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Old 07-28-2014, 01:01 PM   #1
jthomas5062
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Default TopHat - Error: gtf_to_fasta returned an error.

Hello all,

I am trying to perform alignments using TopHat2 and am receiving a gtf_to_fasta error message.

My command is the following:
$tophat -p 8 -G genes.gtf -o C1_R1_thout genome C1_R1_1.fq C1_R1_2.fq

The output:
[2014-07-28 15:40:04] Beginning TopHat run (v2.0.12)
-----------------------------------------------
[2014-07-28 15:40:04] Checking for Bowtie
Bowtie version: 2.2.3.0
[2014-07-28 15:40:05] Checking for Samtools
Samtools version: 0.1.19.0
[2014-07-28 15:40:05] Checking for Bowtie index files (genome)..
[2014-07-28 15:40:05] Checking for reference FASTA file
[2014-07-28 15:40:05] Generating SAM header for genome
[2014-07-28 15:40:05] Reading known junctions from GTF file
[2014-07-28 15:40:09] Preparing reads
left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
[2014-07-28 15:45:43] Building transcriptome data files C1_R1_thout/tmp/genes
[FAILED]
Error: gtf_to_fasta returned an error.

When I look at the output error file "g2f.err", I see the following:
dyld: lazy symbol binding failed: Symbol not found: __ZNKSt5ctypeIcE13_M_widen_initEv
Referenced from: /usr/local/bin/gtf_to_fasta
Expected in: /usr/lib/libstdc++.6.dylib

dyld: Symbol not found: __ZNKSt5ctypeIcE13_M_widen_initEv
Referenced from: /usr/local/bin/gtf_to_fasta
Expected in: /usr/lib/libstdc++.6.dylib


Has anyone had similar problems? Thanks!
James
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Old 07-28-2014, 01:06 PM   #2
jthomas5062
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The following files are in the directory from which I am executing the command:

genome.1.bt2
genome.2.bt2
genome.3.bt2
genome.4.bt2
genome.rev.1.bt2
genome.rev.2.bt2
genome.fa
genes.gtf
C1_R1_1.fq
C1_R1_2.fq
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Old 07-28-2014, 02:16 PM   #3
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Did you download the Mac (this is a Mac I presume) binaries from TopHat site? Do you have Xcode installed on this Mac?

Are you running an older version of OS X? This *may* be a problem of trying to run the 64-bit binary on a 32-bit machine.

Last edited by GenoMax; 07-28-2014 at 02:20 PM.
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Old 07-28-2014, 02:43 PM   #4
jthomas5062
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I am running a 64-Bit Mac OS X 10.9.4; 16 GB RAM; 4 cores and Xcode is installed
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Old 07-28-2014, 02:45 PM   #5
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I'm using TopHat 2

Admins-iMac:~ jamesdthomas$ tophat -v
TopHat v2.0.12
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Old 07-29-2014, 02:56 AM   #6
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Have you checked if the test data supplied with TopHat works without errors?
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Old 07-29-2014, 05:18 AM   #7
jthomas5062
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My command:
tophat -p 8 -o test_thout test_ref reads_1.fq reads_2.fq

Output:
[2014-07-29 09:10:21] Beginning TopHat run (v2.0.12)
-----------------------------------------------
[2014-07-29 09:10:21] Checking for Bowtie
Bowtie version: 2.2.3.0
[2014-07-29 09:10:21] Checking for Samtools
Samtools version: 0.1.19.0
[2014-07-29 09:10:21] Checking for Bowtie index files (genome)..
[2014-07-29 09:10:21] Checking for reference FASTA file
[2014-07-29 09:10:21] Generating SAM header for test_ref
[2014-07-29 09:10:21] 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)
[2014-07-29 09:10:21] Mapping left_kept_reads to genome test_ref with Bowtie2
[2014-07-29 09:10:21] Mapping left_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-07-29 09:10:22] Mapping left_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-07-29 09:10:22] Mapping left_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-07-29 09:10:22] Mapping right_kept_reads to genome test_ref with Bowtie2
[2014-07-29 09:10:22] Mapping right_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-07-29 09:10:22] Mapping right_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-07-29 09:10:22] Mapping right_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-07-29 09:10:22] Searching for junctions via segment mapping
[FAILED]
Error: segment-based junction search failed with err =-5


When I look in segment_juncs log, I see the same error as with my actual dataset:
segment_juncs v2.0.12 (4277)
---------------------------
[samopen] SAM header is present: 1 sequences.
Loading reference sequences...
Loading test_chromosome...done
Loading ...done
>> Performing segment-search:
Loading left segment hits...
dyld: lazy symbol binding failed: Symbol not found: __ZNKSt5ctypeIcE13_M_widen_initEv
Referenced from: /usr/local/bin/segment_juncs
Expected in: /usr/lib/libstdc++.6.dylib

dyld: Symbol not found: __ZNKSt5ctypeIcE13_M_widen_initEv
Referenced from: /usr/local/bin/segment_juncs
Expected in: /usr/lib/libstdc++.6.dylib


It seems like more of a "my computer problem" than a TopHat specific problem. I'm somewhat of a novice when it comes to libraries, etc. When I tried to search for a solution to these "symbol not found" errors, I came up pretty empty handed.

