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  • errors of tophat fusion

    Hi ,all

    I have used tophat-fusion to find the fusion mRNAs with six libs of paired end solexa fastq data, five of them worked well, and one have some errors, bellow is the error information:


    [Mon Jul 25 11:36:56 2011] Checking for Bowtie index files
    [Mon Jul 25 11:36:57 2011] Checking for reference FASTA file
    [Mon Jul 25 11:36:57 2011] Checking for Bowtie
    Bowtie version: 0.12.7.0
    [Mon Jul 25 11:36:57 2011] Checking for Samtools
    Samtools Version: 0.1.16
    [Mon Jul 25 11:36:58 2011] Checking reads
    min read length: 100bp, max read length: 100bp
    format: fastq
    quality scale: phred64 (reads generated with GA pipeline version >= 1.3)

    ...

    [Mon Jul 25 15:24:09 2011] Searching for junctions via segment mapping
    [Mon Jul 25 15:35:34 2011] Retrieving sequences for splices
    [Mon Jul 25 15:35:47 2011] Indexing splices
    [Mon Jul 25 15:37:04 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 15:49:21 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 16:02:00 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 16:15:30 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 16:28:41 2011] Joining segment hits
    [Mon Jul 25 16:36:34 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 16:51:05 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 17:06:34 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 17:21:41 2011] Mapping reads against segment_juncs with Bowtie
    [Mon Jul 25 17:36:42 2011] Joining segment hits
    [Mon Jul 25 17:45:24 2011] Reporting output tracks
    [FAILED]
    Error: Report generation failed with err =-6



    How I can fix this problem?

  • #2
    Hi,

    Have you checked that you do have the right to creat files in the current directory? It seems that tophat is stoped when it wants to output the result.

    Comment


    • #3
      I'm trying to run tophat-fusion with a data set I used with tophat before. However, prep-reads function is quitting and the prep-reads.log file indicates a read has a longer quality length than the read length. I'm surprised since I've used this data set with tophat before.

      The website states "This version does not support color-space reads although we have a plan to support it soon" but there are options to use color space data and this confused me as well. I did try with color space and am wondering if this is the reason.

      I also used a data set of left over reads from a bowtie alignment that were converted into fastq (base space) and got a similar error claiming length mismatch between the read and the quality.

      any ideas? not many tophat-fusion error topics are out there so my google search didn't really bring much either.

      Comment


      • #4
        [FAILED]
        Error: Report generation failed with err =-6

        I got the same error message, too. I checked logs/reports.log and it appears to be a memory allocation issue. I'm using 5GB RAM x 2 CPUs. Does anyone have any clue as to how much memory TopHat Fusion requires??

        ==
        tophat_reports v1.2.0 (2290M)
        ---------------------------------------
        Loading reference sequences...
        Loaded 178105 junctions
        Reporting final accepted alignments...terminate called after throwing an instance of 'std::bad_alloc'
        what(): std::bad_alloc

        Comment


        • #5
          Did you find a solution? I've the same error.

          Comment


          • #6
            Has anyone found a solution to the problem? I am running into a similar error expect for me it is a "Error: Report generation failed with err =1" I've checked that I am able to write to that directory. Full run log below.

            Thanks

            [Thu Nov 10 15:08:33 2011] Beginning TopHat run (v0.1.0 (Beta))
            -----------------------------------------------
            [Thu Nov 10 15:08:33 2011] Preparing output location /share/lustre/gascoyne/WTSS/HL_CELL_LINES/tophat_fusions/A05247/
            [Thu Nov 10 15:08:33 2011] Checking for Bowtie index files
            [Thu Nov 10 15:08:33 2011] Checking for reference FASTA file
            [Thu Nov 10 15:08:33 2011] Checking for Bowtie
            Bowtie version: 0.12.7.0
            [Thu Nov 10 15:08:33 2011] Checking for Samtools
            Samtools Version: 0.1.18
            [Thu Nov 10 15:08:54 2011] Checking reads
            min read length: 76bp, max read length: 76bp
            format: fastq
            quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
            [Thu Nov 10 16:31:14 2011] Mapping reads against hg19 with Bowtie
            [Thu Nov 10 21:43:02 2011] Joining segment hits
            [Thu Nov 10 22:15:31 2011] Mapping reads against hg19 with Bowtie (1/3)
            [Fri Nov 11 02:16:24 2011] Mapping reads against hg19 with Bowtie (2/3)
            [Fri Nov 11 05:44:24 2011] Mapping reads against hg19 with Bowtie (3/3)
            [Fri Nov 11 08:35:53 2011] Mapping reads against hg19 with Bowtie
            [Fri Nov 11 13:31:15 2011] Joining segment hits
            [Fri Nov 11 14:02:01 2011] Mapping reads against hg19 with Bowtie (1/3)
            [Fri Nov 11 18:21:14 2011] Mapping reads against hg19 with Bowtie (2/3)
            [Fri Nov 11 22:35:13 2011] Mapping reads against hg19 with Bowtie (3/3)
            [Sat Nov 12 01:36:36 2011] Searching for junctions via segment mapping
            [Sat Nov 12 05:05:37 2011] Retrieving sequences for splices
            [Sat Nov 12 05:11:51 2011] Indexing splices
            [Sat Nov 12 05:42:28 2011] Mapping reads against segment_juncs with Bowtie
            [Sat Nov 12 08:21:40 2011] Mapping reads against segment_juncs with Bowtie
            [Sat Nov 12 10:48:39 2011] Mapping reads against segment_juncs with Bowtie
            [Sat Nov 12 13:08:03 2011] Joining segment hits
            [Sat Nov 12 15:18:48 2011] Mapping reads against segment_juncs with Bowtie
            [Sat Nov 12 17:57:59 2011] Mapping reads against segment_juncs with Bowtie
            [Sat Nov 12 20:38:36 2011] Mapping reads against segment_juncs with Bowtie
            [Sat Nov 12 22:56:02 2011] Joining segment hits
            [Sun Nov 13 01:01:05 2011] Reporting output tracks
            [FAILED]
            Error: Report generation failed with err =1

