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  • genome1
    Member
    • Oct 2013
    • 12
    #1

    Warning in cuffmerge

    The problem that I am facing is not unique one in the sense that I have seen people to face similar kind of problem from earlier posts. But unfortunately I didn't get any proper solutions from those posts.
    So my problem is like that:
    I am using Cufflinks v2.2.1. I have downloaded human (GRCh37) Bowtie2 index files from iGenome package of Ensembl. I have downloaded separately the GTF file from Ensembl (Homo_sapiens.GRCh37.75.gtf).
    Now after getting accepted_hits.bam files from TopHat2, I moved on to cufflinks (which ran perfectly) for two different samples and thereafter used cuffmerge.
    But while running cuffmerge I am getting warning messages, though I am able to get single merged.gtf file after completion of cuffmerge run.

    My command line for cuffmerge is:
    $ cuffmerge -o /home/cuffmerge_out -g /home/Homo_sapiens.GRCh37.75.gtf -s /home/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome.fa -p 4 /home/assemblies.txt

    Last few lines showing the warning messages are as follows:
    > Map Properties:
    > Normalized Map Mass: 540321.00
    > Raw Map Mass: 540321.00
    > Fragment Length Distribution: Truncated Gaussian (default)
    > Default Mean: 200
    > Default Std Dev: 80
    [02:31:35] Assembling transcripts and estimating abundances.

