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Old 06-05-2013, 08:06 PM   #1
Baoqing
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Default cuffmerge error

Hi, All

I was trying to run the cuffmerge for my two samples. However, there is warning message point to the mitochondrial genome, however, i could not figure out the exact problem, could you please give some hints? I directly download the Saccharomyces cerevisiae/Ensembl/EF4, ref genome.

Thank you very much!

bq@bq-VirtualBox:~/Desktop/rnaseq/trimmed$ cuffmerge -g genes.gtf -s genome.fa -p 4 assemblies.txt

[Wed Jun 5 22:46:37 2013] Beginning transcriptome assembly merge
-------------------------------------------

[Wed Jun 5 22:46:37 2013] Preparing output location ./merged_asm/
[Wed Jun 5 22:46:37 2013] Converting GTF files to SAM
[22:46:37] Loading reference annotation.
[22:46:37] Loading reference annotation.
[Wed Jun 5 22:46:37 2013] Quantitating transcripts
You are using Cufflinks v2.1.1, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g genes.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 4 ./merged_asm/tmp/mergeSam_fileDalcVi
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./merged_asm/tmp/mergeSam_fileDalcVi doesn't appear to be a valid BAM file, trying SAM...
[22:46:37] Loading reference annotation.
[22:46:38] Inspecting reads and determining fragment length distribution.
Processed 2290 loci.
> Map Properties:
> Normalized Map Mass: 6966.00
> Raw Map Mass: 6966.00
> Fragment Length Distribution: Truncated Gaussian (default)
> Default Mean: 200
> Default Std Dev: 80
[22:46:38] Assembling transcripts and estimating abundances.
Processed 2290 loci.
[Wed Jun 5 22:46:43 2013] Comparing against reference file genes.gtf
You are using Cufflinks v2.1.1, which is the most recent release.
Warning: couldn't find fasta record for 'Mito'!
[Wed Jun 5 22:46:44 2013] Comparing against reference file genes.gtf
You are using Cufflinks v2.1.1, which is the most recent release.
Warning: couldn't find fasta record for 'Mito'!
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Old 06-06-2013, 03:04 AM   #2
GenoMax
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The fasta sequence for "Mito" is missing from the genome.fa file.
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Old 06-06-2013, 06:20 AM   #3
Baoqing
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Hi, GenoMax

I should have mentioned that i did some initial troubleshooting, you are right that "Mito" is missing in the genome.fa file. It was named as "MT" instead. So I renamed this file, and reran it, the same error occurred again. Maybe something else has to be done besides renaming, or it is a totally different thing?

Best,
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Old 06-06-2013, 08:51 AM   #4
GenoMax
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So the chromosome names in your genes.gtf are not matching what you had in the original reference file (your alignments inherited those names).

Compare these two commands:
Code:
awk -F "\t" '{print $1}' genes.gtf | uniq
and

Code:
cat genome.fa | grep ">"

If that is true then you could rename the "Mito" from the genes.gtf to "MT" and then try a re-run.

Last edited by GenoMax; 06-06-2013 at 08:53 AM.
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Old 06-06-2013, 09:32 PM   #5
Baoqing
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Thank you so much! The error does go away when I rename the "mito" to "MT". It just never came to me that the two files' name are inconsistent. Really appreciate your help!

Baoqing
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