[Lizex]

Two suggestions. They are not very specific though.

1. Latest Xcode for 10.6.x appears to be v.3.2.6. You could try upgrading and recompiling.

2. Getting the pre-compiled OS X binary from TopHat site. I assume it should work with 10.6.x.

Thanks GenoMax for your suggestions. I finally got the pre-compiled binary to work.

[GenoMax]

Two suggestions. They are not very specific though.

1. Latest Xcode for 10.6.x appears to be v.3.2.6. You could try upgrading and recompiling.

2. Getting the pre-compiled OS X binary from TopHat site. I assume it should work with 10.6.x.

[Lizex]

Is this on OS X? Is yes then what version of OS X and Xcode are you using?

"Image not found" errors *may* to be Xcode related based on some limited googling. Are you sure there were no errors when you built TopHat?

Hi Genomax

I'm using a Mac OS 10.6.8 Xcode 3.2.3

Command to compile tophat-2.0.9
./configure --prefix=/Users/lhusselmann/bin/tophat-2.0.9 --with-boost=/usr/local/Cellar/boost --with-bam=/usr/local/lib --with-gzip=/Users/lhusselmann/bin/gzip-1.6


This is how the compilation looks:lhusselmann$ cd bin/tophat-2.0.9/
8ta-229-97-249:tophat-2.0.9 lhusselmann$ ./configure --prefix=/Users/lhusselmann/bin/tophat-2.0.9 --with-boost=/usr/local/Cellar/boost --with-bam=/usr/local/lib --with-gzip=/Users/lhusselmann/bin/gzip-1.6
checking for a BSD-compatible install... /usr/bin/install -c
checking whether build environment is sane... yes
checking for gawk... no
checking for mawk... no
checking for nawk... no
checking for awk... awk
checking whether make sets $(MAKE)... yes
checking for gawk... (cached) awk
checking for g++... g++
checking for C++ compiler default output file name... a.out
checking whether the C++ compiler works... yes
checking whether we are cross compiling... no
checking for suffix of executables...
checking for suffix of object files... o
checking whether we are using the GNU C++ compiler... yes
checking whether g++ accepts -g... yes
checking for style of include used by make... GNU
checking dependency style of g++... gcc3
checking for gcc... gcc
checking whether we are using the GNU C compiler... yes
checking whether gcc accepts -g... yes
checking for gcc option to accept ANSI C... none needed
checking dependency style of gcc... gcc3
checking whether make sets $(MAKE)... (cached) yes
checking for ranlib... ranlib
checking for a BSD-compatible install... /usr/bin/install -c
checking for a Python interpreter with version >= 2.4... python
checking for python... /Library/Frameworks/Python.framework/Versions/2.7/bin/python
checking for python version... 2.7
checking for python platform... darwin
checking for python script directory... ${prefix}/lib/python2.7/site-packages
checking for python extension module directory... ${exec_prefix}/lib/python2.7/site-packages
checking for boostlib >= 1.38.0... yes
checking for bamlib... yes
checking build system type... i686-apple-darwin10.8.0
checking whether the Boost::Thread library is available... yes
checking for exit in -lboost_thread-mt... yes
checking for exit in -lboost_system-mt... yes
checking how to run the C preprocessor... gcc -E
checking for egrep... grep -E
checking for ANSI C header files... yes
checking for sys/types.h... yes
checking for sys/stat.h... yes
checking for stdlib.h... yes
checking for string.h... yes
checking for memory.h... yes
checking for strings.h... yes
checking for inttypes.h... yes
checking for stdint.h... yes
checking for unistd.h... yes
checking for stdlib.h... (cached) yes
checking for string.h... (cached) yes
checking for unistd.h... (cached) yes
checking for ANSI C header files... (cached) yes
checking for stdbool.h that conforms to C99... yes
checking for _Bool... yes
checking for inline... inline
checking for pid_t... yes
checking for size_t... yes
checking for ptrdiff_t... yes
checking for an ANSI C-conforming const... yes
checking for special C compiler options needed for large files... no
checking for _FILE_OFFSET_BITS value needed for large files... no
checking for _LARGE_FILES value needed for large files... no
checking for gzread in -lz... yes
checking for stdlib.h... (cached) yes
checking for GNU libc compatible malloc... yes
checking for memset... yes
checking for strdup... yes
checking for strrchr... yes
checking for strtol... yes
checking for strsep... yes
checking host system type... i686-apple-darwin10.8.0
checking how to create a pax tar archive... gnutar
checking dependency style of gcc... (cached) gcc3
checking dependency style of g++... (cached) gcc3
configure: creating ./config.status
config.status: creating Makefile
config.status: creating src/Makefile
config.status: creating config.h
config.status: config.h is unchanged
config.status: executing depfiles commands