Thanks,
James
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Old 07-29-2014, 05:35 AM   #8
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Can you try a previous version of TopHat with the test data (say v.2.0.11, http://ccb.jhu.edu/software/tophat/downloads/) to see if this is a problem with the latest Mac binary that is being supplied?
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Old 07-30-2014, 12:38 PM   #9
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I downloaded v.2.0.11 of TopHat and am now getting this error message after running on the test data set:

[2014-07-30 16:36:24] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-07-30 16:36:24] Checking for Bowtie
Bowtie version: 2.2.3.0
[2014-07-30 16:36:24] Checking for Samtools
Samtools version: 0.1.19.0
[2014-07-30 16:36:24] Checking for Bowtie index files (genome)..
[2014-07-30 16:36:24] Checking for reference FASTA file
[2014-07-30 16:36:24] Generating SAM header for test_ref
[2014-07-30 16:36:24] 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)
[2014-07-30 16:36:24] Mapping left_kept_reads to genome test_ref with Bowtie2
[FAILED]
Error running:
/usr/local/bin/bam2fastx --all testdata_thout/tmp/left_kept_reads.bam|/usr/local/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 8 --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 testdata_thout/tmp/left_kept_reads.mapped.bam.index --sam-header testdata_thout/tmp/test_ref_genome.bwt.samheader.sam - testdata_thout/tmp/left_kept_reads.mapped.bam testdata_thout/tmp/left_kept_reads_unmapped.bam
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Old 07-30-2014, 12:39 PM   #10
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My submmitted commands:

mkdir testdata_thout

tophat2 -p 8 -o testdata_thout test_ref reads_1.fq reads_2.fq
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Old 08-14-2014, 07:45 AM   #11
zhangxw
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Default TopHat - Error: gtf_to_fasta returned an error.

has this problem solved? I met the same issue.
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Old 08-14-2014, 08:09 AM   #12
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Quote:
Originally Posted by zhangxw View Post
has this problem solved? I met the same issue.
Just to be specific. You are using a Mac and have had the exact same issue as jthomas5062.

With the test data and/or your own?
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Old 08-14-2014, 08:21 AM   #13
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Yes, I am using Mac OS.
Same problem with the test_data by TopHat run (v2.0.11)
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Old 08-14-2014, 08:26 AM   #14
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Such problem disappears with the test_data by TapHat v2.0.10;

however, in TapHat v2.0.10; it has Error: gtf_to_fasta returned an error. with my own data.
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Old 08-14-2014, 09:21 AM   #15
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It appears that all Mac binaries since 2.0.10 have this problem. -stdlib option to build the binary either needs libstdc++ or libc++ (am not a developer) and it does not appear to be properly linked for Mac binaries. I am surprised that no one has reported this problem to the developers. Until the authors correct this we may be out-of-luck.

Has anyone tried to compile from source?

The last version of TopHat that is functional on OS X (10.9.x) is v.2.0.9.

Last edited by GenoMax; 08-14-2014 at 09:39 AM.
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Old 08-27-2014, 07:45 AM   #16
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Has this issue been resolved? Any updates? I'm dealing the exact same errors/error logs as described above.
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Old 08-27-2014, 07:53 AM   #17
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Unfortunately no. Your only option is to use TopHat 2.0.9 on OS X, unless you want to try compiling from source (not simple).
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Old 08-27-2014, 07:59 AM   #18
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Ok. Thank you. I'll continue with v.2.0.9 then. I'm still waiting for a response from the developers after I reported the issue. Once I get an update I will post it here.
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Old 08-27-2014, 08:13 AM   #19
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Weird. After I replaced Tophat's 2.0.12 version with the 2.0.9 version it doesn't find bowtie anymore. Even though it's in the PATH. I tried with --bowtie1 and --bowtie2. Bowtie2 is present as I can access it from the command line.
What's the catch?

[2014-08-27 09:09:09] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2014-08-27 09:09:09] Checking for Bowtie
Bowtie 2 not found, checking for older version..
Error: Bowtie not found on this system.
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Old 08-27-2014, 08:40 AM   #20
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You may need a corresponding older version of bowtie to go with TopHat 2.0.9. Try v.2.1.0.
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