Question: Testing Tophat installation
1
gravatar for erikfas
4.8 years ago by
erikfas20
Sweden
erikfas20 wrote:

I'm just starting out with RNA-seq, and I'm trying to get the installation of Tophat to work. I'm following the "Getting started" instructions on the site, and am currently testing the installation. When I run

tophat -r 20 test_ref reads_1.fq reads_2.fq

... this is what I get:

[2014-06-20 13:01:22] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-06-20 13:01:22] Checking for Bowtie
          Bowtie version:     2.2.3.0
[2014-06-20 13:01:22] Checking for Samtools
        Samtools version:     0.1.19.0
[2014-06-20 13:01:22] Checking for Bowtie index files (genome)..
    Found both Bowtie1 and Bowtie2 indexes.
[2014-06-20 13:01:22] Checking for reference FASTA file
[2014-06-20 13:01:22] Generating SAM header for test_ref
[2014-06-20 13:01:22] Preparing reads
     left reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
    right reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
[2014-06-20 13:01:22] Mapping left_kept_reads to genome test_ref with Bowtie2
    [FAILED]
Error running:
/Users/erikfasterius/bin/bam2fastx --all ./tophat_out/tmp/left_kept_reads.bam|/Users/erikfasterius/bin/bowtie2 -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --sam-no-hd -x test_ref -|/Users/erikfasterius/bin/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --sam-header ./tophat_out/tmp/test_ref_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.bam

... and I don't really understand the error. It doesn't say what type of error it is, just says [FAILED] and give the long string of whatever caused the error, which I don't understand.

Any help would be greatly appreciated!
Erik

tophat • 3.6k views
ADD COMMENTlink modified 4.8 years ago by doctorbrassica10 • written 4.8 years ago by erikfas20

You'll have to run /Users/erikfasterius/bin/bam2fastx --all ./tophat_out/tmp/left_kept_reads.bam|/Users/erikfasterius/bin/bowtie2 -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --sam-no-hd -x test_ref -|/Users/erikfasterius/bin/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --sam-header ./tophat_out/tmp/test_ref_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.bam from the directory yourself to see what the actual error was.

ADD REPLYlink modified 4.8 years ago • written 4.8 years ago by Devon Ryan89k

Ok, I did that, and this is what it says:

100 reads; of these:
  100 (100.00%) were unpaired; of these:
    59 (59.00%) aligned 0 times
    41 (41.00%) aligned exactly 1 time
    0 (0.00%) aligned >1 times
41.00% overall alignment rate
[sam_read1] missing header? Abort!

ADD REPLYlink written 4.8 years ago by erikfas20

Interesting, given that the command tells bowtie2 to not provide a header, it's surprising that fix_map_ordering would be expecting one. What happens if you remove the --sam-no-hd portion from that line?

ADD REPLYlink written 4.8 years ago by Devon Ryan89k

This:

/Users/erikfasterius/bin/bowtie2-align-s --wrapper basic-0 -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --sam-header ./tophat_out/tmp/test_ref_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.ba
bowtie2-align-s: unrecognized option `--bowtie2-min-score'

Followed by what seems to be the bowtie help (i.e. lots of rows of info on parameters), and then this at the end:

Error: Encountered internal Bowtie 2 exception (#1)
Command: /Users/erikfasterius/bin/bowtie2-align-s --wrapper basic-0 -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --sam-header ./tophat_out/tmp/test_ref_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.ba

ADD REPLYlink written 4.8 years ago by erikfas20

I'm guessing that you missed a pipe, try:

/Users/erikfasterius/bin/bam2fastx --all ./tophat_out/tmp/left_kept_reads.bam|/Users/erikfasterius/bin/bowtie2 -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 -x test_ref -|/Users/erikfasterius/bin/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --sam-header ./tophat_out/tmp/test_ref_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.bam

ADD REPLYlink written 4.8 years ago by Devon Ryan89k

That gives the exact same as above (i.e. "100 reads, of these:" etc). (I'm very much appreciating the help, btw, even tho we/you haven't found the solution yet =P)
 

ADD REPLYlink written 4.8 years ago by erikfas20

At this point I haven't a clue. If you post the reads you're using and let me know the genome they're from then I'll have a look. Barring that, you might try posting this on the google group.