-- tophat 2.0.9 Configuration Results --
C++ compiler: g++ -Wall -Wno-strict-aliasing -g -gdwarf-2 -Wuninitialized -O3 -DNDEBUG -pthread -I/usr/local/Cellar/boost/include -I/usr/local/lib/include -I./SeqAn-1.3
Linker flags:
GCC version: i686-apple-darwin10-gcc-4.2.1 (GCC) 4.2.1 (Apple Inc. build 5666) (dot 3)
Host System type: i686-apple-darwin10.8.0
Install prefix: /Users/lhusselmann/bin/tophat-2.0.9
Install eprefix: ${prefix}

See config.h for further configuration information.
Email <[email protected]> with questions and bug reports.

8ta-229-97-249:tophat-2.0.9 lhusselmann$ make
make all-recursive
Making all in src
make[2]: Nothing to be done for `all'.
8ta-229-97-249:tophat-2.0.9 lhusselmann$ make install
Making install in src
test -z "/Users/lhusselmann/bin/tophat-2.0.9/bin" || /Users/lhusselmann/bin/tophat-2.0.9/build-aux/install-sh -d "/Users/lhusselmann/bin/tophat-2.0.9/bin"
/usr/bin/install -c 'prep_reads' '/Users/lhusselmann/bin/tophat-2.0.9/bin/prep_reads'
/usr/bin/install -c 'gtf_to_fasta' '/Users/lhusselmann/bin/tophat-2.0.9/bin/gtf_to_fasta'
/usr/bin/install -c 'fix_map_ordering' '/Users/lhusselmann/bin/tophat-2.0.9/bin/fix_map_ordering'
/usr/bin/install -c 'bam2fastx' '/Users/lhusselmann/bin/tophat-2.0.9/bin/bam2fastx'
/usr/bin/install -c 'segment_juncs' '/Users/lhusselmann/bin/tophat-2.0.9/bin/segment_juncs'
/usr/bin/install -c 'gtf_juncs' '/Users/lhusselmann/bin/tophat-2.0.9/bin/gtf_juncs'
/usr/bin/install -c 'juncs_db' '/Users/lhusselmann/bin/tophat-2.0.9/bin/juncs_db'
/usr/bin/install -c 'long_spanning_reads' '/Users/lhusselmann/bin/tophat-2.0.9/bin/long_spanning_reads'
/usr/bin/install -c 'bam_merge' '/Users/lhusselmann/bin/tophat-2.0.9/bin/bam_merge'
/usr/bin/install -c 'map2gtf' '/Users/lhusselmann/bin/tophat-2.0.9/bin/map2gtf'
/usr/bin/install -c 'tophat_reports' '/Users/lhusselmann/bin/tophat-2.0.9/bin/tophat_reports'
/usr/bin/install -c 'sam_juncs' '/Users/lhusselmann/bin/tophat-2.0.9/bin/sam_juncs'
test -z "/Users/lhusselmann/bin/tophat-2.0.9/bin" || /Users/lhusselmann/bin/tophat-2.0.9/build-aux/install-sh -d "/Users/lhusselmann/bin/tophat-2.0.9/bin"
/usr/bin/install -c 'tophat2' '/Users/lhusselmann/bin/tophat-2.0.9/bin/tophat2'
/usr/bin/install -c 'tophat' '/Users/lhusselmann/bin/tophat-2.0.9/bin/tophat'
test -z "/Users/lhusselmann/bin/tophat-2.0.9/bin" || /Users/lhusselmann/bin/tophat-2.0.9/build-aux/install-sh -d "/Users/lhusselmann/bin/tophat-2.0.9/bin"
/usr/bin/install -c 'contig_to_chr_coords' '/Users/lhusselmann/bin/tophat-2.0.9/bin/contig_to_chr_coords'
/usr/bin/install -c 'bed_to_juncs' '/Users/lhusselmann/bin/tophat-2.0.9/bin/bed_to_juncs'
/usr/bin/install -c 'sra_to_solid' '/Users/lhusselmann/bin/tophat-2.0.9/bin/sra_to_solid'
/usr/bin/install -c 'tophat-fusion-post' '/Users/lhusselmann/bin/tophat-2.0.9/bin/tophat-fusion-post'
make[2]: Nothing to be done for `install-data-am'.
make[2]: Nothing to be done for `install-exec-am'.
make[2]: Nothing to be done for `install-data-am'.

