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  • tophat 1.3.0 bug??

    any ideas?


    Code:
    matthew@macmanes:~/tophat-1.3.0$ /home/matthew/tophat-1.3.0/tophat -o /media/hd/annotation/tophat/solitary -r 100 -p 16 /media/hd/annotation/tophat/bowtie.index/tuco.mrna.annotate.2June11 /media/hd/tuco/mrna/peruanus/clipped/cp.final.clip_1.fq /media/hd/tuco/mrna/peruanus/clipped/cp.final.clip_2.fq
    
    [Thu Jun  2 17:47:45 2011] Beginning TopHat run (v1.3.0)
    -----------------------------------------------
    [Thu Jun  2 17:47:45 2011] Preparing output location /media/hd/annotation/tophat/solitary/
    [Thu Jun  2 17:47:45 2011] Checking for Bowtie index files
    [Thu Jun  2 17:47:45 2011] Checking for reference FASTA file
    [Thu Jun  2 17:47:45 2011] Checking for Bowtie
    	Bowtie version:			 0.12.7.0
    [Thu Jun  2 17:47:45 2011] Checking for Samtools
    	Samtools Version: 0.1.16
    [Thu Jun  2 17:47:45 2011] Generating SAM header for /media/hd/annotation/tophat/bowtie.index/tuco.mrna.annotate.2June11
    [Thu Jun  2 17:47:49 2011] Preparing reads
    	format:		 fastq
    	quality scale:	 phred33 (default)
    Traceback (most recent call last):
     File "/home/matthew/tophat-1.3.0/tophat", line 2584, in <module>
       sys.exit(main())
     File "/home/matthew/tophat-1.3.0/tophat", line 2511, in main
       "left_kept_reads", "Left ")
     File "/home/matthew/tophat-1.3.0/tophat", line 1362, in prep_reads
       shell_cmd = ' '.join(filter_cmd)
    TypeError: sequence item 50: expected string, NoneType found

  • #2
    get the same error
    Originally posted by peromhc View Post
    any ideas?


    Code:
    matthew@macmanes:~/tophat-1.3.0$ /home/matthew/tophat-1.3.0/tophat -o /media/hd/annotation/tophat/solitary -r 100 -p 16 /media/hd/annotation/tophat/bowtie.index/tuco.mrna.annotate.2June11 /media/hd/tuco/mrna/peruanus/clipped/cp.final.clip_1.fq /media/hd/tuco/mrna/peruanus/clipped/cp.final.clip_2.fq
    
    [Thu Jun  2 17:47:45 2011] Beginning TopHat run (v1.3.0)
    -----------------------------------------------
    [Thu Jun  2 17:47:45 2011] Preparing output location /media/hd/annotation/tophat/solitary/
    [Thu Jun  2 17:47:45 2011] Checking for Bowtie index files
    [Thu Jun  2 17:47:45 2011] Checking for reference FASTA file
    [Thu Jun  2 17:47:45 2011] Checking for Bowtie
    	Bowtie version:			 0.12.7.0
    [Thu Jun  2 17:47:45 2011] Checking for Samtools
    	Samtools Version: 0.1.16
    [Thu Jun  2 17:47:45 2011] Generating SAM header for /media/hd/annotation/tophat/bowtie.index/tuco.mrna.annotate.2June11
    [Thu Jun  2 17:47:49 2011] Preparing reads
    	format:		 fastq
    	quality scale:	 phred33 (default)
    Traceback (most recent call last):
     File "/home/matthew/tophat-1.3.0/tophat", line 2584, in <module>
       sys.exit(main())
     File "/home/matthew/tophat-1.3.0/tophat", line 2511, in main
       "left_kept_reads", "Left ")
     File "/home/matthew/tophat-1.3.0/tophat", line 1362, in prep_reads
       shell_cmd = ' '.join(filter_cmd)
    TypeError: sequence item 50: expected string, NoneType found

    Comment


    • #3
      Yep, same for me.

      Comment


      • #4
        I have another issue about the bowtie indexes :

        Code:
        [Fri Jun  3 14:29:02 2011] Beginning TopHat run (v1.3.0)
        -----------------------------------------------
        [Fri Jun  3 14:29:02 2011] Preparing output location /home/darstr/tmp/rnaSeqTH1.3/P/
        [Fri Jun  3 14:29:02 2011] Checking for Bowtie index files
        Error: Could not find Bowtie index files hg18.*
        darstr@clark-lab:~/tmp$ echo $BOWTIE_INDEXES
        /usr/local/share/indexes/hg18
        darstr@clark-lab:~/tmp$ ls $BOWTIE_INDEXES
        hg18.1.ebwt  hg18.2.ebwt  hg18.3.ebwt  hg18.4.ebwt  hg18.rev.1.ebwt  hg18.rev.2.ebwt
        But the user manual clearly says "... then looks in the directory specified in the BOWTIE_INDEXES environment variable."

