Error in cuff merge? Problem in assemblies to create a single merged transcriptome annotation?

Question

[harshraje19]

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

[dsull]

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.).

[Kevin Blighe]

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

[harshraje19]

Thank you for your suggestions.

[harshraje19]

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

[Kevin Blighe]

I have posted the answer to this specific question below.

Answer 1

[Kevin Blighe]

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