No warnings or errors with compilation and installation.

Thanks in advance.

[GenoMax]

Is this on OS X? Is yes then what version of OS X and Xcode are you using?

"Image not found" errors *may* to be Xcode related based on some limited googling. Are you sure there were no errors when you built TopHat?

[Lizex]

now that we have tophat2 out the door, i'm sure we'll have bunch of tophat2 related error messages and solutions - hopefully.

thank you to the team for releasing the new version. i'm very excited for the combination of tophat and tophat-fusion.

now my problem. I've downloaded and installed bowtie2 and tophat2. everything started out just fine until tophat tried to search for junctions via segment mapping. I got an error 5

tophat2 -o A673_rep1_tophat2 -r 158 --mate-std-dev 44 -p 24 --microexon-search --library-type fr-unstranded --b2-sensitive --fusion-search --fusion-min-dist 50000 --fusion-multireads 5 --fusion-multipairs 5 ~/genome_reference/bowtie_indexed/hg19.bowtie2 A673_rep1_R1.fastq A673_rep1_R2.fastq 

[2012-04-11 14:16:47] Beginning TopHat run (v2.0.0)
-----------------------------------------------
[2012-04-11 14:16:47] Checking for Bowtie
		  Bowtie version:	 2.0.0.5
[2012-04-11 14:16:47] Checking for Samtools
		Samtools version:	 0.1.18.0
[2012-04-11 14:16:47] Checking for Bowtie index files
[2012-04-11 14:16:47] Checking for reference FASTA file
	Warning: Could not find FASTA file /Users/zehira/genome_reference/bowtie_indexed/hg19.bowtie2.fa
[2012-04-11 14:16:47] Reconstituting reference FASTA file from Bowtie index
  Executing: /Users/zehira/local/bin/bowtie2-inspect /Users/zehira/genome_reference/bowtie_indexed/hg19.bowtie2 > A673_rep1_tophat2/tmp/hg19.bowtie2.fa
[2012-04-11 14:19:57] Generating SAM header for /Users/zehira/genome_reference/bowtie_indexed/hg19.bowtie2
	format:		 fastq
	quality scale:	 phred33 (default)
[2012-04-11 14:20:00] Preparing reads
	 left reads: min. length=75, count=12942699
	right reads: min. length=75, count=12927964
[2012-04-11 14:28:54] Mapping left_kept_reads against hg19.bowtie2 with Bowtie2 
[2012-04-11 14:44:43] Mapping left_kept_reads_seg1 against hg19.bowtie2 with Bowtie2 (1/3)
[2012-04-11 14:47:28] Mapping left_kept_reads_seg2 against hg19.bowtie2 with Bowtie2 (2/3)
[2012-04-11 14:50:06] Mapping left_kept_reads_seg3 against hg19.bowtie2 with Bowtie2 (3/3)
[2012-04-11 14:53:27] Mapping right_kept_reads against hg19.bowtie2 with Bowtie2 
[2012-04-11 15:09:38] Mapping right_kept_reads_seg1 against hg19.bowtie2 with Bowtie2 (1/3)
[2012-04-11 15:12:11] Mapping right_kept_reads_seg2 against hg19.bowtie2 with Bowtie2 (2/3)
[2012-04-11 15:14:44] Mapping right_kept_reads_seg3 against hg19.bowtie2 with Bowtie2 (3/3)
[2012-04-11 15:17:26] Searching for junctions via segment mapping
	[FAILED]
Error: segment-based junction search failed with err =-5
  Reason: image not found

I'm hoping it's an easy solution and am sure it's something i did wrong. anyone has any ideas?

Thanks!

Hi Ahmetz
I have compiled and installed Tophat-2.0.9 and got this error on the test_data provided on Tophat website. The same test data run with tophat work, but with tophat2 it gives the error below. I read on Seqanswers http://seqanswers.com/forums/showthread.php?t=19135 , downloading the latest version solved the issue, but this is the latest version. Did you manage to solve your problem?

