[tankman]

tophat-fusion-post

Hi Emilie,

Thanks a lot for your answer. Was your initial alignment call something like

/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

for your MCF7 run?

how many fusions did you get?

I'm still getting nothing!

tophat-fusion-post -p 1 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 indexes/hg19
[Wed Oct 3 10:53:02 2012] Beginning TopHat-Fusion post-processing run (v2.0.3)
-----------------------------------------------
[Wed Oct 3 10:53:02 2012] Extracting 23-mer around fusions and mapping them using Bowtie
samples updated
[Wed Oct 3 10:53:24 2012] Filtering fusions
Processing: tophat_MCF7_final2/fusions.out
0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Wed Oct 3 10:53:32 2012] Blasting 50-mers around fusions
[Wed Oct 3 10:53:32 2012] Generating read distributions around fusions
[Wed Oct 3 10:53:32 2012] Reporting final fusion candidates in html format
num of fusions: 0
-----------------------------------------------
[Wed Oct 3 10:53:33 2012] Run complete [00:00:30 elapsed]

I used as well MCF7 to test TopHat-Fusion but it worked for me.
I am using symlinks for the blast and indexes directories (it works as well if the tophat_MCF7_1 files are symlinks).

The structure I am using to run tophat-fusion-post is the following:

TopHatFusion_MCF7
----blast
----blast_human # symbolic link to the blast directory
----indexes # hg19 indexes / bowtie1
----mcl
----refGene.txt
----ensGene.txt
----tophat_MCF7_1
--------accepted_hits.bam
--------deletions.bed
--------fusions.out
--------insertions.bed
--------junctions.bed
--------logs
--------prep_reads.info
--------unmapped.bam
----tophatfusion_out

The blast directory looks like:

blast
----human_genomic.00.nhd
----human_genomic.00.nhi
[...]
----nt.00.nhd
----nt.00.nhi
[...]
----other_genomic.00.nhd
----other_genomic.00.nhi
[...]

The command I am using:
tophat-fusion-post -p 1 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 indexes/hg19

I am using bowtie/0.12.7, samtools/0.1.18 and blast+/2.2.26
This works with both tophat-2.0.3 (tophat-2.0.3.Linux_x86_64.tar.gz) and tophat-2.0.4 (tophat-2.0.4.Linux_x86_64.tar.gz)

Hope this helps

Emilie

[Emilie]

Hi tankman,

My TopHat2 command:
tophat2 -o tophat_MCF7_1 -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 indexes/hg19 SRR064286_1.fastq SRR064286_2.fastq

fusions.out: 70619 lines
potential_fusion.txt: 84 lines
result.txt: 13 lines

Do you get a similar number of lines in fusions.out?
Is your blast directory organized the same way?
Which files do you obtain when you run tophat-fusion-post?

Emilie

[Emilie]

The corresponding log file (for 2.0.3):

/bin/bash: module: line 1: syntax error: unexpected end of file
/bin/bash: error importing function definition for `module'
[Wed Jun 27 13:23:06 2012] Beginning TopHat-Fusion post-processing run (v2.0.3)
-----------------------------------------------
[Wed Jun 27 13:23:06 2012] Extracting 23-mer around fusions and mapping them using Bowtie
samples updated
[Wed Jun 27 13:24:23 2012] Filtering fusions
Processing: tophat_MCF7_1/fusions.out
14 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Wed Jun 27 13:24:30 2012] Blasting 50-mers around fusions
1. RSBN1 exon7(114354330-114355069) AP4B1 intron5(114441423-114442523)
2. LRP1B exon89(142237963-142238101) PLXDC1 exon12(37265499-37265643)
3. ENSG00000233459 exon1(204499298-204500738) ZNF207 exon8(30692347-30692505)
4. ENSG00000250859 exon1(126847154-126848533) HNRNPK exon3(86585650-86585733)
5. FOXA1 exon1(38058755-38061915) ENSG00000139865 intron7(38184001-38194100)
6. ENSG00000224738 exon1(57183957-57184951) VMP1 exon11(57915654-57915757)
7. VMP1 exon12(57917127-57917951) RPS6KB1 exon4(57991994-57992063)
8. USP32 exon26(58342771-58342834) PPM1D intron1(58678247-58700879)
9. BCAS3 intron23(59161925-59445685) BCAS4 exon1(49411465-49411709)
10. BCAS3 exon24(59445686-59445854) BCAS4 exon1(49411465-49411709)
11. CARM1 exon2(11015625-11015751) SMARCA4 exon4(11096863-11097268)
12. ARFGEF2 exon1(47538273-47538546) SULF2 exon19(46365445-46365685)
13. SULF2 exon21(46414790-46415359) ENSG00000171940 intron4(52199707-52210643)
14. SULF2 exon21(46414790-46415359) ENSG00000171940 exon5(52210644-52210800)
[Wed Jun 27 13:55:46 2012] Generating read distributions around fusions
MCF7_1 (1-14)
chr1-chr1 114354329 114442495 rf
chr2-chr17 142237963 37265642 rr
chr2-chr17 204499953 30692348 rf
chr5-chr9 126847434 86585718 rr
chr14-chr14 38061534 38184710 rr
chr17-chr17 57184951 57915655 ff
chr17-chr17 57917128 57992063 rr
chr17-chr17 58342772 58679978 rr
chr17-chr20 59430948 49411709 rr
chr17-chr20 59445687 49411709 rr
chr19-chr19 11015626 11097268 rr
chr20-chr20 47538546 46365685 fr
chr20-chr20 46415148 52210294 rf
chr20-chr20 46415148 52210645 rf
[Wed Jun 27 14:08:48 2012] Reporting final fusion candidates in html format
num of fusions: 11
-----------------------------------------------
[Wed Jun 27 14:08:50 2012] Run complete [00:45:43 elapsed]


