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  • HSV-1
    Member
    • Jul 2012
    • 38

    #1

    Please diagnose my RNA-seq data.

    I have processed a few public available RNA-seq data I downloaded from NCBI SRA. Most are ok. Except these two below:
    GSM849855: Total RNA extracted from mock infected NIH-3T3 cells; Mus musculus; RNA-Seq
    GSM849856: Total RNA extracted from MCMV infected NIH-3T3 cells; Mus musculus; RNA-Seq

    I use tophat to map rna-seq data. Normally 4G raw data will produce an ~800M BAM file. But the above two data give 20-30M. I always use the same parameters! I have run the flagstat to analyse the BAM files and the reports are below:

    1373321 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    1373321 + 0 mapped (100.00%:-nan%)
    0 + 0 paired in sequencing
    0 + 0 read1
    0 + 0 read2
    0 + 0 properly paired (-nan%:-nan%)
    0 + 0 with itself and mate mapped
    0 + 0 singletons (-nan%:-nan%)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)

    271331 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    271331 + 0 mapped (100.00%:-nan%)
    0 + 0 paired in sequencing
    0 + 0 read1
    0 + 0 read2
    0 + 0 properly paired (-nan%:-nan%)
    0 + 0 with itself and mate mapped
    0 + 0 singletons (-nan%:-nan%)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)


    What could be wrong with these two data? Please help me out!

    (The title doesn't mean these data were generated in my lab but mean I got these data from NCBI and I processed them.)
    sorry for the inaccuracy
    Last edited by HSV-1; 07-21-2012, 09:44 PM. Reason: to be accurate
  • Richard Finney
    Senior Member
    • Feb 2009
    • 701

    #2
    Look at SRR390297

    example.
    -bash-3.00$ head -50000 SRR390297.fastq | awk '{if ((NR%4)==2) print $0}' | tail
    GAGGTAGTAGGTTGTATGGTTATCGTATGCCGTCTT
    TGAGGTAGTAGATTGTATAGTTTCGTATGCCGTCTT
    AGAGGTAGTAGGTTGCATAGTTTCGTATGCCGACTT
    CTGTGCGTGTGACAGCGGCTGAATTCGTATGCCGCC
    TGAGGTAGTAGGTTGTATGGCTTCGTATGCCGTCTT
    TGAGGTAGTAGTTTGTGCTGTTTCGTATGCCGTCTT
    TGAGGTAGTAGGTTGTATGGTTTCGTATGCCGTCTT
    AGCTACATCTGGCTACTGGGCCTCTTCGTATGCCGT
    TGAGGTAGTAGGTTGTGTGGTTTCGTATGCCGTCTT
    ACAGTAGTCTGCACATTGGTTATCGTATGCCGTCTT

    There all "variations on a theme".

    They have the same motif over and over -- most with TATGCCGTCTT at the end.

    Anybody recognize this? Wet lab guys? Adapters?

    Counts of most common transcripts :
    185154 TGAGGTAGTAGTTTGTGCTGTTATCGTATGCCGTCT
    250743 TAGCTTATCAGACTGATGTTGATCGTATGCCGTCTT
    312964 TGAGGTAGTAGGTTGTGTGGTTTCGTATGCCGTCTT
    388556 TAGCTTATCAGACTGATGTTGACTCGTATGCCGTCT
    414442 ACAGTAGTCTGCACATTGGTTATCGTATGCCGTCTT
    448175 TGAGATGAAGCACTGTAGCTCTTCGTATGCCGTCTT
    546039 TGAGGTAGTAGGTTGTATAGTTTCGTATGCCGTCTT
    652936 TGAGGTAGTAGTTTGTGCTGTTTCGTATGCCGTCTT
    908341 TGAGGTAGTAGATTGTATAGTTTCGTATGCCGTCTT
    1218229 TGAGGTAGTAGGTTGTATGGTTTCGTATGCCGTCTT

    Comment

    • HSV-1
      Member
      • Jul 2012
      • 38

      #3
      Hi, I have found that the last 8 base pairs in low quality but I didn't go to insight. Thanks for your pointing out.
      Do you think trimming the last 8 bp pairs will help?
      I have done blast and found that the first 20bp are MicroRNAs!

      Thanks!

      Originally posted by Richard Finney View Post
      Look at SRR390297

      example.
      -bash-3.00$ head -50000 SRR390297.fastq | awk '{if ((NR%4)==2) print $0}' | tail
      GAGGTAGTAGGTTGTATGGTTATCGTATGCCGTCTT
      TGAGGTAGTAGATTGTATAGTTTCGTATGCCGTCTT
      AGAGGTAGTAGGTTGCATAGTTTCGTATGCCGACTT
      CTGTGCGTGTGACAGCGGCTGAATTCGTATGCCGCC
      TGAGGTAGTAGGTTGTATGGCTTCGTATGCCGTCTT
      TGAGGTAGTAGTTTGTGCTGTTTCGTATGCCGTCTT
      TGAGGTAGTAGGTTGTATGGTTTCGTATGCCGTCTT
      AGCTACATCTGGCTACTGGGCCTCTTCGTATGCCGT
      TGAGGTAGTAGGTTGTGTGGTTTCGTATGCCGTCTT
      ACAGTAGTCTGCACATTGGTTATCGTATGCCGTCTT

      There all "variations on a theme".

      They have the same motif over and over -- most with TATGCCGTCTT at the end.

      Anybody recognize this? Wet lab guys? Adapters?

      Counts of most common transcripts :
      185154 TGAGGTAGTAGTTTGTGCTGTTATCGTATGCCGTCT
      250743 TAGCTTATCAGACTGATGTTGATCGTATGCCGTCTT
      312964 TGAGGTAGTAGGTTGTGTGGTTTCGTATGCCGTCTT
      388556 TAGCTTATCAGACTGATGTTGACTCGTATGCCGTCT
      414442 ACAGTAGTCTGCACATTGGTTATCGTATGCCGTCTT
      448175 TGAGATGAAGCACTGTAGCTCTTCGTATGCCGTCTT
      546039 TGAGGTAGTAGGTTGTATAGTTTCGTATGCCGTCTT
      652936 TGAGGTAGTAGTTTGTGCTGTTTCGTATGCCGTCTT
      908341 TGAGGTAGTAGATTGTATAGTTTCGTATGCCGTCTT
      1218229 TGAGGTAGTAGGTTGTATGGTTTCGTATGCCGTCTT
      Last edited by HSV-1; 07-21-2012, 11:26 PM. Reason: update

      Comment

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