Tophat can't build transcriptome files

[stickleback]

Hello all,

I am attempting to build a three-spined stickleback transcriptome index with TopHat v2.0.14 but I keep getting an error. I've already indexed the reference genome using bowtie and Tophat aligns without to the genome. However as soon as I use the following command:

tophat -G Gac_genes.gtf --transcriptome-index=gac_transcript/known_gac gac


I get this output after a few seconds:


[2015-06-01 15:57:08] Building transcriptome files with TopHat v2.0.14
-----------------------------------------------
[2015-06-01 15:57:08] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-06-01 15:57:08] Checking for Bowtie index files (genome)..
[2015-06-01 15:57:08] Checking for reference FASTA file
[2015-06-01 15:57:08] Building transcriptome data files gac_transcript/known_gac
[2015-06-01 15:57:26] Building Bowtie index from known_gac.fa
[FAILED]
Error: Couldn't build bowtie index with err = -6

I have checked that the first column of the GTF and the genome indexes are identical. Can anyone shed any light on what a -6 error indicates?

Many thanks in advance

[GenoMax]

I think you may need to provide the full path to the genome index if it is not in the local directory.

Have you tried this?

$ tophat -G Gac_genes.gtf --transcriptome-index=gac_transcript/known_gac /path_to/gac

Are you using the most recent bowtie2 with tophat? I see that you are using the latest bowtie2.

[stickleback]

Thanks for the quick reply!

The genome index is in the local directory but I added the path just in case. The command runs a bit further but now I get this output:

[2015-06-04 11:49:26] Building transcriptome files with TopHat v2.0.14
-----------------------------------------------
[2015-06-04 11:49:26] Checking for Bowtie
Bowtie version: 2.2.5.0
[2015-06-04 11:49:31] Checking for Bowtie index files (genome)..
[2015-06-04 11:49:31] Checking for reference FASTA file
[2015-06-04 11:49:31] Building transcriptome data files gac_transcript/known_gac
[2015-06-04 11:49:42] Building Bowtie index from known_gac.fa
*** glibc detected *** bowtie2-build: double free or corruption (out): 0x00000000048848c0 ***
======= Backtrace: =========
/lib64/libc.so.6[0x37d9c760e6]
/lib64/libc.so.6[0x37d9c78c13]
bowtie2-build[0x40f155]
bowtie2-build[0x409f74]
bowtie2-build[0x40bf56]
bowtie2-build[0x4515f0]
/lib64/libc.so.6(__libc_start_main+0xfd)[0x37d9c1ecdd]
bowtie2-build[0x404029]
======= Memory map: ========
00400000-0045d000 r-xp 00000000 216:5c050 144116364813035716 /lustre4/home/markravinet/local/bin/bowtie2-build-s
0065c000-00661000 rw-p 0005c000 216:5c050 144116364813035716 /lustre4/home/markravinet/local/bin/bowtie2-build-s
00661000-04664000 rw-p 00000000 00:00 0
0487f000-0691e000 rw-p 00000000 00:00 0 [heap]
37d9400000-37d9420000 r-xp 00000000 08:03 70385816 /lib64/ld-2.12.so
37d961f000-37d9620000 r--p 0001f000 08:03 70385816 /lib64/ld-2.12.so
37d9620000-37d9621000 rw-p 00020000 08:03 70385816 /lib64/ld-2.12.so
37d9621000-37d9622000 rw-p 00000000 00:00 0
37d9800000-37d9883000 r-xp 00000000 08:03 70385731 /lib64/libm-2.12.so
37d9883000-37d9a82000 ---p 00083000 08:03 70385731 /lib64/libm-2.12.so
37d9a82000-37d9a83000 r--p 00082000 08:03 70385731 /lib64/libm-2.12.so
37d9a83000-37d9a84000 rw-p 00083000 08:03 70385731 /lib64/libm-2.12.so
37d9c00000-37d9d8a000 r-xp 00000000 08:03 70385808 /lib64/libc-2.12.so
37d9d8a000-37d9f89000 ---p 0018a000 08:03 70385808 /lib64/libc-2.12.so
37d9f89000-37d9f8d000 r--p 00189000 08:03 70385808 /lib64/libc-2.12.so
37d9f8d000-37d9f8e000 rw-p 0018d000 08:03 70385808 /lib64/libc-2.12.so
37d9f8e000-37d9f93000 rw-p 00000000 00:00 0
37da400000-37da417000 r-xp 00000000 08:03 70385864 /lib64/libpthread-2.12.so
37da417000-37da617000 ---p 00017000 08:03 70385864 /lib64/libpthread-2.12.so
37da617000-37da618000 r--p 00017000 08:03 70385864 /lib64/libpthread-2.12.so
37da618000-37da619000 rw-p 00018000 08:03 70385864 /lib64/libpthread-2.12.so
37da619000-37da61d000 rw-p 00000000 00:00 0
7f0025946000-7f0025a09000 rw-p 00000000 00:00 0
7f0025c9b000-7f0025ee3000 rw-p 00000000 00:00 0
7f0025ee3000-7f0025ef8000 r-xp 00000000 08:03 69609115 /usr/local/lib64/libgcc_s.so.1
7f0025ef8000-7f00260f8000 ---p 00015000 08:03 69609115 /usr/local/lib64/libgcc_s.so.1
7f00260f8000-7f00260f9000 rw-p 00015000 08:03 69609115 /usr/local/lib64/libgcc_s.so.1
7f00260f9000-7f00261e4000 r-xp 00000000 08:03 69609156 /usr/local/lib64/libstdc++.so.6.0.18
7f00261e4000-7f00263e3000 ---p 000eb000 08:03 69609156 /usr/local/lib64/libstdc++.so.6.0.18
7f00263e3000-7f00263eb000 r--p 000ea000 08:03 69609156 /usr/local/lib64/libstdc++.so.6.0.18
7f00263eb000-7f00263ed000 rw-p 000f2000 08:03 69609156 /usr/local/lib64/libstdc++.so.6.0.18
7f00263ed000-7f0026403000 rw-p 00000000 00:00 0
7f002642e000-7f0026430000 rw-p 00000000 00:00 0
7ffffc2ca000-7ffffc305000 rw-p 00000000 00:00 0 [stack]
7ffffc3fa000-7ffffc3fb000 r-xp 00000000 00:00 0 [vdso]
ffffffffff600000-ffffffffff601000 r-xp 00000000 00:00 0 [vsyscall]
[FAILED]
Error: Couldn't build bowtie index with err = -6

A quick google suggests this might be due to a contig with all Ns. I will post again if I can find a solution.

https://github.com/ibest/ARC/issues/33

[stickleback]

Problem solved. Repeat masking in the reference sequence meant that some scaffolds consisted of Ns only. Removing these scaffolds, reindexing the edited genome and rerunning the command allowed the transcriptome files to be built without issue.