Tophat Running Error

[Wei-HD]

Hi All,

I met a problem when running Tophat. I just copy tophat, bowtie and cufflinks Unix executable file into local/bin file (I use Mac 10.5). Then I put the prebuilt M. musculus, UCSC mm9 index in the data file, but when I run tophat, could not find bowtie index.

I must have made mistakes, can someone give me some hints.

Thanks in advance!
wei



053:~ wei$ cd /usr/local/bin
053:bin wei$ ls
bed_to_juncs closure_juncs juncs_db tophat_reports
bowtie contig_to_chr_coords library_stats tophat~
bowtie-build cuffcompare long_spanning_reads vpn_uninstall
bowtie-build-debug cufflinks mask_sam vpnclient
bowtie-debug extract_reads prep_reads wish8.5
bowtie-inspect fix_map_ordering segment_juncs
bowtie-inspect-debug gff_juncs tclsh8.5
cisco_cert_mgr ipseclog tophat
053:bin wei$ cd
053:~ wei$ cd desktop/seq/data
053:data wei$ ls -l
total 31530120
-rwxr-xr-x 1 wei staff 1342932855 Jan 8 17:39 42YAVAAX_051109BIC_BasOrth_s_6_sequence.rar
-rwxr-xr-x 1 wei staff 1358512988 Jan 8 17:28 42YAVAAX_051109Ctrl_BasOrth_s_5_sequence.rar
-rwxrwxrwx 1 wei staff 3937882178 Dec 18 14:19 Bic.txt
-rw-r--r-- 1 wei staff 1385985820 Jan 26 19:34 Bic.txt.zip
-rwxrwxrwx 1 wei staff 3966044812 Dec 18 14:19 control.txt
-rw-r--r-- 1 wei staff 1402339205 Jan 26 19:33 control.txt.zip
-rw-r--r-- 1 wei staff 1902 Nov 13 20:56 make_mm9.sh
-rw-r--r--@ 1 wei staff 735209933 Nov 13 16:57 mm9.1.ebwt
-rw-r--r--@ 1 wei staff 319815728 Nov 13 16:57 mm9.2.ebwt
-rw-r--r--@ 1 wei staff 5858 Nov 13 14:35 mm9.3.ebwt
-rw-r--r--@ 1 wei staff 639631445 Nov 13 14:35 mm9.4.ebwt
-rw-r--r--@ 1 wei staff 735209933 Nov 13 19:20 mm9.rev.1.ebwt
-rw-r--r--@ 1 wei staff 319815728 Nov 13 19:20 mm9.rev.2.ebwt
drwxr-xr-x 4 wei staff 136 Jan 27 11:37 tophat_out
053:data wei$ tophat ../../seq/data bic.txt

[Wed Jan 27 11:49:28 2010] Beginning TopHat run (v1.0.11)
-----------------------------------------------
[Wed Jan 27 11:49:28 2010] Preparing output location ./tophat_out/
[Wed Jan 27 11:49:28 2010] Checking for Bowtie index files
Error: Could not find Bowtie index files ../../seq/data.*
053:data wei$

[dawe]

053:data wei$ tophat ../../seq/data bic.txt

[Wed Jan 27 11:49:28 2010] Beginning TopHat run (v1.0.11)
-----------------------------------------------
[Wed Jan 27 11:49:28 2010] Preparing output location ./tophat_out/
[Wed Jan 27 11:49:28 2010] Checking for Bowtie index files
Error: Could not find Bowtie index files ../../seq/data.*
053:data wei$

You should provide the basename of the index not only the directory:

$ tophat ../../seq/data/INDEXBASENAME bic.txt

where INDEXBASENAME is the filename without the ".ebwt" extension, I guess it is just "mm9" for you.
d

[Wei-HD]

Thanks Dawe! I did it like you told me!

It seems that I have to rebuilt the bowtie index since there is no mm9.fa file. Should I?

[dawe]

Thanks Dawe! I did it like you told me!