        Also, does anyone send questions or bug reports to the official [email protected] address ? It doesn't seem to be monitored.

        Comment


        • #5
          new bug
          tophat --no-novel-juncs --phred64-quals -G ensembl_refGene_ucscGene.gtf -o output genome_index/bowtie/mm9/mm9 s_3_sequence.txt
          Traceback (most recent call last):
          File "/home/liuxq/mybin/tools/tophat", line 2584, in <module>
          sys.exit(main())
          File "/home/liuxq/mybin/tools/tophat", line 2543, in main
          user_supplied_deletions)
          File "/home/liuxq/mybin/tools/tophat", line 2376, in spliced_alignment
          os.remove(unspl_bwtfile)
          UnboundLocalError: local variable 'unspl_bwtfile' referenced before assignment

          Comment


          • #6
            what's your tophat command? use "hg18" for the <bowtie_index> parameter
            Originally posted by Dario1984 View Post
            I have another issue about the bowtie indexes :

            Code:
            [Fri Jun  3 14:29:02 2011] Beginning TopHat run (v1.3.0)
            -----------------------------------------------
            [Fri Jun  3 14:29:02 2011] Preparing output location /home/darstr/tmp/rnaSeqTH1.3/P/
            [Fri Jun  3 14:29:02 2011] Checking for Bowtie index files
            Error: Could not find Bowtie index files hg18.*
            darstr@clark-lab:~/tmp$ echo $BOWTIE_INDEXES
            /usr/local/share/indexes/hg18
            darstr@clark-lab:~/tmp$ ls $BOWTIE_INDEXES
            hg18.1.ebwt  hg18.2.ebwt  hg18.3.ebwt  hg18.4.ebwt  hg18.rev.1.ebwt  hg18.rev.2.ebwt
            But the user manual clearly says "... then looks in the directory specified in the BOWTIE_INDEXES environment variable."

            Also, does anyone send questions or bug reports to the official [email protected] address ? It doesn't seem to be monitored.

            Comment


            • #7
              Originally posted by peromhc View Post
              any ideas?


              Code:
              matthew@macmanes:~/tophat-1.3.0$ /home/matthew/tophat-1.3.0/tophat -o /media/hd/annotation/tophat/solitary -r 100 -p 16 /media/hd/annotation/tophat/bowtie.index/tuco.mrna.annotate.2June11 /media/hd/tuco/mrna/peruanus/clipped/cp.final.clip_1.fq /media/hd/tuco/mrna/peruanus/clipped/cp.final.clip_2.fq
              
              [Thu Jun  2 17:47:45 2011] Beginning TopHat run (v1.3.0)
              -----------------------------------------------
              [Thu Jun  2 17:47:45 2011] Preparing output location /media/hd/annotation/tophat/solitary/
              [Thu Jun  2 17:47:45 2011] Checking for Bowtie index files
              [Thu Jun  2 17:47:45 2011] Checking for reference FASTA file
              [Thu Jun  2 17:47:45 2011] Checking for Bowtie
              	Bowtie version:			 0.12.7.0
              [Thu Jun  2 17:47:45 2011] Checking for Samtools
              	Samtools Version: 0.1.16
              [Thu Jun  2 17:47:45 2011] Generating SAM header for /media/hd/annotation/tophat/bowtie.index/tuco.mrna.annotate.2June11
              [Thu Jun  2 17:47:49 2011] Preparing reads
              	format:		 fastq
              	quality scale:	 phred33 (default)
              Traceback (most recent call last):
               File "/home/matthew/tophat-1.3.0/tophat", line 2584, in <module>
                 sys.exit(main())
               File "/home/matthew/tophat-1.3.0/tophat", line 2511, in main
                 "left_kept_reads", "Left ")
               File "/home/matthew/tophat-1.3.0/tophat", line 1362, in prep_reads
                 shell_cmd = ' '.join(filter_cmd)
              TypeError: sequence item 50: expected string, NoneType found

              This should now be fixed. We pushed an update to the 1.3.0 tarballs late last night.

              Comment


              • #8
                Thanks Cole for the quick fix. Version 1.3.0 is running successfully now.

                Originally posted by Cole Trapnell View Post
                This should now be fixed. We pushed an update to the 1.3.0 tarballs late last night.

                Comment


                • #9
                  Originally posted by liuxq View Post
                  what's your tophat command? use "hg18" for the <bowtie_index> parameter
                  Yeah, I am using that. Also, it works if I cd into the directory where the indexes are. It just doesn't work from outside the directory, when the environment variable set to the path of the indexes, as the TopHat documentation says to do.

                  Comment


                  • #10
                    I also get the same error message in post#5. an update on the binaries would be much appreciated.

