2/fusions.out
0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Wed Oct 3 07:06:56 2012] Blasting 50-mers around fusions
[Wed Oct 3 07:06:56 2012] Generating read distributions around fusions
[Wed Oct 3 07:06:56 2012] Reporting final fusion candidates in html format
num of fusions: 0
-----------------------------------------------
[Wed Oct 3 07:06:56 2012] Run complete [00:00:31 elapsed]



QUOTE=fjrossello;85530]Hi Tankman,

Find below my directory structure:

Total 48K
32K drwxr-xr-x 2 fernandr hpcmimr 16K Jun 22 12:33 blast
0 -rw-r--r-- 1 fernandr hpcmimr 38M Jun 22 12:33 ensGene.txt
0 -rw-r--r-- 1 fernandr hpcmimr 7.2M Jun 22 12:33 ensGtp.txt
0 -rw-r--r-- 1 fernandr hpcmimr 398K Jun 22 12:33 mcl
0 drwxr-xr-x 3 fernandr hpcmimr 161 Jun 22 15:18 old.tophat_MCF7
0 -rw-r--r-- 1 fernandr hpcmimr 11M Jun 22 12:33 refGene_sorted.txt
0 -rw-r--r-- 1 fernandr hpcmimr 12M Jun 22 12:33 refGene.txt
0 -rw-r--r-- 1 fernandr hpcmimr 1.7G May 7 2011 SRR064286_1.fastq
0 -rw-r--r-- 1 fernandr hpcmimr 1.7G Mar 15 2011 SRR064286_2.fastq
4.0K -rw-r--r-- 1 fernandr hpcmimr 3.5K Jun 30 19:25 tophat2_fusion_post.e3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.o3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.pe3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.po3330717
4.0K -rw-r--r-- 1 fernandr hpcmimr 2.0K Jun 22 15:18 tophat2_test.e3327346
4.0K -rw-r--r-- 1 fernandr hpcmimr 854 Jun 22 12:06 tophat2_test.job
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.o3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.pe3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.po3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Oct 3 14:34 tophat_fusion.out
0 drwxr-xr-x 7 fernandr hpcmimr 223 Jun 30 19:25 tophatfusion_out
4.0K -rw-r--r-- 1 fernandr hpcmimr 763 Jun 30 18:07 tophat_fusion_post.job

As you can see, I have not used symlinks at all. I know it may sound counter intuitive but you should give it a go when all the necessary files are in the directory where the program will be run.

Find below the commands used to generated the output - similar, if not identical - to yours:

/nfs/home/fernandr/biotools/tophat-2.0.3.Linux_x86_64/tophat -o tophat_MCF7 -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /nfs/home/fernandr/biotools/references/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome SRR064286_1.fastq SRR064286_2.fastq

/nfs/home/fernandr/biotools/bin/tophat-fusion-post -p 24 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 /nfs/home/fernandr/biotools/references/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome

I hope it helps.

Cheers,

Fernando[/QUOTE]
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Old 10-03-2012, 03:56 AM   #20
fjrossello
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Location: Melbourne (Victoria) Australia

Join Date: Sep 2011
Posts: 30
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Hi Tankman,

Q1: Yes, I tested the output of that test-sample and I then processed my own.
Q2: Yes, I used tophat 2.0.3 for those reads and I am currently using TopHat 2.0.4. Unfortunately, I will not be able to send you the tophat-fusion-post output for the MCF7 sample because I deleted the tophat-fusion_out folder for that sample.
At the same time, I could try to repeat the process and let you know the outcome. Is this OK?

Cheers,

Fernando

Quote:
Originally Posted by tankman View Post
Hi Fernando,

Thanks a lot for your help. 2 questions:

1) Did you run only align the (SRR064286) MCF7 reads using tophat-fusion and then use tophat-fusion-post on just that lone fusions.out file (as opposed to all of KPL, SKPR, BT474, and MCF7)?

2) Did you also use tophat 2.0.3 to align your reads?

I still can't get the damn thing to work, even after removing the sym links (which really shouldn't matter!) and trying tophat-fusion-post version 2.0.3

ahhhhh!

Would it be trouble to pass me the exact output of tophat-fusion-post?

thanks,
tm

tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 index/hg19
[Wed Oct 3 07:06:25 2012] Beginning TopHat-Fusion post-processing run (v2.0.3)
-----------------------------------------------
[Wed Oct 3 07:06:25 2012] Extracting 23-mer around fusions and mapping them using Bowtie
samples updated
[Wed Oct 3 07:06:48 2012] Filtering fusions
Processing: tophat_MCF7_final2/fusions.out
0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Wed Oct 3 07:06:56 2012] Blasting 50-mers around fusions
[Wed Oct 3 07:06:56 2012] Generating read distributions around fusions
[Wed Oct 3 07:06:56 2012] Reporting final fusion candidates in html format
num of fusions: 0
-----------------------------------------------
[Wed Oct 3 07:06:56 2012] Run complete [00:00:31 elapsed]



QUOTE=fjrossello;85530]Hi Tankman,

Find below my directory structure:

Total 48K
32K drwxr-xr-x 2 fernandr hpcmimr 16K Jun 22 12:33 blast
0 -rw-r--r-- 1 fernandr hpcmimr 38M Jun 22 12:33 ensGene.txt
0 -rw-r--r-- 1 fernandr hpcmimr 7.2M Jun 22 12:33 ensGtp.txt
0 -rw-r--r-- 1 fernandr hpcmimr 398K Jun 22 12:33 mcl
0 drwxr-xr-x 3 fernandr hpcmimr 161 Jun 22 15:18 old.tophat_MCF7
0 -rw-r--r-- 1 fernandr hpcmimr 11M Jun 22 12:33 refGene_sorted.txt
0 -rw-r--r-- 1 fernandr hpcmimr 12M Jun 22 12:33 refGene.txt
0 -rw-r--r-- 1 fernandr hpcmimr 1.7G May 7 2011 SRR064286_1.fastq
0 -rw-r--r-- 1 fernandr hpcmimr 1.7G Mar 15 2011 SRR064286_2.fastq
4.0K -rw-r--r-- 1 fernandr hpcmimr 3.5K Jun 30 19:25 tophat2_fusion_post.e3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.o3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.pe3330717
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 30 18:39 tophat2_fusion_post.po3330717
4.0K -rw-r--r-- 1 fernandr hpcmimr 2.0K Jun 22 15:18 tophat2_test.e3327346
4.0K -rw-r--r-- 1 fernandr hpcmimr 854 Jun 22 12:06 tophat2_test.job
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.o3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.pe3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Jun 22 13:12 tophat2_test.po3327346
0 -rw-r--r-- 1 fernandr hpcmimr 0 Oct 3 14:34 tophat_fusion.out
0 drwxr-xr-x 7 fernandr hpcmimr 223 Jun 30 19:25 tophatfusion_out
4.0K -rw-r--r-- 1 fernandr hpcmimr 763 Jun 30 18:07 tophat_fusion_post.job

As you can see, I have not used symlinks at all. I know it may sound counter intuitive but you should give it a go when all the necessary files are in the directory where the program will be run.

Find below the commands used to generated the output - similar, if not identical - to yours:

/nfs/home/fernandr/biotools/tophat-2.0.3.Linux_x86_64/tophat -o tophat_MCF7 -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /nfs/home/fernandr/biotools/references/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome SRR064286_1.fastq SRR064286_2.fastq

/nfs/home/fernandr/biotools/bin/tophat-fusion-post -p 24 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 /nfs/home/fernandr/biotools/references/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome

I hope it helps.

Cheers,

Fernando
[/QUOTE]
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