The resulting tophatfusion_out files contains (no empty files or folders):

blast_genomic
blast_nt
check
fusion_seq.bwtout
fusion_seq.fa
fusion_seq.map
logs
potential_fusion.txt
result.html
result.txt
sample_list.txt
tmp

[tankman]

Hi Emilie,

my tophat 2.0.4 command was identical to yours however I obtained 74484 lines in my fusions.out file, so somewhat more than you strangely enough. My potential_fusion.txt and result.txt files are both empty!

I obtain these files from tophatfusion_output

total 453248
-rw-rw-r--. 1 tankman01 tankman01a 51 Oct 2 13:31 sample_list.txt
-rw-rw-r--. 1 tankman01 tankman01a 37305758 Oct 2 13:31 fusion_seq.fa
-rw-rw-r--. 1 tankman01 tankman01a 355780646 Oct 2 13:32 fusion_seq.bwtout
-rw-rw-r--. 1 tankman01 tankman01a 69632205 Oct 2 13:32 fusion_seq.map
drwxrwxr-x. 2 tankman01 tankman01a 32768 Oct 2 13:32 tmp
-rw-rw-r--. 1 tankman01 tankman01a 0 Oct 2 13:33 potential_fusion.txt
drwxrwxr-x. 2 tankman01 tankman01a 32768 Oct 2 13:33 blast_genomic
drwxrwxr-x. 2 tankman01 tankman01a 32768 Oct 2 13:33 blast_nt
drwxrwxr-x. 2 tankman01 tankman01a 32768 Oct 2 13:33 check
-rw-rw-r--. 1 tankman01 tankman01a 0 Oct 2 13:33 result.txt
-rw-rw-r--. 1 tankman01 tankman01a 1120380 Oct 2 13:33 result.html
drwxrwxr-x. 2 tankman01 tankman01a 32768 Oct 2 13:33 logs
-rw-rw-r--. 1 tankman01 tankman01a 0 Oct 3 21:56 file

Hi tankman,

My TopHat2 command:
tophat2 -o tophat_MCF7_1 -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 indexes/hg19 SRR064286_1.fastq SRR064286_2.fastq

fusions.out: 70619 lines
potential_fusion.txt: 84 lines
result.txt: 13 lines

Do you get a similar number of lines in fusions.out?
Is your blast directory organized the same way?
Which files do you obtain when you run tophat-fusion-post?