ADD REPLYlink written 4.8 years ago by Devon Ryan89k

The reads are reads_1.fq and reads_2.fq from the test_data that can be downloaded from the Tophat tutorial (http://ccb.jhu.edu/software/tophat/tutorial.shtml), which also contains the reference (test_ref.*.bt2).

ADD REPLYlink written 4.8 years ago by erikfas20

For what it's worth, I can also reproduce this. Presumably this is specific to the test dataset, since otherwise I imagine someone would have reported this already. Either way, do ask on the google group that I linked to, since this really does seem to be a bug.

As an aside, I can recommend using STAR instead. It's vastly faster and produces equivalent to superior results.

ADD REPLYlink written 4.8 years ago by Devon Ryan89k
1
gravatar for doctorbrassica
4.8 years ago by
United Kingdom
doctorbrassica10 wrote:

I ran into this problem too and found a suggested fix here.  It is a on-line amendment to the tophat script that worked for me and others: https://groups.google.com/forum/#!topic/tuxedo-tools-users/vG5Rn0IgxoA

 

ADD COMMENTlink written 4.8 years ago by doctorbrassica10

Ah, interesting. Exactly how do I change the Tophat code? Which file, where is it, etc? Do I have to somehow change the source code and compile myself, or is it some other way to do it?

ADD REPLYlink written 4.8 years ago by erikfas20

You can edit the tophat file itself in any text editor (it's just a python script). At line 1415, change this:

        if read_params.color:
            bowtie_header_cmd.append('-C')

to this:

        if read_params.color:
            bowtie_header_cmd.append('-C')
        bowtie_header_cmd += ["-x"]

Note that indentation is important.

ADD REPLYlink written 4.8 years ago by Devon Ryan89k

Do you mean the Tophat executable or some other "the" Tophat file? Because I was under the impression that you can't edit executables.

ADD REPLYlink written 4.8 years ago by erikfas20

The executable is just a text file in this case. You're correct that you can't edit binary files in a text editor (well you can, but you'll just break the file).

ADD REPLYlink written 4.8 years ago by Devon Ryan89k

It says "Unix Executable File" for me, so not a text file. Am I looking at the wrong file?

ADD REPLYlink written 4.8 years ago by erikfas20

Just head the file. If it doesn't look like text, then you're looking at the wrong file.

ADD REPLYlink written 4.8 years ago by Devon Ryan89k

Ah, I just couldn't open it because of security options in OSX. Fixed that and added the line, it works now, even with 2.2.3, so thanks for the help!

ADD REPLYlink written 4.8 years ago by erikfas20

In tophat 2.0.12  the file tophay.py has this

if not params.bowtie2:
            bowtie_header_cmd += ["--sam"]

        if read_params.color:
            bowtie_header_cmd.append('-C')

        if params.bowtie2:
            bowtie_header_cmd += ["-x"]

Is this equivalent to what you are saying (there is no condition in your case). Does bowtie_header_cmd have to be "-x" all the time?

ADD REPLYlink modified 4.8 years ago • written 4.8 years ago by Bharat Iyengar260
0
gravatar for Hyun Jin
4.8 years ago by
Hyun Jin20
United States
Hyun Jin20 wrote:

Hi guys, 

I'm also trying to set up Tophat in my MAC, and I encountered the exactly same problem. 

