Cuffmerge BAM error

Question

[jenkelly]

Dear forum

I have run cuffmerge on multiple gtf files generated by cufflinks. I am getting the following error:

  [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).
    File cufflinks_frsecondstrand/cuffmerge/trout_cuffmerge/tmp/mergeSam_fileMhPT93 doesn't appear to be a valid BAM file, trying SAM...

but it seems to complete ok, and produces a merged.gtf file at the end. Is this running correctly or is there something wrong with the input gtf files? (see full output below)

Thank you for your help, Kate

[

Thu Apr 28 11:34:15 2016] Preparing output location cufflinks_frsecondstrand/cuffmerge/trout_cuffmerge/
[Thu Apr 28 11:34:19 2016] Converting GTF files to SAM
[11:34:19] Loading reference annotation.
[11:34:21] Loading reference annotation.
[11:34:23] Loading reference annotation.
[11:34:25] Loading reference annotation.
[11:34:26] Loading reference annotation.
[Thu Apr 28 11:34:29 2016] Quantitating transcripts
You are using Cufflinks v2.2.1, which is the most recent release.
Command line:
cufflinks -o cufflinks_frsecondstrand/cuffmerge/trout_cuffmerge/ -F 0.05 -g ref_genomes/Oncorhynchus_mykiss_chr_annot_rename_sort.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 16 cufflinks_frsecondstrand/cuffmerge/trout_cuffmerge/tmp/mergeSam_fileMhPT93 
[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).
File cufflinks_frsecondstrand/cuffmerge/trout_cuffmerge/tmp/mergeSam_fileMhPT93 doesn't appear to be a valid BAM file, trying SAM...
[11:34:29] Loading reference annotation.
[11:34:32] Inspecting reads and determining fragment length distribution.
Processed 44693 loci.                       
> Map Properties:
>   Normalized Map Mass: 232925.00
>   Raw Map Mass: 232925.00
>   Fragment Length Distribution: Truncated Gaussian (default)
>                 Default Mean: 200
>              Default Std Dev: 80
[11:34:34] Assembling transcripts and estimating abundances.
Processed 44693 loci.                       
[Thu Apr 28 11:35:44 2016] Comparing against reference file ref_genomes/Oncorhynchus_mykiss_chr_annot_rename_sort.gtf
You are using Cufflinks v2.2.1, which is the most recent release.
[Thu Apr 28 11:36:06 2016] Comparing against reference file ref_genomes/Oncorhynchus_mykiss_chr_annot_rename_sort.gtf
You are using Cufflinks v2.2.1, which is the most recent release.

[Benn]

It seems to complain about the bam file, which is probably not a bam file but a sam file.

Did you use a sam file instead of bam? If so, the program automatically tried sam after it discovered it was not a bam (I don't know if this is a real error, seems more like a warning).

[jenkelly]

Sorry I should have also posted the command I used:

cuffmerge -p 16 -o cufflinks_frsecondstrand/cuffmerge/trout_cuffmerge -g ref_genomes/Oncorhynchus_mykiss_chr_annot_rename_sort.gtf -s ref_genomes/bowtie_index/rainbow_trout.fa cufflinks_frsecondstrand/cuffmerge/trout_gft_list.txt

As you can see there was no BAM or SAM given as an input file.

[Benn]

You're right, there is no sam or bam file needed for cuffmerge. I don't know why it is complaining about it.

You say that you get right results, so I would ignore the warning about the bam and sam files...