Emilie

[tankman]

fusions.out file might be different

Hi Emilie,

The only think I can think of now is that the fusions.out file is somehow very different than yours. Is there a way we could exchange fusions.out files and you try it on mine and try it on yours to see if there's any difference in result?

thanks
tm

The corresponding log file (for 2.0.3):

/bin/bash: module: line 1: syntax error: unexpected end of file
/bin/bash: error importing function definition for `module'
[Wed Jun 27 13:23:06 2012] Beginning TopHat-Fusion post-processing run (v2.0.3)
-----------------------------------------------
[Wed Jun 27 13:23:06 2012] Extracting 23-mer around fusions and mapping them using Bowtie
samples updated
[Wed Jun 27 13:24:23 2012] Filtering fusions
Processing: tophat_MCF7_1/fusions.out
14 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Wed Jun 27 13:24:30 2012] Blasting 50-mers around fusions
1. RSBN1 exon7(114354330-114355069) AP4B1 intron5(114441423-114442523)
2. LRP1B exon89(142237963-142238101) PLXDC1 exon12(37265499-37265643)
3. ENSG00000233459 exon1(204499298-204500738) ZNF207 exon8(30692347-30692505)
4. ENSG00000250859 exon1(126847154-126848533) HNRNPK exon3(86585650-86585733)
5. FOXA1 exon1(38058755-38061915) ENSG00000139865 intron7(38184001-38194100)
6. ENSG00000224738 exon1(57183957-57184951) VMP1 exon11(57915654-57915757)
7. VMP1 exon12(57917127-57917951) RPS6KB1 exon4(57991994-57992063)
8. USP32 exon26(58342771-58342834) PPM1D intron1(58678247-58700879)
9. BCAS3 intron23(59161925-59445685) BCAS4 exon1(49411465-49411709)
10. BCAS3 exon24(59445686-59445854) BCAS4 exon1(49411465-49411709)
11. CARM1 exon2(11015625-11015751) SMARCA4 exon4(11096863-11097268)
12. ARFGEF2 exon1(47538273-47538546) SULF2 exon19(46365445-46365685)
13. SULF2 exon21(46414790-46415359) ENSG00000171940 intron4(52199707-52210643)
14. SULF2 exon21(46414790-46415359) ENSG00000171940 exon5(52210644-52210800)
[Wed Jun 27 13:55:46 2012] Generating read distributions around fusions
MCF7_1 (1-14)
chr1-chr1 114354329 114442495 rf
chr2-chr17 142237963 37265642 rr
chr2-chr17 204499953 30692348 rf
chr5-chr9 126847434 86585718 rr
chr14-chr14 38061534 38184710 rr
chr17-chr17 57184951 57915655 ff
chr17-chr17 57917128 57992063 rr
chr17-chr17 58342772 58679978 rr
chr17-chr20 59430948 49411709 rr
chr17-chr20 59445687 49411709 rr
chr19-chr19 11015626 11097268 rr
chr20-chr20 47538546 46365685 fr
chr20-chr20 46415148 52210294 rf
chr20-chr20 46415148 52210645 rf
[Wed Jun 27 14:08:48 2012] Reporting final fusion candidates in html format
num of fusions: 11
-----------------------------------------------
[Wed Jun 27 14:08:50 2012] Run complete [00:45:43 elapsed]


The resulting tophatfusion_out files contains (no empty files or folders):

blast_genomic
blast_nt
check
fusion_seq.bwtout
fusion_seq.fa
fusion_seq.map
logs
potential_fusion.txt
result.html
result.txt
sample_list.txt
tmp

[Emilie]

Sure, I just sent you a private message with my email address.

Hi Emilie,

The only think I can think of now is that the fusions.out file is somehow very different than yours. Is there a way we could exchange fusions.out files and you try it on mine and try it on yours to see if there's any difference in result?

thanks
tm

[yingzhang]

extreme memory requirement for Tophat-fusion-post

After 3 failed tophat-fusion-post runs on the sample MCF data, I finally got the program to work.

This is the setting of my successful job:

#PBS -l mem=64gb,nodes=1pn=8,walltime=24:00:00

It finished in 21 minutes.

The last failed job used 32G mem.

[kokonech]

Hi,
does anybody have a response from the authors or any clues how the filtering of juncions.out occur?

In my simulation experiments I have a large list of fusion candidates (about 500), and this list contains the simulated fusions. These fusions should pass the basic filtering: number of fusions reads, number of read pairs etc. But after running tophat-fusion-post I have 0 fusions.

[Charitra]

tophat-fusion-post result empty

Answer is:
My experience.. There is only one reason for empty tophat fusion post empty and that is: you did not prefix tophat_(your sample name) to your output directory while commanding tophat (pre)fusion i.e. first command. That is why tophat can not read your output file after first tophat (pre)fusion run....if not sure then try changing your output file name and again run tophat fusion post (not pre fusion)... you will get empty result file with 0 fusion gene.
If there is other reason, tophat will give you error... if there is no error that mean it can not read your output file.

