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  • tophat problem: no accepted_hits.bam generated

    I am using tophat to map the SOLiD single-end 50mer reads to
    S.cerevisae genome. The commands I used are:

    $ bowtie-build -C reads/sgd_genome.fa reads/sgd_genome
    $ qsub -V -o WT.1_tophat.O -e WT.1_tophat.E -N WT.1_tophat -l
    nodes=1pn1,walltime=10:00:00 <<< " tophat --color --output-dir
    cufflinks/WT.1 --GTF reads/gene_features.gff --quals reads/genome
    reads/WT.1.csfasta reads/WT.1.qual"

    The job ran without any error but there is no accepted_hits.bam
    generated and all the BED files are empty.
    $ ls -l WT.1
    total 16K
    -rw-rw-r-- 1 zxu7 zxu7 0 07/19 15:15:24 deletions.bed
    -rw-rw-r-- 1 zxu7 zxu7 0 07/19 15:15:24 insertions.bed
    -rw-rw-r-- 1 zxu7 zxu7 0 07/19 15:15:24 junctions.bed
    -rw-rw-r-- 1 zxu7 zxu7 70 07/19 12:30:42 left_kept_reads.info
    drwxrwxr-x 2 zxu7 zxu7 8.0K 07/18 14:06:23 logs

    $ cat WT.1_tophat.E
    [Mon Jul 18 10:36:07 2011] Beginning TopHat run (v1.3.1)
    -----------------------------------------------
    [Mon Jul 18 10:36:07 2011] Preparing output location cufflinks/WT.1/
    [Mon Jul 18 10:36:07 2011] Checking for Bowtie index files
    [Mon Jul 18 10:36:07 2011] Checking for reference FASTA file
    Warning: Could not find FASTA file cufflinks/genome.fa
    [Mon Jul 18 10:36:07 2011] Reconstituting reference FASTA file from Bowtie index
    Executing: /home/zxu7/Softwares/bowtie-0.12.7/bowtie-inspect
    cufflinks/genome > cufflinks/WT.1/tmp/genome.fa
    [Mon Jul 18 10:36:08 2011] Checking for Bowtie
    Bowtie version: 0.12.7.0
    [Mon Jul 18 10:36:08 2011] Checking for Samtools
    Samtools Version: 0.1.14
    [Mon Jul 18 10:36:08 2011] Generating SAM header for cufflinks/genome
    [Mon Jul 18 10:36:11 2011] Preparing reads
    format: fasta
    [Mon Jul 18 10:36:11 2011] Reading known junctions from GTF file
    Left reads: min. length=50, count=32186882
    [Mon Jul 18 10:46:11 2011] Mapping left_kept_reads against genome with Bowtie
    [Mon Jul 18 11:22:32 2011] Processing bowtie hits
    [Mon Jul 18 11:56:23 2011] Mapping left_kept_reads_seg1 against genome
    with Bowtie (1/2)
    [Mon Jul 18 12:26:07 2011] Mapping left_kept_reads_seg2 against genome
    with Bowtie (2/2)
    [Mon Jul 18 13:01:47 2011] Searching for junctions via segment mapping
    [Mon Jul 18 13:13:27 2011] Retrieving sequences for splices
    [Mon Jul 18 13:13:28 2011] Indexing splices
    [Mon Jul 18 13:13:29 2011] Mapping left_kept_reads_seg1 against
    segment_juncs with Bowtie (1/2)
    [Mon Jul 18 13:38:03 2011] Mapping left_kept_reads_seg2 against
    segment_juncs with Bowtie (2/2)
    [Mon Jul 18 13:48:21 2011] Joining segment hits
    [Mon Jul 18 14:06:53 2011] Reporting output tracks
    -----------------------------------------------
    Run complete [03:32:48 elapsed]

    By the way, the gff files is in GFF version 3 format. Does anyone know
    what's wrong with my tophat command?

  • #2
    Based on your bowtie-build command, shouldn't the reference genome for your tophat run be "reads/sgd_genome" not "reads/genome"?

