For people doing high molecular weight DNA sequencing library prep, do you see a steep drop off in the fragment sizes before and after the library prep?
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3 months ago
Mark • 0

Specifically, I'm wondering does anyone have trouble with magnetic bead clean up on high molecular weight DNA? I extracted some DNA using a High Molecular Weight Kit and got got a characteristic fragment length of 163,457bp when measured on a FEMTO. However after preparing that DNA using a ligation kit for Nanopore sequencing my N50 was only around 5kb. I noticed that during the first magnetic bead wash step the DNA seemed to bind really tight to the beads and formed like a visible, stringy bead mix that was super hard to resuspend. I tried to resuspend it by flicking it. I tried to do it gently to not break the DNA but had to apply a little more force than usually because the beads would not resuspend. Even with more force I couldn't resuspend the beads fully. I tried incubating the DNA/bead mixture at 37*C to loosen the DNA but I was still only able to recover 33% of the DNA I inputted after the bead wash was complete. My 2 potential hypotheses are that either my DNA got severely sheared during this process or that the longer fragments remained intertwined in the magnetic bead mix and only the shorter fragments got eluded. Both of these could explain the steep dropoff in fragment size found before and after library prep. I'm wondering if anyone else has experienced anything like this or has an estimate on how much fragmentation can be expected during sequencing library prep.

library-prep sequencing ont nanopore ngs • 947 views
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