Please let me explain you in detail.

Follow these commands: (available online at broadinstitute) How to run Tophat-fusion? https://confluence.broadinstitute.or...ageId=46531375

Step 1: Install
bowtie i.e. bowtie1
tophat2
samtools
ncbi.blast

Step 2: download
ensGene.txt
refGene.txt

Step 3:
make a directory of your sample, for example your sample name is John then your directory is John. Place your .fastq files (n=2, for pair end).

Step 4: transfer the downloaded files ensGene.txt and refGene.txt into John directory which is your sample directory.

Step 4: Login to putty
PATH=$PATHplease give the path of bowtie)
PATH=$PATHplease give the path of samtols)
PATH=$PATHplease give the path of tophat2)
PATH=$PATHplease give the path of blast)
export PATH

Step 5: change directory and come to your sample directory i.e. John
cd (please give the path of John)
Now you are here in you working directory like below:
John$

Step 6:run the tophat fusion using following standard commands. Most important thing is -o command which makes the your output directory, So always make your output folder name starting from tophat_(your output name). For example; sample name John so I will give command like this: -o tophat_John. Thats all.

Standard commands are: for sample name John (remember now I have sample name John which has two .fastq files i.e. John_1.fastq John_2.fastq).

tophat -o tophat_John -p 8 --fusion-search --keep-fasta-order --bowtie 1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromoso mes chrM (Path of hg19 which should be in bowtie1) John_1.fastq John_2.fastq
run it....

Step 7:Post Fusion
tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 (Path of hg19 which should be in bowtie1)
run it....

You will get the fusion genes.
If still there is proble... reply to me please....

[Charitra]

Answer is:
My experience.. There is only one reason for empty tophat fusion post empty and that is: you did not prefix tophat_(your sample name) to your output directory while commanding tophat (pre)fusion i.e. first command. That is why tophat can not read your output file after first tophat (pre)fusion run....if not sure then try changing your output file name and again run tophat fusion post (not pre fusion)... you will get empty result file with 0 fusion gene.
If there is other reason, tophat will give you error... if there is no error that mean it can not read your output file.

Please let me explain you in detail.

Follow these commands: (available online at broadinstitute) How to run Tophat-fusion? https://confluence.broadinstitute.or...ageId=46531375

Step 1: Install
bowtie i.e. bowtie1
tophat2
samtools
ncbi.blast

Step 2: download
ensGene.txt
refGene.txt

Step 3:
make a directory of your sample, for example your sample name is John then your directory is John. Place your .fastq files (n=2, for pair end).

Step 4: transfer the downloaded files ensGene.txt and refGene.txt into John directory which is your sample directory.

Step 4: Login to putty
PATH=$PATHplease give the path of bowtie)
PATH=$PATHplease give the path of samtols)
PATH=$PATHplease give the path of tophat2)
PATH=$PATHplease give the path of blast)
export PATH

Step 5: change directory and come to your sample directory i.e. John
cd (please give the path of John)
Now you are here in you working directory like below:
John$

Step 6:run the tophat fusion using following standard commands. Most important thing is -o command which makes the your output directory, So always make your output folder name starting from tophat_(your output name). For example; sample name John so I will give command like this: -o tophat_John. Thats all.

Standard commands are: for sample name John (remember now I have sample name John which has two .fastq files i.e. John_1.fastq John_2.fastq).

tophat -o tophat_John -p 8 --fusion-search --keep-fasta-order --bowtie 1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromoso mes chrM (Path of hg19 which should be in bowtie1) John_1.fastq John_2.fastq
run it....

Step 7:Post Fusion
tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 (Path of hg19 which should be in bowtie1)
run it....

You will get the fusion genes.

[samar]

tophat fusion post 0 fusions

Hi,
I have similar problem, but when I run tophat-fusion-post with the developers samples, I am getting fusions, but looks dufferent.
However, when I tried many samples all are giving me 0 fusions.
Any idea , please!

Thanks

Answer is:
My experience.. There is only one reason for empty tophat fusion post empty and that is: you did not prefix tophat_(your sample name) to your output directory while commanding tophat (pre)fusion i.e. first command. That is why tophat can not read your output file after first tophat (pre)fusion run....if not sure then try changing your output file name and again run tophat fusion post (not pre fusion)... you will get empty result file with 0 fusion gene.
If there is other reason, tophat will give you error... if there is no error that mean it can not read your output file.