    6:126102306-130686570 Warning: Skipping large bundle.
    Processed 47147 loci.
    [Thu May 15 02:48:37 2014] Comparing against reference file /home/Homo_sapiens.GRCh37.75.gtf
    You are using Cufflinks v2.2.1, which is the most recent release.
    Warning: couldn't find fasta record for 'GL000191.1'!
    Warning: couldn't find fasta record for 'GL000192.1'!
    Warning: couldn't find fasta record for 'GL000193.1'!
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    Warning: couldn't find fasta record for 'GL000209.1'!
    Warning: couldn't find fasta record for 'GL000211.1'!
    Warning: couldn't find fasta record for 'GL000212.1'!
    Warning: couldn't find fasta record for 'GL000213.1'!
    Warning: couldn't find fasta record for 'GL000215.1'!
    Warning: couldn't find fasta record for 'GL000216.1'!
    Warning: couldn't find fasta record for 'GL000218.1'!
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    Warning: couldn't find fasta record for 'HG1007_PATCH'!
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    Warning: couldn't find fasta record for 'HG1063_PATCH'!
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    Warning: couldn't find fasta record for 'HG1350_HG959_PATCH'!
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    Warning: couldn't find fasta record for 'HG142_HG150_NOVEL_TEST'!
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    Warning: couldn't find fasta record for 'HSCHR10_1_CTG2'!
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    Warning: couldn't find fasta record for 'HSCHR2_1_CTG12'!
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    Warning: couldn't find fasta record for 'HSCHR3_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR3_1_CTG2_1'!
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    Warning: couldn't find fasta record for 'HSCHR4_1_CTG6'!
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    Warning: couldn't find fasta record for 'HSCHR5_1_CTG1'!
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    Warning: couldn't find fasta record for 'HSCHR5_1_CTG5'!
    Warning: couldn't find fasta record for 'HSCHR5_2_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR5_3_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR6_1_CTG5'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_APD'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_COX'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_DBB'!
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    Warning: couldn't find fasta record for 'HSCHR6_MHC_MCF'!
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    [Thu May 15 02:50:55 2014] Comparing against reference file /home/Homo_sapiens.GRCh37.75.gtf
    You are using Cufflinks v2.2.1, which is the most recent release.
    Warning: couldn't find fasta record for 'GL000191.1'!
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    Warning: couldn't find fasta record for 'HG281_PATCH'!
    Warning: couldn't find fasta record for 'HG299_PATCH'!
    Warning: couldn't find fasta record for 'HG29_PATCH'!
    Warning: couldn't find fasta record for 'HG305_PATCH'!
    Warning: couldn't find fasta record for 'HG306_PATCH'!
    Warning: couldn't find fasta record for 'HG311_PATCH'!
    Warning: couldn't find fasta record for 'HG325_PATCH'!
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    Warning: couldn't find fasta record for 'HG339_PATCH'!
    Warning: couldn't find fasta record for 'HG344_PATCH'!
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    Warning: couldn't find fasta record for 'HG375_PATCH'!
    Warning: couldn't find fasta record for 'HG385_PATCH'!
    Warning: couldn't find fasta record for 'HG388_HG400_PATCH'!
    Warning: couldn't find fasta record for 'HG414_PATCH'!
    Warning: couldn't find fasta record for 'HG417_PATCH'!
    Warning: couldn't find fasta record for 'HG418_PATCH'!
    Warning: couldn't find fasta record for 'HG444_PATCH'!
    Warning: couldn't find fasta record for 'HG480_HG481_PATCH'!
    Warning: couldn't find fasta record for 'HG497_PATCH'!
    Warning: couldn't find fasta record for 'HG506_HG507_HG1000_PATCH'!
    Warning: couldn't find fasta record for 'HG50_PATCH'!
    Warning: couldn't find fasta record for 'HG531_PATCH'!
    Warning: couldn't find fasta record for 'HG536_PATCH'!
    Warning: couldn't find fasta record for 'HG544_PATCH'!
    Warning: couldn't find fasta record for 'HG686_PATCH'!
    Warning: couldn't find fasta record for 'HG706_PATCH'!
    Warning: couldn't find fasta record for 'HG729_PATCH'!
    Warning: couldn't find fasta record for 'HG730_PATCH'!
    Warning: couldn't find fasta record for 'HG736_PATCH'!
    Warning: couldn't find fasta record for 'HG745_PATCH'!
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    Warning: couldn't find fasta record for 'HG75_PATCH'!
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    Warning: couldn't find fasta record for 'HG858_PATCH'!
    Warning: couldn't find fasta record for 'HG865_PATCH'!
    Warning: couldn't find fasta record for 'HG871_PATCH'!
    Warning: couldn't find fasta record for 'HG873_PATCH'!
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    Warning: couldn't find fasta record for 'HG905_PATCH'!
    Warning: couldn't find fasta record for 'HG944_PATCH'!
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    Warning: couldn't find fasta record for 'HG953_PATCH'!
    Warning: couldn't find fasta record for 'HG957_PATCH'!
    Warning: couldn't find fasta record for 'HG962_PATCH'!
    Warning: couldn't find fasta record for 'HG971_PATCH'!
    Warning: couldn't find fasta record for 'HG979_PATCH'!
    Warning: couldn't find fasta record for 'HG987_PATCH'!
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    Warning: couldn't find fasta record for 'HG996_PATCH'!
    Warning: couldn't find fasta record for 'HG998_1_PATCH'!
    Warning: couldn't find fasta record for 'HG998_2_PATCH'!
    Warning: couldn't find fasta record for 'HG999_1_PATCH'!
    Warning: couldn't find fasta record for 'HG999_2_PATCH'!
    Warning: couldn't find fasta record for 'HSCHR10_1_CTG2'!
    Warning: couldn't find fasta record for 'HSCHR10_1_CTG5'!
    Warning: couldn't find fasta record for 'HSCHR12_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR12_1_CTG2_1'!
    Warning: couldn't find fasta record for 'HSCHR12_1_CTG5'!
    Warning: couldn't find fasta record for 'HSCHR12_2_CTG2'!
    Warning: couldn't find fasta record for 'HSCHR12_2_CTG2_1'!
    Warning: couldn't find fasta record for 'HSCHR12_3_CTG2_1'!
    Warning: couldn't find fasta record for 'HSCHR15_1_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR15_1_CTG8'!
    Warning: couldn't find fasta record for 'HSCHR16_1_CTG3_1'!
    Warning: couldn't find fasta record for 'HSCHR16_2_CTG3_1'!
    Warning: couldn't find fasta record for 'HSCHR17_1'!
    Warning: couldn't find fasta record for 'HSCHR17_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR17_1_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR17_2_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR17_3_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR17_4_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR17_5_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR17_6_CTG4'!
    Warning: couldn't find fasta record for 'HSCHR18_1_CTG1_1'!
    Warning: couldn't find fasta record for 'HSCHR18_1_CTG2_1'!
    Warning: couldn't find fasta record for 'HSCHR18_2_CTG2'!
    Warning: couldn't find fasta record for 'HSCHR18_2_CTG2_1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_COX1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_COX2_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_LRC_I_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_LRC_J_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_LRC_S_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_LRC_T_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_PGF1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19LRC_PGF2_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR19_1_CTG3'!
    Warning: couldn't find fasta record for 'HSCHR19_1_CTG3_1'!
    Warning: couldn't find fasta record for 'HSCHR19_2_CTG3'!
    Warning: couldn't find fasta record for 'HSCHR19_3_CTG3'!
    Warning: couldn't find fasta record for 'HSCHR1_1_CTG31'!
    Warning: couldn't find fasta record for 'HSCHR1_2_CTG31'!
    Warning: couldn't find fasta record for 'HSCHR1_3_CTG31'!
    Warning: couldn't find fasta record for 'HSCHR20_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR21_2_CTG1_1'!
    Warning: couldn't find fasta record for 'HSCHR21_3_CTG1_1'!
    Warning: couldn't find fasta record for 'HSCHR21_4_CTG1_1'!
    Warning: couldn't find fasta record for 'HSCHR22_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR22_1_CTG2'!
    Warning: couldn't find fasta record for 'HSCHR22_2_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR2_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR2_1_CTG12'!
    Warning: couldn't find fasta record for 'HSCHR2_2_CTG12'!
    Warning: couldn't find fasta record for 'HSCHR3_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR3_1_CTG2_1'!
    Warning: couldn't find fasta record for 'HSCHR4_1'!
    Warning: couldn't find fasta record for 'HSCHR4_1_CTG12'!
    Warning: couldn't find fasta record for 'HSCHR4_1_CTG6'!
    Warning: couldn't find fasta record for 'HSCHR4_2_CTG9'!
    Warning: couldn't find fasta record for 'HSCHR5_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR5_1_CTG2'!
    Warning: couldn't find fasta record for 'HSCHR5_1_CTG5'!
    Warning: couldn't find fasta record for 'HSCHR5_2_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR5_3_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR6_1_CTG5'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_APD'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_COX'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_DBB'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_MANN'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_MCF'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_QBL'!
    Warning: couldn't find fasta record for 'HSCHR6_MHC_SSTO'!
    Warning: couldn't find fasta record for 'HSCHR7_1_CTG6'!
    Warning: couldn't find fasta record for 'HSCHR9_1_CTG1'!
    Warning: couldn't find fasta record for 'HSCHR9_1_CTG35'!
    Warning: couldn't find fasta record for 'HSCHR9_2_CTG35'!
    Warning: couldn't find fasta record for 'HSCHR9_3_CTG35'!

