Newbie Help for 1D^2 library shearing

yiyi_Z · 03-25-2019, 01:48 AM

Hi, all!
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 (see https://imgur.com/O7PVgF3), which wasn't seen in the QC gel photo before (see https://imgur.com/xrnX09v) and after library preparation (see https://imgur.com/P1pTC6e). The bands had no significant low molecular weight fragments.
Anyone using nanopore has encountered this before? Any explanation? And how to avoid it? Since I could not find the problem in my QC steps...

Thanks!
Yiyi

Femta · 03-28-2019, 05:57 AM

Hi,

as far as I've seen you have clean up steps in LSK308. These steps are risky to fragment your DNA.
So try to avoid as many as possible to pipet your DNA during the library preparation. To mix just flick your tube, no vortexing.

And for sure, do not degrade your DNA ;-)

yiyi_Z · 04-02-2019, 01:47 AM

Hi, Femta. Thank you for your answer and suggestion first.
As I didn't find too much smeared DNA on the gel run after library preparation, library fragmentation may not be the case here. In combination with the low portion of Sequencing pores in MinKNOW report (not shown above), the most possible reason is the failure of adaptor ligation step. That is, I had enough DNA, but only a small part of them (especially the shorter part) was ligated to adaptors successfully, and only ligated DNA can be sequenced.
And I have asked for help from nanopore support and other nanopore users. They suggested that a longer ligation time under temperature lower than RM would increase ligation efficiency. I would try that in the next trial.

Femta · 04-02-2019, 07:31 AM

Hi,

well you initially asked why there are these small fragments ;-) And since you didn't see them in your QC before the library prep it's most likely that that happen due to pipetting during the library prep.

But yes, the adapter ligation step is the most critical step during this protocol. You might also loose more than you expect, so it can be helpful to increase the DNA input in advance.

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03-25-2019, 01:48 AM	#1
yiyi_Z Junior Member Location: HK Join Date: Mar 2019 Posts: 2	Newbie Help for 1D^2 library shearing Hi, all! 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 (see https://imgur.com/O7PVgF3), which wasn't seen in the QC gel photo before (see https://imgur.com/xrnX09v) and after library preparation (see https://imgur.com/P1pTC6e). The bands had no significant low molecular weight fragments. Anyone using nanopore has encountered this before? Any explanation? And how to avoid it? Since I could not find the problem in my QC steps... Thanks! Yiyi Last edited by yiyi_Z; 03-25-2019 at 01:51 AM.

03-28-2019, 05:57 AM	#2
Femta Junior Member Location: Germany Join Date: Oct 2018 Posts: 4	Hi, as far as I've seen you have clean up steps in LSK308. These steps are risky to fragment your DNA. So try to avoid as many as possible to pipet your DNA during the library preparation. To mix just flick your tube, no vortexing. And for sure, do not degrade your DNA ;-)

04-02-2019, 01:47 AM	#3
yiyi_Z Junior Member Location: HK Join Date: Mar 2019 Posts: 2	Hi, Femta. Thank you for your answer and suggestion first. As I didn't find too much smeared DNA on the gel run after library preparation, library fragmentation may not be the case here. In combination with the low portion of Sequencing pores in MinKNOW report (not shown above), the most possible reason is the failure of adaptor ligation step. That is, I had enough DNA, but only a small part of them (especially the shorter part) was ligated to adaptors successfully, and only ligated DNA can be sequenced. And I have asked for help from nanopore support and other nanopore users. They suggested that a longer ligation time under temperature lower than RM would increase ligation efficiency. I would try that in the next trial.

04-02-2019, 07:31 AM	#4
Femta Junior Member Location: Germany Join Date: Oct 2018 Posts: 4	Hi, well you initially asked why there are these small fragments ;-) And since you didn't see them in your QC before the library prep it's most likely that that happen due to pipetting during the library prep. But yes, the adapter ligation step is the most critical step during this protocol. You might also loose more than you expect, so it can be helpful to increase the DNA input in advance.