TOPHAT EMPTY accepted_hits.bam ISSUE

[waterboy]

Folks..!

I've been running tophat since a couple of weeks and I've seen no success so far
I have colorspace transcriptome data having 97 million reads. I've built Bowtie colorspace index as well. When I run tophat everything works fine, no error displays. The run completed perfectly all right; however, the output accepted_hits.bam file is COMPLETELY empty. The junctions.bed file is fine and is of 1.7 MB.

On the other hand if I take 500,000 reads dataset from this same data, there is no error at all and it gives bam file that too not empty..!! and I'm running it on cluster head node with 64GB RAM so it should not be a memory problem I guess.

Here is the command prompt and output display that tophat shows:

$ tophat -G refgene_main.gtf --color --quals /color_index/oceano_c Solid97M_F3.csfasta Solid97M_F3_QV.qual

[Mon Nov 15 18:14:47 2010] Beginning TopHat run (v1.1.2)
-----------------------------------------------
[Mon Nov 15 18:14:47 2010] Preparing output location ./tophat_out/
[Mon Nov 15 18:14:47 2010] Checking for Bowtie index files
[Mon Nov 15 18:14:47 2010] Checking for reference FASTA file
[Mon Nov 15 18:14:47 2010] Checking for Bowtie
Bowtie version: 0.12.7.0
[Mon Nov 15 18:14:47 2010] Checking for Samtools
Samtools version: 0.1.9.0
[Mon Nov 15 18:14:47 2010] Checking reads
min read length: 50bp, max read length: 50bp
format: fasta
[Mon Nov 15 18:22:18 2010] Reading known junctions from GFF file
Warning: TopHat did not find any junctions in GFF file
[Mon Nov 15 18:46:59 2010] Mapping reads against oceano_c with Bowtie
[Mon Nov 15 20:47:35 2010] Joining segment hits
[Mon Nov 15 21:32:26 2010] Mapping reads against oceano_c with Bowtie(1/2)
[Mon Nov 15 22:10:52 2010] Mapping reads against oceano_c with Bowtie(2/2)
[Mon Nov 15 22:45:59 2010] Searching for junctions via segment mapping
[Mon Nov 15 23:10:36 2010] Retrieving sequences for splices
[Mon Nov 15 23:10:51 2010] Indexing splices
[Mon Nov 15 23:30:30 2010] Mapping reads against segment_juncs with Bowtie
[Tue Nov 16 02:11:44 2010] Mapping reads against segment_juncs with Bowtie
[Tue Nov 16 03:58:20 2010] Joining segment hits
[Tue Nov 16 04:34:25 2010] Reporting output tracks
-----------------------------------------------
Run complete [11:07:08 elapsed]


Looks so good, isn't it?

the logs folder is fine except one file "accepted_hits_sam_to_bam.log" where it shows "fail to open "./tophat_out/tmp/accepted_hits.sam" for reading" even though the folder has chmod 777 permissions ???????


Any help please?

Any help is much appreciated

[Daehwan]

I just sent you an email. One thing you can try is to use "--keep-tmp" option not to delete the temporary files including tmp/accepted_hit.sam, and taking a look at them will probably give you some hints about what's going on there.