Cuffmerge error coultn't finda data file for Mt or Pt?

[Richard Barker]

I'm tring to merge my transcript data produced from 8 samples analysed using TopHat and compiled using Cufflinks. Unfortunately when i run cuffmerge I get an error message about "missing fasta data files for 'Mt' and 'Pt'" but can't find reference to these files anywhere else in the literature or on-line?
Any advice greatfully received.

richard@ubuntu:~/RNA_seq_analysis/Cuffmerge$ cuffmerge -g arabidopsis_thaliana.TAIR10.60.gtf -s TAIR10_chr_all.fas -p 6 run297_transcript_cuffmerge.txt

[Thu Aug 9 15:36:29 2012] Beginning transcriptome assembly merge
-------------------------------------------

[Thu Aug 9 15:36:29 2012] Preparing output location ./merged_asm/
[Thu Aug 9 15:36:36 2012] Converting GTF files to SAM
[15:36:36] Loading reference annotation.
[15:36:37] Loading reference annotation.
[15:36:38] Loading reference annotation.
[15:36:39] Loading reference annotation.
[15:36:40] Loading reference annotation.
[15:36:41] Loading reference annotation.
[15:36:42] Loading reference annotation.
[15:36:44] Loading reference annotation.
[Thu Aug 9 15:36:45 2012] Quantitating transcripts
You are using Cufflinks v2.0.2, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g arabidopsis_thaliana.TAIR10.60.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 6 ./merged_asm/tmp/mergeSam_filejHHJWI
[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_filejHHJWI doesn't appear to be a valid BAM file, trying SAM...
[15:36:45] Loading reference annotation.
[15:36:47] Inspecting reads and determining fragment length distribution.
Processed 26332 loci.
> Map Properties:
> Normalized Map Mass: 194074.00
> Raw Map Mass: 194074.00
> Fragment Length Distribution: Truncated Gaussian (default)
> Default Mean: 200
> Default Std Dev: 80
[15:36:48] Assembling transcripts and estimating abundances.
Processed 26332 loci.
[Thu Aug 9 15:44:22 2012] Comparing against reference file arabidopsis_thaliana.TAIR10.60.gtf
You are using Cufflinks v2.0.2, which is the most recent release.
No fasta index found for TAIR10_chr_all.fas. Rebuilding, please wait..
Fasta index rebuilt.
Warning: couldn't find fasta record for 'Mt'!
Warning: couldn't find fasta record for 'Pt'!
[Thu Aug 9 15:44:34 2012] Comparing against reference file arabidopsis_thaliana.TAIR10.60.gtf
You are using Cufflinks v2.0.2, which is the most recent release.
Warning: couldn't find fasta record for 'Mt'!
Warning: couldn't find fasta record for 'Pt'!

[kmcarr]

Richard,

This is most likely caused by a mismatch between the reference names between the fasta file and the gtf file you are using.

The official TAIR10 genome sequence release names the mitochondrial and plastid (chloroplast) chromsomes ChrM and ChrC respectively. These are the names used in TAIR10_chr_all.fas. It would appear that your reference GTF file, arabidopsis_thaliana.TAIR10.60.gtf, is from a different source and uses different names (Mt and Pt). You will need to make sure the chromosome names match exactly between your FASTA and GTF files.

[Richard Barker]

Thanks for the swift response (again)

Your advice worked perfectly, i searched the directories near where i downloaded the TAIR10.fasta file and found a TAIR10_GFF3_genes.gff file.

The following script appears to be working, but whats the difference between a GFF and GTF file?

cuffmerge -g TAIR10_GFF3_genes -s TAIR10_chr_all.fas -p 6 run297_transcript_cuffmerge.txt

[kmcarr]

The following script appears to be working, but whats the difference between a GFF and GTF file?

Cufflinks documentation

[Richard Barker]

Ooops spoke too soon now i get the following error message?

richard@ubuntu:~/RNA_seq_analysis/Cuffmerge$ cuffmerge -g TAIR10_GFF3_genes.gff -s TAIR10_chr_all.fas -p 6 run297_transcript_cuffmerge.txt

[Fri Aug 10 07:41:48 2012] Beginning transcriptome assembly merge
-------------------------------------------

[Fri Aug 10 07:41:48 2012] Preparing output location ./merged_asm/
[Fri Aug 10 07:41:52 2012] Converting GTF files to SAM
[07:41:52] Loading reference annotation.
[07:41:53] Loading reference annotation.
[07:41:54] Loading reference annotation.
[07:41:56] Loading reference annotation.
[07:41:57] Loading reference annotation.
[07:41:58] Loading reference annotation.
[07:41:59] Loading reference annotation.
[07:42:00] Loading reference annotation.
[Fri Aug 10 07:42:02 2012] Quantitating transcripts
You are using Cufflinks v2.0.2, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g TAIR10_GFF3_genes.gff -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 6 ./merged_asm/tmp/mergeSam_filefWraGs
[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_filefWraGs doesn't appear to be a valid BAM file, trying SAM...
[07:42:02] Loading reference annotation.
[07:42:03] Inspecting reads and determining fragment length distribution.
Processed 47416 loci.
> Map Properties:
> Normalized Map Mass: 194074.00
> Raw Map Mass: 194074.00
> Fragment Length Distribution: Truncated Gaussian (default)
> Default Mean: 200
> Default Std Dev: 80
[07:42:05] Assembling transcripts and estimating abundances.
Processed 47416 loci.
[Fri Aug 10 07:53:04 2012] Comparing against reference file TAIR10_GFF3_genes.gff
You are using Cufflinks v2.0.2, which is the most recent release.
Warning: couldn't find fasta record for 'Chr1'!
Warning: couldn't find fasta record for 'Chr2'!
Warning: couldn't find fasta record for 'Chr3'!
Warning: couldn't find fasta record for 'Chr4'!
Warning: couldn't find fasta record for 'Chr5'!
Warning: couldn't find fasta record for 'ChrC'!
Warning: couldn't find fasta record for 'ChrM'!
[Fri Aug 10 07:53:20 2012] Comparing against reference file TAIR10_GFF3_genes.gff
You are using Cufflinks v2.0.2, which is the most recent release.
Warning: couldn't find fasta record for 'Chr1'!
Warning: couldn't find fasta record for 'Chr2'!
Warning: couldn't find fasta record for 'Chr3'!
Warning: couldn't find fasta record for 'Chr4'!
Warning: couldn't find fasta record for 'Chr5'!
Warning: couldn't find fasta record for 'ChrC'!
Warning: couldn't find fasta record for 'ChrM'!