$ tophat2 -o Tophat2 -r 20 test_ref reads_1.fq reads_2.fq

[2013-09-03 13:53:17] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2013-09-03 13:53:17] Checking for Bowtie
Bowtie version: 2.1.0.0
[2013-09-03 13:53:17] Checking for Samtools
Samtools version: 0.1.19.0
[2013-09-03 13:53:17] Checking for Bowtie index files (genome)..
Found both Bowtie1 and Bowtie2 indexes.
[2013-09-03 13:53:17] Checking for reference FASTA file
[2013-09-03 13:53:17] Generating SAM header for test_ref
format: fastq
quality scale: phred33 (default)
[2013-09-03 13:53:17] 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)
[2013-09-03 13:53:17] Mapping left_kept_reads to genome test_ref with Bowtie2
[2013-09-03 13:53:17] Mapping left_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2013-09-03 13:53:17] Mapping left_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2013-09-03 13:53:17] Mapping left_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2013-09-03 13:53:17] Mapping right_kept_reads to genome test_ref with Bowtie2
[2013-09-03 13:53:17] Mapping right_kept_reads_seg1 to genome test_ref with Bowtie2 (1/3)
[2013-09-03 13:53:17] Mapping right_kept_reads_seg2 to genome test_ref with Bowtie2 (2/3)
[2013-09-03 13:53:17] Mapping right_kept_reads_seg3 to genome test_ref with Bowtie2 (3/3)
[2013-09-03 13:53:17] Searching for junctions via segment mapping
[FAILED]
Error: segment-based junction search failed with err =-5
Reason: image not found

[NicoBxl]

I tried with the fix, and it's been two days that tophat is trapped in :

[2012-04-21 18:34:05] Searching for junctions via segment mapping

it's strange :

here's my command :

tophat -p 8 --coverage-search -r 70 -G gff.gtf --fusion-search --b2-sensitive --mate-std-dev 50 --library-type fr-unstranded --read-mismatches 3 --output-dir tophat_2 --segment-mismatches 2 BowtieDB R1.fastq.gz R2.fastq.gz

[labrat73]

I'm sorry, but what is the point of version numbers if you are not going to differentiate a bug fix release? Please reconsider and release it as 2.0.1. Even 2.0.0.1 would be OK.

Mistakes happen in all software. Rails is famous for releasing a version, and releasing another version 1 day later due to a major bug or security hole they found after the release.

For more on the topic, see http://semver.org/

+2. Also, I'm still hoping for a fix to the problem of segment_juncs process hanging for >24 hours. Saw a post earlier where user was able to shorten the process by not using certain flags. For now, back to using Tophat 1.4.0 and TopHatFusion.

[dvanic]

I'm sorry, but what is the point of version numbers if you are not going to differentiate a bug fix release? Please reconsider and release it as 2.0.1. Even 2.0.0.1 would be OK.

Mistakes happen in all software. Rails is famous for releasing a version, and releasing another version 1 day later due to a major bug or security hole they found after the release.

For more on the topic, see http://semver.org/

+1 to this.
It's just that there are probably people who are using tophat who haven't checked this thread in the last week, which may mean they'll be using a version without this bug fixed.
Thank you in advance!

[Dario1984]

I have a new problem. For one samples, the mapped reads between TopHat 1.33 and 2 went from 21 million to 12 million reads. I decided to BLAT some of the reads in the unmapped.bam file.

I wonder why reads like this are excluded ?

No code has to be inserted here.The first alignment is better.

[delagoya]

why not up the version to 2.0.1?

There are quite a few different problems reported in this thread - I apologize for them. We are aware of a couple of these problems and we are trying to fix them, though we are encountering some difficulties reproducing some of these errors.
We just updated the packages on the website with a couple of last minute fixes so please re-download the tophat-2.0.0 package of your choice (yes, it's the same version, same file names) from http://tophat.cbcb.umd.edu and try running the new version again on your test data. If tophat 2 still fails at any stage in the process please report the failure at [email protected] - it would also help if you attach a tarball or zip file with the content of the "logs" directory when you post a bug report there.

Thank you,
-Geo

I'm sorry, but what is the point of version numbers if you are not going to differentiate a bug fix release? Please reconsider and release it as 2.0.1. Even 2.0.0.1 would be OK.

Mistakes happen in all software. Rails is famous for releasing a version, and releasing another version 1 day later due to a major bug or security hole they found after the release.

For more on the topic, see http://semver.org/

[vyellapa]

Have you tried indexing your reference using

bowtie2-build <reference.fa> <bt2_base>

I ran into the same error when I downloaded the pre-built indexes, however, building my own worked ok.

