<p>Short template DNA is a big problem with nanopore sequencing; it doesn't easily show up on a gel if it's well distributed across different lengths (as appears to be the case here from the read length plot), and sucks up both adapter and sequencing capacity.</p>
<p>The usual way to deal with it (for reads in the size ranges seen here) is to reduce the bead concentration when doing magnetic bead cleanup. Try something like 0.6X beads (e.g. 30μl of Ampure XP beads added to 50μl template DNA solution), which should still allow most of the long template sequences to bind, but leave shorter reads in solution.</p>

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2021-05-06T10:09:19.373Z
2021-05-06T10:09:19.373Z
gringer
<p>I've done my 1st trial of 1D^2 whole genome sequencing using LSK-SQK308 kit with R9.5 flowcell. Without doing library fragmentation, I encountered the unexpected library shearing during sequencing.</p>

<p><a href="https://i.sstatic.net/MdRPp.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/MdRPp.png" alt="enter image description here"></a></p>

<p>But I didn't see significant portion of low-molecular-weight fragments in the QC gel photo before and after library preparation.</p>

<p><a href="https://i.sstatic.net/MQuY6.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/MQuY6.png" alt="enter image description here"></a> </p>

<p>Anyone using nanopore has encountered this before? Any explanation? And how to avoid it? I could not find evidence of the problem in my QC steps...</p>

<p>Thanks in advance!</p>

<nanopore><quality-control><1d2-reads>
Help with 1D^2 library shearing
# Help with 1D^2 library shearing

(Asked by: username_0 on 2019-03-27)

I've done my 1st trial of 1D^2 whole genome sequencing using LSK-SQK308 kit with R9.5 flowcell. Without doing library fragmentation, I encountered the unexpected library shearing during sequencing.

[![enter image description here](https://i.sstatic.net/MdRPp.png)](https://i.sstatic.net/MdRPp.png)

But I didn't see significant portion of low-molecular-weight fragments in the QC gel photo before and after library preparation.

[![enter image description here](https://i.sstatic.net/MQuY6.png)](https://i.sstatic.net/MQuY6.png)

Anyone using nanopore has encountered this before? Any explanation? And how to avoid it? I could not find evidence of the problem in my QC steps...

Thanks in advance!

**Tags:** nanopore, quality-control, 1d2-reads

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## 1 answers

### Answer by username_1 on 2021-05-06. Score: 1

Short template DNA is a big problem with nanopore sequencing; it doesn't easily show up on a gel if it's well distributed across different lengths (as appears to be the case here from the read length plot), and sucks up both adapter and sequencing capacity.

The usual way to deal with it (for reads in the size ranges seen here) is to reduce the bead concentration when doing magnetic bead cleanup. Try something like 0.6X beads (e.g. 30μl of Ampure XP beads added to 50μl template DNA solution), which should still allow most of the long template sequences to bind, but leave shorter reads in solution.


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