    Comment


    • #3
      Originally posted by thurisaz View Post
      Based on your bowtie-build command, shouldn't the reference genome for your tophat run be "reads/sgd_genome" not "reads/genome"?
      Sorry, it's a typo. I was indeed using "reads/sgd_genome"

      Comment


      • #4
        I've been having the same problem- I'm aligning a published data set of single reads to the drosophila genome, using the iGenome bowtie index and gtf file. Tophat runs with no errors in the log files, but the bed files are empty and accepted_hits.bam is absent. I've tried using tophat 1.3.2 and 1.4.0 and have gotten similar results.

        It's been awhile since the original post, but I was curious to hear if/how you'd resolved the problem.

        Thanks!


        I've been running tophat from the following shell script:
        tophat -p 8 -G genes2.gtf -o C1_R1_thout2 genome GSM794483_C1_R1_1.fq

        Here's the error log file:

        [Sat Apr 21 17:11:25 2012] Beginning TopHat run (v1.4.0)
        -----------------------------------------------
        [Sat Apr 21 17:11:25 2012] Preparing output location C1_R1_thout2/
        [Sat Apr 21 17:11:25 2012] Checking for Bowtie index files
        [Sat Apr 21 17:11:25 2012] Checking for reference FASTA file
        [Sat Apr 21 17:11:25 2012] Checking for Bowtie
        Bowtie version: 0.12.7.0
        [Sat Apr 21 17:11:25 2012] Checking for Samtools
        Samtools Version: 0.1.17
        [Sat Apr 21 17:11:25 2012] Generating SAM header for genome
        format: fastq
        quality scale: phred33 (default)
        [Sat Apr 21 17:11:26 2012] Reading known junctions from GTF file
        [Sat Apr 21 17:11:28 2012] Preparing reads
        left reads: min. length=75, count=11607353
        [Sat Apr 21 17:12:41 2012] Creating transcriptome data files..
        [Sat Apr 21 17:12:45 2012] Building Bowtie index from genes2.fa
        [Sat Apr 21 17:17:56 2012] Mapping left_kept_reads against transcriptome genes2 with Bowtie
        [Sat Apr 21 17:20:46 2012] Converting left_kept_reads.m2g to genomic coordinates (map2gtf)
        [Sat Apr 21 17:23:37 2012] Reporting output tracks

        Comment


        • #5
          Hi jenright, sorry I still don't find a solution. I just changed to other softwares. I also emailed to the email the developers posted online but unfortunately didn't get any reply.

          Comment


          • #6
            Hi there
            I got the same problem (no bam file generated by tophat). any idea ?
            Results
            [2013-08-01 14:27:41] Reporting output tracks
            -----------------------------------------------
            [2013-08-01 14:54:53] A summary of the alignment counts can be found in 1_sample1/align_summary.txt
            Alilgn_summary.txt file cotains:
            Left reads:
            Input: 42586551
            Mapped: 38891429 (91.3% of input)
            of these: 5953741 (15.3%) have multiple alignments (278645 have >20)
            Right reads:
            Input: 42586551
            Mapped: 38959655 (91.5% of input)
            of these: 6012542 (15.4%) have multiple alignments (283163 have >20)
            91.4% overall read alignment rate.

            Aligned pairs: 36632986
            of these: 4547311 (12.4%) have multiple alignments
            and: 251712 ( 0.7%) are discordant alignments
            85.4% concordant pair alignment rate.

            Comment


            • #7
              Hello,

              The main problem I was having seemed to be related to not allocating enough memory for the job. I run tophat through a cluster, and now request either 8GB or 16GB of memory (depending on the size of the reads files).

              Hope this helps, and good luck!

              Comment


              • #8
                Hi
                My user id has 30GB mem on server, but I still not gettin .bam files. There is some problem with system configuration.... I guess... can you guess...something ?

                Comment


                • #9
                  I ran into similar problem: tophat generated all files except accepted_hits.bam.
                  I made some more disk space available and then it ran fine.

                  Comment


                  • #10
                    My user location on Linux Server has the problem. I have full/max control permission but tophat does not produce .bam file. When I changed output location (shared by all user+root), it can generate the .bam
                    Any idea what to change to file permission in nodes?
                    --keep tmp doesnt work ?


                    Originally posted by blanco View Post
                    I ran into similar problem: tophat generated all files except accepted_hits.bam.
                    I made some more disk space available and then it ran fine.

                    Comment

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