Question: Error in cuff merge? Problem in assemblies to create a single merged transcriptome annotation?
0
gravatar for harshraje19
11 months ago by
harshraje1930
harshraje1930 wrote:

Hello everyone, I am performing the RNA-Seq data analysis in linux. I have successfully mapped reads to the genome using the tophat, and also prepared the transcript assembly using the cufflink. But whenever, I am running the cuffmerge on all assemblies to create a single merged transcriptome annotation (using this code- cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt) I am getting following error.

harshraj@harshraj-XPS-8930:~/ArabidopsisExpt$ cuffmerge -g annotation.gff3 -s genome.fa -p 8 assemblies.txt

[Sun Dec 22 19:03:52 2019] Beginning transcriptome assembly merge
-------------------------------------------

[Sun Dec 22 19:03:52 2019] Preparing output location ./merged_asm/
[Sun Dec 22 19:04:06 2019] Converting GTF files to SAM
[19:04:06] Loading reference annotation.
[19:04:07] Loading reference annotation.
[19:04:07] Loading reference annotation.
[19:04:08] Loading reference annotation.
[Sun Dec 22 19:04:09 2019] Quantitating transcripts
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g annotation.gff3 -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 8 ./merged_asm/tmp/mergeSam_fileHvRpOD 
[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 ./merged_asm/tmp/mergeSam_fileHvRpOD doesn't appear to be a valid BAM file, trying SAM...
[19:04:09] Loading reference annotation.
[19:04:11] Inspecting reads and determining fragment length distribution.
Processed 25108 loci.                       
> Map Properties:
> Normalized Map Mass: 105564.00
> Raw Map Mass: 105564.00
> Fragment Length Distribution: Truncated Gaussian (default)
>              Default Mean: 200
>           Default Std Dev: 80
[19:04:12] Assembling transcripts and estimating abundances.
Processed 25108 loci.                       
[Sun Dec 22 19:04:30 2019] Comparing against reference file annotation.gff3
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
[Sun Dec 22 19:04:38 2019] Comparing against reference file annotation.gff3
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).

Can anyone help to solve this error Thank you in advance Harshraj

rna-seq alignment assembly • 306 views
ADD COMMENTlink modified 11 months ago by Kevin Blighe67k • written 11 months ago by harshraje1930
1

Not an answer but: Please replace your tophat+cufflinks pipeline. Those tools are antiquated and there are far better ones out there today (STAR, kallisto, etc.).

ADD REPLYlink written 11 months ago by dsull1.7k

What is the content of assemblies.txt? Also note the comment of dsull

ADD REPLYlink written 11 months ago by Kevin Blighe67k

Thank you for your suggestions.

ADD REPLYlink modified 11 months ago • written 11 months ago by harshraje1930

Can you suggest me the recent and best pipeline for RNA seq data analysis

ADD REPLYlink modified 11 months ago • written 11 months ago by harshraje1930

I have posted the answer to this specific question below.

ADD REPLYlink written 11 months ago by Kevin Blighe67k
0
gravatar for Kevin Blighe
11 months ago by
Kevin Blighe67k
Republic of Ireland
Kevin Blighe67k wrote:

For cuffmerge, your assemblies.txt file must contain GTF files. Please see the manual page.

ADD COMMENTlink written 11 months ago by Kevin Blighe67k
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