Please let me explain you in detail.

Follow these commands: (available online at broadinstitute) How to run Tophat-fusion? https://confluence.broadinstitute.or...ageId=46531375

Step 1: Install
bowtie i.e. bowtie1
tophat2
samtools
ncbi.blast

Step 2: download
ensGene.txt
refGene.txt

Step 3:
make a directory of your sample, for example your sample name is John then your directory is John. Place your .fastq files (n=2, for pair end).

Step 4: transfer the downloaded files ensGene.txt and refGene.txt into John directory which is your sample directory.

Step 4: Login to putty
PATH=$PATHplease give the path of bowtie)
PATH=$PATHplease give the path of samtols)
PATH=$PATHplease give the path of tophat2)
PATH=$PATHplease give the path of blast)
export PATH

Step 5: change directory and come to your sample directory i.e. John
cd (please give the path of John)
Now you are here in you working directory like below:
John$

Step 6:run the tophat fusion using following standard commands. Most important thing is -o command which makes the your output directory, So always make your output folder name starting from tophat_(your output name). For example; sample name John so I will give command like this: -o tophat_John. Thats all.

Standard commands are: for sample name John (remember now I have sample name John which has two .fastq files i.e. John_1.fastq John_2.fastq).

tophat -o tophat_John -p 8 --fusion-search --keep-fasta-order --bowtie 1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromoso mes chrM (Path of hg19 which should be in bowtie1) John_1.fastq John_2.fastq
run it....

Step 7:Post Fusion
tophat-fusion-post -p 8 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 (Path of hg19 which should be in bowtie1)
run it....

You will get the fusion genes.

[Charitra]

Please read my last post. check the output directory name, if it is different then you will get 0 fusion. and.. try chimerascan is the best for fusion.
Please reply if you get error again.
Charitra

[samar]

Thank you so much for replying!
Yes, what I did is very similar to yours.
And, I did the prefix:
tophat -o /path/to/sample/tophat_PRO -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /../UCSC/hg19/Sequence/BowtieIndex/genome sample.fastq

The odd thing is that when I run the test sample from tophat fusion website, I did get the fusions.
However, with the 4 samples I did not get anything.

Please read my last post. check the output directory name, if it is different then you will get 0 fusion. and.. try chimerascan is the best for fusion.
Please reply if you get error again.
Charitra

[Charitra]

Hi there,
Can you compare the two commands:

tophat -o /path/to/sample/tophat_PRO -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /../UCSC/hg19/Sequence/BowtieIndex/genome sample.fastq

tophat -o tophat_John -p 8 --fusion-search --keep-fasta-order --bowtie 1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromoso mes chrM (Path of hg19 which should be in bowtie1) John_1.fastq John_2.fastq

Well, the problems may be sorted out if you:
1. -o tophat_PRO
2. remove spaces from file or dir names i.e. genome_sample.fastq (genome sample.fastq ?)
3. Where is your two .fastq files. pair end ?

Thank you so much for replying!
Yes, what I did is very similar to yours.
And, I did the prefix:
tophat -o /path/to/sample/tophat_PRO -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /../UCSC/hg19/Sequence/BowtieIndex/genome sample.fastq

The odd thing is that when I run the test sample from tophat fusion website, I did get the fusions.
However, with the 4 samples I did not get anything.

[samar]

Hi Charita,,
1. Yeah. This is only the path for the output directory
2. The space because the genome is a completion from the index directory path, and the sample.fastq is the sample.
3. Yeah I think this is an important point, it is single end. And it's looks that I am running paired end. However, I am not sure if should I run it in the same way or should it be different?
In the manual it says that tophat-fusion can run single or paired end.

I am really appreciating your help!!
Thank you Charita

Hi there,
Can you compare the two commands:

tophat -o /path/to/sample/tophat_PRO -p 8 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /../UCSC/hg19/Sequence/BowtieIndex/genome sample.fastq

tophat -o tophat_John -p 8 --fusion-search --keep-fasta-order --bowtie 1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromoso mes chrM (Path of hg19 which should be in bowtie1) John_1.fastq John_2.fastq

Well, the problems may be sorted out if you:
1. -o tophat_PRO
2. remove spaces from file or dir names i.e. genome_sample.fastq (genome sample.fastq ?)
3. Where is your two .fastq files. pair end ?