tophat & samtools: accepted_hits0_sorted.0000.bam

[esiefker]

I am encountering an error when running tophat v2.0.8b with samtools 0.1.19+. At the end of a run, during tophat_reports, I get the following error:

Error: [Errno 2] No such file or directory: 'EGFR-tophat/tmp/accepted_hits0_sorted.bam'

The directory 'EGFR-tophat/tmp' has a bunch of files that look like this:

accepted_hits0_sorted.0000.bam
accepted_hits0_sorted.0001.bam
accepted_hits0_sorted.0002.bam
..
accepted_hits7_sorted.0004.bam
accepted_hits7_sorted.0005.bam

On checking 'run.log', it appears that these files are being created with samtools, invoked like this:

/usr/local/bin/samtools sort EGFR-tophat/tmp/accepted_hits0.bam EGFR-tophat/tmp/accepted_hits0_sorted

And the error is being thrown by samtools, invode like this:

/usr/local/bin/samtools merge -f -h EGFR-tophat/tmp/Homo_sapiens_GRCh37_71_genome.bwt.samheader.sam EGFR-tophat/accepted_hits.bam EGFR-tophat/tmp/accepted_hits0_sorted.bam EGFR-tophat/tmp/accepted_hits1_sorted.bam EGFR-tophat/tmp/accepted_hits2_sorted.bam EGFR-tophat/tmp/accepted_hits3_sorted.bam EGFR-tophat/tmp/accepted_hits4_sorted.bam EGFR-tophat/tmp/accepted_hits5_sorted.bam EGFR-tophat/tmp/accepted_hits6_sorted.bam EGFR-tophat/tmp/accepted_hits7_sorted.bam

Checking the samtools manual, I see:

This command may also create temporary files <out.prefix>.%d.bam when  the  whole  alignment  cannot  be fitted into memory (controlled by option -m).

Apparently tophat is not aware that samtools may segment the alignments, and fails to properly merge them. Is there a way to direct tophat to do this? Should I just use --no-sort-bam with tophat? What are the implications of an unsorted bam?

Meanwhile, can I merge these semented alignments myself and resume tophat? Something like:

samtools merge accepted_hits0_sorted.bam accepted_hits0_sorted.*.bam

and then resume tophat with -R? Since memory was a limit during the sort, will I run out of memory during the merge? Is there a reason samtools doesn't automatically merge the segmented alignments?

[esiefker]

Meanwhile, can I merge these semented alignments myself and resume tophat? Something like:

samtools merge accepted_hits0_sorted.bam accepted_hits0_sorted.*.bam

and then resume tophat with -R?

I tried this, it complained about truncated files.

[ebs15242@soresearch tmp]$ samtools merge accepted_hits0_sorted.bam accepted_hits0_sorted.*.bam
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
Segmentation fault
[ebs15242@soresearch tmp]$ du -hsc !$
du -hsc accepted_hits0_sorted.*.bam
228M accepted_hits0_sorted.0000.bam
226M accepted_hits0_sorted.0001.bam
177M accepted_hits0_sorted.0002.bam
236K accepted_hits0_sorted.0003.bam
16K accepted_hits0_sorted.0004.bam
0 accepted_hits0_sorted.0005.bam
0 accepted_hits0_sorted.0006.bam
630M total

Why is samtools outputting truncated files? Should I pass it the -m flag? How do I instruct tophat to do that?

[esiefker]

Oh, never mind. I was running out of disk space. I was not aware, because tophat removed some temporary files after samtools failed. Making space has gotten me past this error. Hope this experience can help someone else...

[NKAkers]

Dear esiefker,

I've just encountered the same error, your post was indeed a big help for me. Thanks!