                    Comment


                    • #11
                      Originally posted by berath View Post
                      I also get the same error message in post#5. an update on the binaries would be much appreciated.
                      I also got this error message. My parameter is
                      -p 8 -g 1 -G Mus_musculus.NCBIM37.62.gtf.new --no-novel-juncs --no-novel-indels --solexa1.3-qual

                      Comment


                      • #12
                        I have a different error message. This was using tophat 1.3.0 on paired-end solid reads, which had been successfully mapped using tophat 1.2.0. The error message reads:

                        Error: qual length (57) differs from seq length (51) for fastq record 83_1325_28_F3!

                        Comment


                        • #13
                          bug fix

                          Originally posted by liuxq View Post
                          new bug
                          tophat --no-novel-juncs --phred64-quals -G ensembl_refGene_ucscGene.gtf -o output genome_index/bowtie/mm9/mm9 s_3_sequence.txt

                          Here is a fix for
                          "UnboundLocalError: local variable 'unspl_samfile' referenced before assignment"
                          error.

                          Edit tophat and comment out ( usually it is in /usr/bin/tophat )
                          #os.remove(unspl_bwtfile) #os.remove(unspl_samfile)
                          my_dummy = 0 # to keep the if statment intact

                          The real fix should come from Cole!

                          Ramu

                          Comment


                          • #14
                            Originally posted by ramu View Post
                            Here is a fix for
                            "UnboundLocalError: local variable 'unspl_samfile' referenced before assignment"
                            error.

                            Edit tophat and comment out ( usually it is in /usr/bin/tophat )
                            #os.remove(unspl_bwtfile) #os.remove(unspl_samfile)
                            my_dummy = 0 # to keep the if statment intact

                            The real fix should come from Cole!

                            Ramu
                            Another work around is to specify the "--keep-tmp" option to keep temporary files. The error is associated with the source code attempting to delete a temporary file before it is referenced. After the run is finished, you can delete the "tmp/" subdirectory.

                            Or just wait for the actual fix

                            -C

                            Comment


                            • #15
                              Originally posted by babbitt View Post
                              I have a different error message. This was using tophat 1.3.0 on paired-end solid reads, which had been successfully mapped using tophat 1.2.0. The error message reads:

                              Error: qual length (57) differs from seq length (51) for fastq record 83_1325_28_F3!
                              I also receive this error when running pair-end reads from the SOLiD. This error shows up in the segment_juncs.log

                              Code:
                              segment_juncs v1.3.0 (2398:2399)
                              ---------------------------
                              Loading reference sequences...
                              	Loading chr1...done
                              	Loading chr2...done
                              	Loading chr3...done
                              	Loading chr4...done
                              	Loading chr5...done
                              	Loading chr6...done
                              	Loading chr7...done
                              	Loading chr8...done
                              	Loading chr9...done
                              	Loading chr10...done
                              	Loading chr11...done
                              	Loading chr12...done
                              	Loading chr13...done
                              	Loading chr14...done
                              	Loading chr15...done
                              	Loading chr16...done
                              	Loading chr17...done
                              	Loading chr18...done
                              	Loading chr19...done
                              	Loading chr20...done
                              	Loading chr21...done
                              	Loading chr22...done
                              	Loading chrX...done
                              	Loading chrY...done
                              Loading left segment hits...
                              Error: qual length (56) differs from seq length (51) for fastq record 9_274_760_F3!
                              Edit: I should mention that doing a grep on the csfasta and qual shows seq length to be 51 (due to colorspace) with a qual length of 50.

                              That is all after receiving "err =1" from Tophat's stdout:
                              Code:
                              [Thu Jun  9 13:48:18 2011] Preparing reads
                              	format:		 fasta
                              	Left  reads: min. length=51, count=151286332
                              	Right reads: min. length=36, count=151112639
                              [Thu Jun  9 15:33:59 2011] Mapping left_kept_reads against hg19All.fa with Bowtie 
                              [Thu Jun  9 16:49:23 2011] Processing bowtie hits
                              [Thu Jun  9 18:31:50 2011] Mapping left_kept_reads_seg1 against hg19All.fa with Bowtie (1/2)
                              [Thu Jun  9 21:19:13 2011] Mapping left_kept_reads_seg2 against hg19All.fa with Bowtie (2/2)
                              [Fri Jun 10 00:11:09 2011] Mapping right_kept_reads against hg19All.fa with Bowtie 
                              [Fri Jun 10 01:26:25 2011] Processing bowtie hits
                              [Fri Jun 10 02:40:22 2011] Mapping right_kept_reads_seg1 against hg19All.fa with Bowtie (1/1)
                              [Fri Jun 10 03:39:36 2011] Searching for junctions via segment mapping
                              	[FAILED]
                              Error: segment-based junction search failed with err =1
                              Using these options:
                              Code:
                              tophat --color --quals --mate-inner-dist 200 --num-threads 8 --tmp-dir $tmp --output-dir $out --library-type fr-secondstrand $colorspaceindex $f3read $f5read $f3qual $f5qual
                              Last edited by jbrwn; 06-10-2011, 07:25 AM.

                              Comment

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