            Comment


            • #7
              Though I like to refer to computers and algorithms as inanimate objects this one seems to have a mind of its own. We have found it to be completely chaotic in run success. Often some of these fails work if you just relaunch or fail again but at a different point. I've actually found that the more reads results in more fails and longer reads also result in more fails.

              The good news is when it actually works I'm pretty happy with the results.

              Comment


              • #8
                I got the same errors for some of my runs. It turned out to be running out of memory. For some reason, the last step of tophat-fusion to output the final results requires large memory. About half of my Hiseq runs went through with memory 72GB of memory.

                Comment


                • #9
                  Same here. Systematic crash at the last step of tophat-fusion (tophat-reports).
                  The sample comes from an HiSeq2000 and the fastq files are 21Go (compressed. ~140M reads)
                  The 4 files it uses are each between 23Go (*_kept_reads.fq) and 47Go (*_kept_reads.candidate_hits.sam).

                  reports.log indicates:

                  tophat_reports v1.2.0 (2290M)
                  ---------------------------------------
                  Loading reference sequences...
                  and that's it!

                  I am running it on a server with 512Go of RAM and 2.5 To of HD space. How can it be unsufficient??? What kind of configuration is necessary to run this script? Is it necessary to get a huge /tmp folder?

                  Comment


                  • #10
                    Hi guys,

                    This is a memory issue that the tophat_reports program uses too much memory for storing mate pair information that support fusions. A temporary solution is to use a smaller number of reads (e.g., 30M instead of 140M). We'll release a fixed version as a part of TopHat2 soon.

                    Thanks,
                    Daehwan Kim

                    Comment


                    • #11
                      Thanks a lot.
                      I am really looking forward for Tophat2 ;-)

                      Comment


                      • #12
                        I found a workaround to all these problems - instead of using all available threads I used n-1 (where n is number of available threads). To make it run smoothly, when RAM is restricted (eg 8 or 16Gb) I would suggest to make swap ~30Gb. In my case, 170million reads sample, on 4 threads (3) and 16gb takes about 13h. On 7 threads with only 8gb and approx same number of reads it takes about the same (13h).
                        Hope that workaround help someone : )

                        Comment


                        • #13
                          Hi,
                          I tried tophat fusion prebuild binaries to align my SOLID reads to oryza sativa.
                          It gave the following error.
                          /opt/tophatfusion-0.1.0-bin/tophat-fusion -p 4 -o fusion -G ~/03_Genomes/Oryza_sativa_Indica/osa.gtf -C -Q ~/03_Genomes/Oryza_sativa_Indica/bowtie_C/osa test.csfasta test_QV.qual

                          [Thu Mar 22 16:13:06 2012] Beginning TopHat run (v0.1.0 (Beta))
                          -----------------------------------------------
                          [Thu Mar 22 16:13:06 2012] Preparing output location fusion/
                          [Thu Mar 22 16:13:06 2012] Checking for Bowtie index files
                          [Thu Mar 22 16:13:06 2012] Checking for reference FASTA file
                          [Thu Mar 22 16:13:06 2012] Checking for Bowtie
                          Bowtie version: 0.12.7.0
                          [Thu Mar 22 16:13:06 2012] Checking for Samtools
                          Samtools Version: 0.1.16
                          [Thu Mar 22 16:13:06 2012] Checking reads
                          min read length: 50bp, max read length: 50bp
                          format: fasta
                          [Thu Mar 22 16:13:11 2012] Reading known junctions from GTF file
                          Traceback (most recent call last):
                          File "/opt/tophatfusion-0.1.0-bin/tophat-fusion", line 2610, in <module>
                          sys.exit(main())
                          File "/opt/tophatfusion-0.1.0-bin/tophat-fusion", line 2566, in main
                          user_supplied_deletions)
                          File "/opt/tophatfusion-0.1.0-bin/tophat-fusion", line 2251, in spliced_alignment
                          left_reads_map = maps[left_reads].unspliced_bwt
                          AttributeError: 'list' object has no attribute 'unspliced_bwt'

                          Please help
                          Anurag

                          Comment

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