I basically followed the instructions and ran "test file" provided in TopHat getting start page (http://ccb.jhu.edu/software/tophat/tutorial.shtml). Error message as follow

-------------

[2014-06-20 22:27:46] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-06-20 22:27:46] Checking for Bowtie
          Bowtie version:     2.2.3.0
[2014-06-20 22:27:46] Checking for Samtools
        Samtools version:     0.1.19.0
[2014-06-20 22:27:46] Checking for Bowtie index files (genome)..
    Found both Bowtie1 and Bowtie2 indexes.
[2014-06-20 22:27:46] Checking for reference FASTA file
[2014-06-20 22:27:46] Generating SAM header for test_ref
[2014-06-20 22:27:46] Preparing reads
     left reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
    right reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
[2014-06-20 22:27:47] Mapping left_kept_reads to genome test_ref with Bowtie2 
    [FAILED]
Error running:
/usr/local/bin/bam2fastx --all ./tophat_out/tmp/left_kept_reads.bam|/usr/local/Cellar/bowtie2-2.2.3/bowtie2 -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --sam-no-hd -x test_ref -|/usr/local/bin/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --sam-header ./tophat_out/tmp/test_ref_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.bam

----------

I noticed that version of TopHat(v2.0.11) Bowtie(2.2.3) and Samtools(0.1.19.0) are identical, so I just wonder this is some sort of bug. 

Please post if you find any solution, I appreciate it

hyj

 

 

ADD COMMENTlink modified 4.8 years ago • written 4.8 years ago by Hyun Jin20
2

Hi Guys, 

Downgrade of bowtie2 (2.2.3 --> 2.2.2)solved the problem!

--------

[2014-06-20 23:19:32] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-06-20 23:19:32] Checking for Bowtie
          Bowtie version:     2.2.2.0
[2014-06-20 23:19:32] Checking for Samtools
        Samtools version:     0.1.19.0
[2014-06-20 23:19:32] Checking for Bowtie index files (genome)..
    Found both Bowtie1 and Bowtie2 indexes.
[2014-06-20 23:19:32] Checking for reference FASTA file
[2014-06-20 23:19:32] Generating SAM header for test_ref
[2014-06-20 23:19:32] Preparing reads
     left reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
    right reads: min. length=75, max. length=75, 100 kept reads (0 discarded)
[2014-06-20 23:19:32] Mapping left_kept_reads to genome test_ref with Bowtie2 
[2014-06-20 23:19:33] Mapping left_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-06-20 23:19:33] Mapping left_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-06-20 23:19:33] Mapping left_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-06-20 23:19:33] Mapping right_kept_reads to genome test_ref with Bowtie2 
[2014-06-20 23:19:33] Mapping right_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-06-20 23:19:33] Mapping right_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-06-20 23:19:33] Mapping right_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-06-20 23:19:33] Searching for junctions via segment mapping
[2014-06-20 23:19:34] Retrieving sequences for splices
[2014-06-20 23:19:34] Indexing splices
Building a SMALL index
[2014-06-20 23:19:34] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2014-06-20 23:19:35] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2014-06-20 23:19:35] Mapping left_kept_reads_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2014-06-20 23:19:35] Joining segment hits
[2014-06-20 23:19:35] Mapping right_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2014-06-20 23:19:35] Mapping right_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2014-06-20 23:19:35] Mapping right_kept_reads_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2014-06-20 23:19:35] Joining segment hits
[2014-06-20 23:19:35] Reporting output tracks
-----------------------------------------------
[2014-06-20 23:19:35] A summary of the alignment counts can be found in ./tophat_out/align_summary.txt
[2014-06-20 23:19:35] Run complete: 00:00:03 elapsed

 

 

ADD REPLYlink written 4.8 years ago by Hyun Jin20

Thanks, downgrading did it!

ADD REPLYlink written 4.8 years ago by erikfas20

Hi.. I am running a slightly different command.
tophat -r 350 --mate-std-dev 150 --no-discordant --no-mixed --no-coverage-search --transcriptome-index ../transcriptome_merged/merged2 -T -M -p 20 -o blood ../bwtindex/Dre_nuclear_2 Blood_1.fq Blood_2.fq

I am getting the same error as you guys. This did not happen when I allowed genomic mappings (i.e not used the option -T), although it was a different file. I had sporadic problems with --no-discordant before but that error comes very late in the process. I used old version of bowtie2, 2.2.3 and 2.2.2; nothing works.

ADD REPLYlink written 4.8 years ago by Bharat Iyengar260
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