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**liuxq** · 06-02-2011, 06:06 PM

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

**jmartin127** · 06-02-2011, 07:07 PM

Yep, same for me.

**Dario1984** · 06-02-2011, 09:00 PM

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.

**liuxq** · 06-03-2011, 12:28 AM

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

**liuxq** · 06-03-2011, 12:35 AM

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.

**Cole Trapnell** · 06-03-2011, 05:17 AM

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.

**jmartin127** · 06-03-2011, 07:31 AM

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.

**Dario1984** · 06-03-2011, 07:00 PM

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.

**berath** · 06-06-2011, 06:29 AM

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

**Auction** · 06-07-2011, 01:32 PM

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

**babbitt** · 06-08-2011, 07:40 AM

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!

**ramu** · 06-09-2011, 02:15 AM

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

**cwng** · 06-09-2011, 08:24 AM

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

**jbrwn** · 06-10-2011, 07:18 AM

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

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