Question: Cuffmerge Error
1
gravatar for mad.cichlids
5.9 years ago by
mad.cichlids100
Texas
mad.cichlids100 wrote:

Hi, All

I am running into a problem while I was executing the cuffmerge. According to error message, it seems i missed some index for the mitochondria gene index, and the bam file is invalid. But When i check the ref genome i downloaded, it seems everything is fine. Any thoughts or ideas will be much appreciated!

bq@bq-VirtualBox:~/Desktop/rnaseq/trimmed$ cuffmerge -g genes.gtf -s genome.fa -p 2 assemblies.txt 
[Mon Jun  3 23:44:55 2013] Beginning transcriptome assembly merge
-------------------------------------------
[Mon Jun  3 23:44:55 2013] Preparing output location ./merged_asm/
[Mon Jun  3 23:44:55 2013] Converting GTF files to SAM
[23:44:55] Loading reference annotation.
[23:44:55] Loading reference annotation.
[Mon Jun  3 23:44:55 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 2 ./merged_asm/tmp/mergeSam_file76O1Gs 

[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_file76O1Gs doesn't appear to be a valid BAM file, trying SAM...
[23:44:56] Loading reference annotation.
[23:44:56] Inspecting reads and determining fragment length distribution.
Processed 2316 loci.                        
> Map Properties:
>    Normalized Map Mass: 7048.00
>    Raw Map Mass: 7048.00
>    Fragment Length Distribution: Truncated Gaussian (default)
>                  Default Mean: 200
>               Default Std Dev: 80
[23:44:56] Assembling transcripts and estimating abundances.
Processed 2316 loci.                        
[Mon Jun  3 23:45:05 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'!
[Mon Jun  3 23:45:06 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'
cuffmerge rna-seq • 4.0k views
ADD COMMENTlink modified 5.9 years ago by Neilfws48k • written 5.9 years ago by mad.cichlids100
1

what's the exact name for the mitochondrial sequence in your fasta ?

ADD REPLYlink written 5.9 years ago by Pierre Lindenbaum119k

Hi, Pierre

I downloaded the ref genome of S. cerevisiae and opened the genome.fa file, the name of the mitochondria genome is "MT", hopefully this is the info you need. Really appreciate your help!

Best,

ADD REPLYlink modified 5.9 years ago • written 5.9 years ago by mad.cichlids100

ah, might be the naming issue, but i went into the genome.fa file and changed the name "MT" to "Mito" The problem still did not go away. Do you think I should rerun the tophat with the re-edited genome ("Mito" instead of "MT") and see if it works? since the tophat used the ref genome to align the read, it might consequentially gave the name to all of the reads name "MT" instead of "Mito"? Anyway I can troubleshoot this?

ADD REPLYlink modified 5.9 years ago • written 5.9 years ago by mad.cichlids100

if your genome is indexed just changing the name in the fasta reference is not enough (and will cause some errors)

ADD REPLYlink written 5.9 years ago by Pierre Lindenbaum119k

ok, so the problem could be in the bowtie2index file i was using?

any solutions or suggestions on this?

do i have to reindex the genome?

I thought the ref genome downloaded from the Tophat website should work, however, this is apparently not the case. thanks!

ADD REPLYlink modified 5.9 years ago • written 5.9 years ago by mad.cichlids100

Ok. it turned out the mitochondrial name is different from the genome and gene annotation files, after changed the name, everything works, by the way, i was using the ensemble ref genome.

ADD REPLYlink written 5.9 years ago by mad.cichlids100
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