Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

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

    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'!

  • #2
    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.

    Comment


    • #3
      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

      Comment


      • #4
        The following script appears to be working, but whats the difference between a GFF and GTF file?
        Cufflinks documentation

        Comment


        • #5
          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'!

          Comment


          • #6
            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!

            Comment


            • #7
              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?

              Comment


              • #8
                I have the same problem, How you solve it?






                Originally posted by Richard Barker View Post
                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'!

                Comment


                • #9
                  I used the pipeline that was made in the CyVerse Discovery environment. It's easy to use and really fast!

                  Comment


                  • #10
                    Can you tell me what that pipeline is, do not I know it?
                    regards

                    Comment


                    • #11
                      They have the HTprocess and Kalisto if you're in a rush

                      Comment


                      • #12
                        But what was the problem, the inputs gff anda fasta? I need the output merged.gtf without warnings

                        Comment


                        • #13
                          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.

                          Comment


                          • #14
                            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

                            Comment

                            Latest Articles

                            Collapse

                            • seqadmin
                              Choosing Between NGS and qPCR
                              by seqadmin



                              Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...
                              10-18-2024, 07:11 AM
                            • seqadmin
                              Non-Coding RNA Research and Technologies
                              by seqadmin




                              Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.

                              Nobel Prize for MicroRNA Discovery
                              This week,...
                              10-07-2024, 08:07 AM

                            ad_right_rmr

                            Collapse

                            News

                            Collapse

                            Topics Statistics Last Post
                            Started by seqadmin, 11-01-2024, 06:09 AM
                            0 responses
                            12 views
                            0 likes
                            Last Post seqadmin  
                            Started by seqadmin, 10-30-2024, 05:31 AM
                            0 responses
                            14 views
                            0 likes
                            Last Post seqadmin  
                            Started by seqadmin, 10-24-2024, 06:58 AM
                            0 responses
                            24 views
                            0 likes
                            Last Post seqadmin  
                            Started by seqadmin, 10-23-2024, 08:43 AM
                            0 responses
                            52 views
                            0 likes
                            Last Post seqadmin  
                            Working...
                            X