It seems that I have to rebuilt the bowtie index since there is no mm9.fa file. Should I?

No need to do that... the fasta file will be extracted from ebwt files on the fly.

[Wei-HD]

Thanks dawe!

And the result is:

Last login: Wed Jan 27 12:30:54 on ttys000
053:~ wei$ cd desktop/seq/data
053:data wei$ tophat ../../seq/data/mm9 bic.txt

[Wed Jan 27 12:51:21 2010] Beginning TopHat run (v1.0.11)
-----------------------------------------------
[Wed Jan 27 12:51:21 2010] Preparing output location ./tophat_out/
[Wed Jan 27 12:51:21 2010] Checking for Bowtie index files
[Wed Jan 27 12:51:21 2010] Checking for reference FASTA file
Warning: Could not find FASTA file ../../seq/data/mm9.fa
[Wed Jan 27 12:51:21 2010] Reconstituting reference FASTA file from Bowtie index

Is the fasta file extracting from ebwt?

[ECHo]

Well, that's why we must set PATH including bowtie-build
bowtie-build can build *.ebwt through *.fa/*.fna

I think these steps can do the reverse...
So, it shows the same messages on fa/fna/ebwt
And Tophat could do the transform automatically...

[Wei-HD]

Hi All,

Thanks for your help! I have solved this problem. Just like ECHo mentioned, bowtie will build *.fa to *.ebwt.

[thinkRNA]

Wei-HD,
I am running tophat and right now staring at this message on my 64bit 12 GB RAM for a while:
"Reconstituting reference FASTA file from Bowtie index".

How long does it typically take and can we bypass this?

Thanks
Priyam

[Wei-HD]

No, you could not skip this step. For my mac, just 64 bit 4 GB ,it takes less than 30 min, I guess it would be on a fly in your computer with 12 GB.

By the way, you change the extra memory card on your mac? I don't know mac can be 12 Gb right now.

[nilshomer]

No, you could not skip this step. For my mac, just 64 bit 4 GB ,it takes less than 30 min, I guess it would be on a fly in your computer with 12 GB.

By the way, you change the extra memory card on your mac? I don't know mac can be 12 Gb right now.

Mac Pro (Desktop).

[Xi Wang]

Wei-HD,
I am running tophat and right now staring at this message on my 64bit 12 GB RAM for a while:
"Reconstituting reference FASTA file from Bowtie index".

How long does it typically take and can we bypass this?

Thanks
Priyam

You can put the genome fasta sequence, say mm9.fa, or symbol link into the Bowtie index directory to skip the reconstruction. Tophat will check this file, if it exists, no need to reconstruct.

[Wei-HD]

Thanks Xi! That is an easy way!

[thinkRNA]

Does anyone know why I get this warning when running tophat

./tophat ./tophat_out/mm9 ../../../bowtie/Mikkelson/SRX001940/SRR006806.fastq
[Thu Feb 11 12:51:47 2010] Beginning TopHat run (v1.0.13)
-----------------------------------------------
[Thu Feb 11 12:51:47 2010] Preparing output location ./tophat_out/
[Thu Feb 11 12:51:47 2010] Checking for Bowtie index files
[Thu Feb 11 12:51:47 2010] Checking for reference FASTA file
[Thu Feb 11 12:51:47 2010] Checking for Bowtie
Bowtie version: 0.11.3.0
[Thu Feb 11 12:51:47 2010] Checking reads
seed length: 27bp
format: fastq
quality scale: phred33 (default)
[Thu Feb 11 12:54:38 2010] Mapping reads against mm9 with Bowtie
^T[Thu Feb 11 14:12:47 2010] Joining segment hits
[Thu Feb 11 14:15:10 2010] Searching for junctions via segment mapping
Warning: junction database is empty!
[Thu Feb 11 14:16:40 2010] Joining segment hits
[Thu Feb 11 14:19:05 2010] Reporting output tracks
-----------------------------------------------