    I have also tried using gtf file provided in iGenome package: /home/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf
    But in both cases getting same warning messages.
    Can anyone help me to solve this out. Perhaps it might also be beneficial for many other people facing similar kind of problem.
    Thank you.
    Tags: cufflinks-2.2.1, cuffmerge, rna-seq
    • AddDNAse
      Junior Member
      • May 2014
      • 3
      #2
      Originally posted by genome1 View Post
      The problem that I am facing is not unique one in the sense that I have seen people to face similar kind of problem from earlier posts. But unfortunately I didn't get any proper solutions from those posts.
      So my problem is like that:
      I am using Cufflinks v2.2.1. I have downloaded human (GRCh37) Bowtie2 index files from iGenome package of Ensembl. I have downloaded separately the GTF file from Ensembl (Homo_sapiens.GRCh37.75.gtf).
      Now after getting accepted_hits.bam files from TopHat2, I moved on to cufflinks (which ran perfectly) for two different samples and thereafter used cuffmerge.
      But while running cuffmerge I am getting warning messages, though I am able to get single merged.gtf file after completion of cuffmerge run.

      My command line for cuffmerge is:
      $ cuffmerge -o /home/cuffmerge_out -g /home/Homo_sapiens.GRCh37.75.gtf -s /home/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome.fa -p 4 /home/assemblies.txt

      Last few lines showing the warning messages are as follows:
      > Map Properties:
      > Normalized Map Mass: 540321.00
      > Raw Map Mass: 540321.00
      > Fragment Length Distribution: Truncated Gaussian (default)
      > Default Mean: 200
      > Default Std Dev: 80

      I have also tried using gtf file provided in iGenome package: /home/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf
      But in both cases getting same warning messages.
      Can anyone help me to solve this out. Perhaps it might also be beneficial for many other people facing similar kind of problem.
      Thank you.
      Open up the annotation file you're using (.gtf) and check how the chromosomes are labeled. Make sure you see the same thing in your sequence file (genome.fa). Specifically, if you are naming your chromosome in your annotation as "HSChr1" make sure in your genome.fa chromosome 1 is labeled: >HSChr1. You can manually change this.

      I've used the annotation and sequence files from http://hgdownload.cse.ucsc.edu/downloads.html#human
      With success.

      Hope this helps!

        Comment

        • genome1
          Member
          • Oct 2013
          • 12
          #3
          Thanks AddDNAse.

          But my concern is why it should be needed to change manually. As I am using both .gtf file and genome.fa file from Ensembl, hence there is no chance of error in the the nomenclature of chromosome. In Ensembl chromosomes are named as "1", "2", "3",.. instead of "chr1", "chr2", "chr3", etc.
          Is it a bug in the "Cufflinks-2.2.1"?
          Do anyone know about it?

            Comment

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