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  • #46
    I know that this post is dead for 3 years now, but I had the same problem and I know that many people are looking for an answer. What they are telling me is that tagmentase is an extremely labile enzyme and it is enough for it to be a little bit outdated or to have several freeze-thaw cycles to lose most of its activity. Hence very large library sizes - tagmentation is just not good enough, and increasing its duration will not help because the enzyme is inactive. Better switch to other methods of DNA shearing.

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    • #47
      Originally posted by rudzik79 View Post
      I know that this post is dead for 3 years now, but I had the same problem and I know that many people are looking for an answer. What they are telling me is that tagmentase is an extremely labile enzyme and it is enough for it to be a little bit outdated or to have several freeze-thaw cycles to lose most of its activity. Hence very large library sizes - tagmentation is just not good enough, and increasing its duration will not help because the enzyme is inactive. Better switch to other methods of DNA shearing.
      Eh? Who are the "they" to whom you refer? Nextera doesn't seem particularly labile to me.

      One point of possible confusion: after tagmentation, but before PCR, everything tends to look very big because the Tn5 has inserted but apparently it still tethering both DNA ends to each other. See here for more details:
      Haplotype-resolved genome sequencing enables the accurate interpretation of medically relevant genetic variation, deep inferences regarding population history and non-invasive prediction of fetal genomes. We describe an approach for genome-wide haplotyping based on contiguity-preserving transpositio …

      This paper describes a "contiguity preserving transposition based sequencing" method that takes advantage of this behavior of Tn5.

      --
      Phillip

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      • #48
        Originally posted by rudzik79 View Post
        I know that this post is dead for 3 years now
        Really Last posting in this thread was on 20th April of this year by @fanli

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        • #49
          Originally posted by GenoMax View Post
          Really Last posting in this thread was on 20th April of this year by @fanli
          Yes, I think it is possible that rudzik79 did not read the entire thread prior to posting. Which one could understand, I suppose. But in this case led to an erroneous assumption as to the "aliveness" of the thread...

          --
          Phillip

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