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  • AdrianP
    Senior Member
    • Apr 2011
    • 130

    #1

    MiSeq Confusion

    Hello, I am involved in a project where an organism (eukaryote) was sent for paired end sequencing. Usually when we do that we get something like XX_SN132_B_s_7_2_seq_GDR-14.txt and XX_SN132_B_s_7_1_seq_GDR-14.txt back indicating the forward file and reverse file. So I use the velvet provided script called shuffle sequences, get one file and start assembling.

    But last week we got something in the format: XX_SNXX_M_L001_GDR-24_R1.fastq.gz and XX_SNXX_M_L001_GDR-24_R2.fastq.gz. At first I saw the files and thought it was no big deal. I extracted them, shuffled them and started assembly. What I noticed is that although the sequencing was paired end with 500 bp inserts, velvetoptimiser was telling me that the library length is 250, which is shorter than the insert. I then checked and saw that the forward reads overlap their mate pairs, almost entirely, 190-240 bp overlap. Read length is 250.

    I am not sure I followed the correct procedure with the data this time. overlapping paired end reads are not normal as far as i understand.
  • GenoMax
    Senior Member
    • Feb 2008
    • 7142

    #2
    Are you certain your library had 500 bp inserts?

    In case of MiSeq with 2 x 250 bp reads it is possible to get paired-end reads that overlap (extent of overlap will depend on the length of the insert).

    If all the reads are overlapping to a large extent (sounds like you have confirmed that) then is it possible that the facility that did the sequencing fragmented the DNA before making the libraries leading to the short(er) than expected inserts?

    Comment

    • pmiguel
      Senior Member
      • Aug 2008
      • 2328

      #3
      Yes, I agree with GenoMax. "Insert Length" is not just some parameter guys in a lab can plug into their calculator and generate a library with that insert length. It requires some care to pull it off. Not that it is extremely difficult, but neither is is "plug and chug".

      --
      Phillip

      Comment

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