[Richard Barker]

I've found a TAIR10_GFF file (ftp://ftp.arabidopsis.org/home/tair/...enome_release/) which was also near the location where i downloaded my genome fasta file (ftp://ftp.arabidopsis.org/home/tair/...omosome_files/) and one was able to completed the alignment!
Thanks for your help!

[Richard Barker]

Shouldn't the cuffmerge out put have the gene names (Arabidopsis ATG codes?). What methods are there for adding your genome annotation, i thought that was the reason for using the GFF/gtf files during TopHat and/or cuffmerge?

[shinigam123]

I have the same problem, How you solve it?

Ooops spoke too soon now i get the following error message?

richard@ubuntu:~/RNA_seq_analysis/Cuffmerge$ cuffmerge -g TAIR10_GFF3_genes.gff -s TAIR10_chr_all.fas -p 6 run297_transcript_cuffmerge.txt

[Fri Aug 10 07:41:48 2012] Beginning transcriptome assembly merge
-------------------------------------------

[Fri Aug 10 07:41:48 2012] Preparing output location ./merged_asm/
[Fri Aug 10 07:41:52 2012] Converting GTF files to SAM
[07:41:52] Loading reference annotation.
[07:41:53] Loading reference annotation.
[07:41:54] Loading reference annotation.
[07:41:56] Loading reference annotation.
[07:41:57] Loading reference annotation.
[07:41:58] Loading reference annotation.
[07:41:59] Loading reference annotation.
[07:42:00] Loading reference annotation.
[Fri Aug 10 07:42:02 2012] Quantitating transcripts
You are using Cufflinks v2.0.2, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g TAIR10_GFF3_genes.gff -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 6 ./merged_asm/tmp/mergeSam_filefWraGs
[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_filefWraGs doesn't appear to be a valid BAM file, trying SAM...
[07:42:02] Loading reference annotation.
[07:42:03] Inspecting reads and determining fragment length distribution.
Processed 47416 loci.
> Map Properties:
> Normalized Map Mass: 194074.00
> Raw Map Mass: 194074.00
> Fragment Length Distribution: Truncated Gaussian (default)
> Default Mean: 200
> Default Std Dev: 80
[07:42:05] Assembling transcripts and estimating abundances.
Processed 47416 loci.
[Fri Aug 10 07:53:04 2012] Comparing against reference file TAIR10_GFF3_genes.gff
You are using Cufflinks v2.0.2, which is the most recent release.
Warning: couldn't find fasta record for 'Chr1'!
Warning: couldn't find fasta record for 'Chr2'!
Warning: couldn't find fasta record for 'Chr3'!
Warning: couldn't find fasta record for 'Chr4'!
Warning: couldn't find fasta record for 'Chr5'!
Warning: couldn't find fasta record for 'ChrC'!
Warning: couldn't find fasta record for 'ChrM'!
[Fri Aug 10 07:53:20 2012] Comparing against reference file TAIR10_GFF3_genes.gff
You are using Cufflinks v2.0.2, which is the most recent release.
Warning: couldn't find fasta record for 'Chr1'!
Warning: couldn't find fasta record for 'Chr2'!
Warning: couldn't find fasta record for 'Chr3'!
Warning: couldn't find fasta record for 'Chr4'!
Warning: couldn't find fasta record for 'Chr5'!
Warning: couldn't find fasta record for 'ChrC'!
Warning: couldn't find fasta record for 'ChrM'!

[Richard Barker]

I used the pipeline that was made in the CyVerse Discovery environment. It's easy to use and really fast!

[shinigam123]

Can you tell me what that pipeline is, do not I know it?
regards

[Richard Barker]

They have the HTprocess and Kalisto if you're in a rush

[shinigam123]

But what was the problem, the inputs gff anda fasta? I need the output merged.gtf without warnings

[vivekkeshri]

Cuffmerge output

I am trying to execute Cuffmerge (cuffmerge -p 5 -g Homo.gtf assemblies.txt), but unable to get FPKM values in output file ("merged.gtf).
Please let me know how to solve this problem.

[vivekkeshri]

Please let me know about how "Cuffdiff -L" [-L/--labels: comma-separated list of condition labels] command works. How it is labeling / merging the bam files.
Thanks