Error running test in Tophat

[Alessandro_S]

Hi everybody, I'm new to the RNA-seq data analysis world and I really need your help.
Just one premise: I have poor experience in coding/programming.

I'm using a Mac Pro (2 x 2.8 GHz Quad-core, 18GB RAM). I've installed SAM tools, Boost 1_55_0, Bowtie 2.2.3 and Tophat-2.0.12 (pre compiled for Mac OSX 64bit). I followed the installation instructions that I found in the corresponding manuals and I tested successfully Bowtie2.

But when I tested Tophat (using the test_data file as suggested in the "getting started" manual), I came across this error:

[2014-08-23 14:03:53] Beginning TopHat run (v2.0.12)
-----------------------------------------------
[2014-08-23 14:03:53] Checking for Bowtie
Bowtie version: 2.2.2.0
[2014-08-23 14:03:53] Checking for Samtools
Samtools version: 0.1.16.0
[2014-08-23 14:03:53] Checking for Bowtie index files (genome)..
[2014-08-23 14:03:53] Checking for reference FASTA file
[2014-08-23 14:03:53] Generating SAM header for test_ref
[2014-08-23 14:03:53] 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)
[2014-08-23 14:03:53] Mapping left_kept_reads to genome test_ref with Bowtie2
[2014-08-23 14:03:53] Mapping left_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-08-23 14:03:53] Mapping left_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-08-23 14:03:53] Mapping left_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-08-23 14:03:53] Mapping right_kept_reads to genome test_ref with Bowtie2
[2014-08-23 14:03:54] Mapping right_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-08-23 14:03:54] Mapping right_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-08-23 14:03:54] Mapping right_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-08-23 14:03:54] Searching for junctions via segment mapping
[2014-08-23 14:03:54] Retrieving sequences for splices
[2014-08-23 14:03:54] Indexing splices
[2014-08-23 14:03:54] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2014-08-23 14:03:54] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2014-08-23 14:03:55] Mapping left_kept_reads_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2014-08-23 14:03:55] Joining segment hits
[FAILED]
Error running 'long_spanning_reads':

And this is the long_spanning_reads.segs.log file:

long_spanning_reads v2.0.12 (4277)
--------------------------------------------
Opening /dev/null for reading
[samopen] SAM header is present: 1 sequences.
Loading reference sequences...
reference sequences loaded.
Loading junctions...done
Loading deletions...done
Loading insertions...done
dyld: lazy symbol binding failed: Symbol not found: __ZNSt15_List_node_base7_M_hookEPS_
Referenced from: /***/***/***/***/long_spanning_reads
Expected in: /usr/lib/libstdc++.6.dylib

dyld: Symbol not found: __ZNSt15_List_node_base7_M_hookEPS_
Referenced from: /***/***/***/***/long_spanning_reads
Expected in: /usr/lib/libstdc++.6.dylib

I've read several threads but I couldn't find an answer or a solution.

Could you please help me? I have no idea of what is going on.

Thank you,

Alessandro.

[GenoMax]

There *may* be a problem with published TopHat Mac binaries for some time, in terms of how they are linked to libraries. See this thread for related information: http://seqanswers.com/forums/showthread.php?t=45349

Do you have the latest Xcode tools installed?

[Alessandro_S]

Hi GenoMax,

Thank you so much for your help.

Switching to TopHat v.2.0.9 and Bowtie 2.1.0.0 solved the problem:

[2014-08-25 09:08:17] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2014-08-25 09:08:17] Checking for Bowtie
Bowtie version: 2.1.0.0
[2014-08-25 09:08:17] Checking for Samtools
Samtools version: 0.1.19.0
[2014-08-25 09:08:17] Checking for Bowtie index files (genome)..
Found both Bowtie1 and Bowtie2 indexes.
[2014-08-25 09:08:17] Checking for reference FASTA file
[2014-08-25 09:08:17] Generating SAM header for test_ref
format: fastq
quality scale: phred33 (default)
[2014-08-25 09:08:18] 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)
[2014-08-25 09:08:18] Mapping left_kept_reads to genome test_ref with Bowtie2
[2014-08-25 09:08:18] Mapping left_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-08-25 09:08:18] Mapping left_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-08-25 09:08:18] Mapping left_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-08-25 09:08:19] Mapping right_kept_reads to genome test_ref with Bowtie2
[2014-08-25 09:08:19] Mapping right_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2014-08-25 09:08:19] Mapping right_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2014-08-25 09:08:19] Mapping right_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2014-08-25 09:08:19] Searching for junctions via segment mapping
[2014-08-25 09:08:19] Retrieving sequences for splices
[2014-08-25 09:08:19] Indexing splices
[2014-08-25 09:08:19] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2014-08-25 09:08:19] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2014-08-25 09:08:19] Mapping left_kept_reads_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2014-08-25 09:08:19] Joining segment hits
[2014-08-25 09:08:19] Mapping right_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2014-08-25 09:08:19] Mapping right_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2014-08-25 09:08:19] Mapping right_kept_reads_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2014-08-25 09:08:19] Joining segment hits
[2014-08-25 09:08:19] Reporting output tracks
-----------------------------------------------
[2014-08-25 09:08:19] A summary of the alignment counts can be found in ./tophat_out/align_summary.txt
[2014-08-25 09:08:19] Run complete: 00:00:02 elapsed

I'm still wondering if it's a problem related to my machine (my Xcode was updated) or with Tophat 2.0.12 pre-compiled release for MacOSX.

[GenoMax]

AFAIK the problem is with the latest pre-compiled OS X binaries for TopHat. Unfortunately no one from Tuxedo suite team seem to actively participate on this forum.

[dpryan]

They aren't even active in their own google group or in replying to bug reports. My rule of thumb is to give an issue 3-4 months before expecting a reply from them.