[Dario1984]

Index Locations

The location of the indexes doesn't work like the documentation says.

darstr@clark-lab:~/bowtie2$ echo $BOWTIE_INDEXES
/home/darstr/bowtie2/indexes/
darstr@clark-lab:~/bowtie2$ ls $BOWTIE_INDEXES
hg18.1.bt2  hg18.2.bt2  hg18.3.bt2  hg18.4.bt2  hg18.rev.1.bt2  hg18.rev.2.bt2

[2012-04-18 15:43:14] Beginning TopHat run (v2.0.0)
-----------------------------------------------
[2012-04-18 15:43:14] Checking for Bowtie
                  Bowtie version:        2.0.0.5
[2012-04-18 15:43:14] Checking for Samtools
                Samtools version:        0.1.18.0
[2012-04-18 15:43:14] Checking for Bowtie index files
Error: Could not find Bowtie 2 index files (hg18.*.bt2)

Not only is the environment variable ignored, but it also doesn't look in the indexes directory in the directory of the bowtie instance.

The only was it works is if I change directories to /home/darstr/bowtie2/indexes/ and run it from there.

[DineshCyanam]

So I was able to reduce my runtime to ~4 hrs from ~30 hrs by using the --no-coverage-search and --no-novel-indels flags.

Yes... Tophat 2 is taking extremely long (~ 30 hrs)... particularly the Searching for junctions via segment mapping step as NicoBxl above observed.
Previous versions of Tophat for the same data took ~8 hours to run

[turnersd]

I'm also getting an error when using the --transcript-index option, "GFF transcripts file not found or invalid". The file exists, and it appears valid.

[DineshCyanam]

Yes... Tophat 2 is taking extremely long (~ 30 hrs)... particularly the Searching for junctions via segment mapping step as NicoBxl above observed. Here is my log.

$ tophat -r 150 --solexa1.3-quals -p 24 -g 20 --library-type fr-unstranded -o tophat_out $BOWTIE2_INDEXES/genome 61T4D/s_1_1_sequence.txt.gz,6286U/s_1_1_sequence.txt.gz 61T4D/s_1_2_sequence.txt.gz,6286U/s_1_2_sequence.txt.gz

[2012-04-13 18:14:51] Beginning TopHat run (v2.0.0)
-----------------------------------------------
[2012-04-13 18:14:51] Checking for Bowtie
Bowtie version: 2.0.0.5
[2012-04-13 18:14:51] Checking for Samtools
Samtools version: 0.1.18.0
[2012-04-13 18:14:51] Checking for Bowtie index files
[2012-04-13 18:14:51] Checking for reference FASTA file
[2012-04-13 18:14:51] Generating SAM header for /Homo_sapiens/Ensembl/GRCh37/Sequence/BowtieIndex/genome
format: fastq
quality scale: phred64 (reads generated with GA pipeline version >= 1.3)
[2012-04-13 18:14:53] Preparing reads
left reads: min. length=60, count=44338793
right reads: min. length=60, count=44098248
[2012-04-13 18:29:52] Mapping left_kept_reads against genome with Bowtie2
[2012-04-13 19:17:01] Mapping left_kept_reads_seg1 against genome with Bowtie2 (1/2)
[2012-04-13 19:28:48] Mapping left_kept_reads_seg2 against genome with Bowtie2 (2/2)
[2012-04-13 19:47:50] Mapping right_kept_reads against genome with Bowtie2
[2012-04-13 20:33:37] Mapping right_kept_reads_seg1 against genome with Bowtie2 (1/2)
[2012-04-13 20:47:07] Mapping right_kept_reads_seg2 against genome with Bowtie2 (2/2)
[2012-04-13 21:08:17] Searching for junctions via segment mapping
[2012-04-14 22:36:08] Retrieving sequences for splices
[2012-04-14 22:38:05] Indexing splices
[2012-04-14 22:51:34] Mapping left_kept_reads_seg1 against segment_juncs with Bowtie2 (1/2)
[2012-04-14 23:01:42] Mapping left_kept_reads_seg2 against segment_juncs with Bowtie2 (2/2)
[2012-04-14 23:15:09] Joining segment hits
[2012-04-14 23:37:53] Mapping right_kept_reads_seg1 against segment_juncs with Bowtie2 (1/2)
[2012-04-14 23:49:11] Mapping right_kept_reads_seg2 against segment_juncs with Bowtie2 (2/2)
[2012-04-15 00:04:38] Joining segment hits
[2012-04-15 00:28:13] Reporting output tracks
-----------------------------------------------
[2012-04-15 00:43:47] Run complete: 1 days 06:28:56 elapsed

Previous versions of Tophat for the same data took ~8 hours to run