Jan 11, 2018
One-pot ligation protocol for Oxford Nanopore libraries
- 1University of Birmingham
Protocol Citation: Josh Quick 2018. One-pot ligation protocol for Oxford Nanopore libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.k9acz2e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
This protocol is not recommend for use with the latest LSK109 kits
Created: December 13, 2017
Last Modified: August 06, 2018
Protocol Integer ID: 9218
Keywords: Oxford Nanopore, MinION, GridION, PromethION, Ultra II, one-pot
Abstract
This is our ‘one-pot ligation’ protocol for Oxford Nanopore ligation libraries. It benefits from increased recovery of library and faster preparation time by using the Ultra II ligation module in conjunction with the Ultra II end repair/dA-tailing module therefore removing a clean-up step. It can be used with or without the optional FFPE DNA repair step which shoudl help to improve read-lengths in nicked DNA. The incubation times given here are sufficient to generate high quality libraries in under one hour. In the past we have cut these times in half without noticably impacting performance but we will leave this up to you.
Materials
MATERIALS
NEBNext FFPE DNA Repair Mix - 96 rxnsNew England BiolabsCatalog #M6630L
Agencourt AmPure XP beadsCatalog #A63880
NEBNext Ultra II End Repair/dA-Tailing Module - 96 rxnsNew England BiolabsCatalog #E7546L
NEBNext Ultra II Ligation Module - 96 rxnsNew England BiolabsCatalog #E7595L
Ligation Sequencing Kit 1DCatalog #SQK-LSK108
Set up either the following end-prep reaction:
| DNA (200-400 fmol) | 25 μl | |
| Ultra II End Prep Reaction Buffer | 3.5 μl | |
| Ultra II End Prep Enzyme Mix | 1.5 μl | |
| Total | 30 μl |
Or to include FFPE DNA repair set up the following combined reaction:
| DNA (200-400 fmol) | 24 μl | |
| Ultra II End Prep Reaction Buffer | 1.75 μl | |
| FFPE DNA Repair Buffer | 1.75 μl | |
| Ultra II End Prep Enzyme Mix | 1.5 μl | |
| FFPE DNA Repair Mix | 1 μl | |
| Total | 30 μl |
Incubate at RT for 10 minutes.
00:10:00
20 °C
Then incubate at 65°C for 10 minutes.
00:10:00
65 °C
Place on ice for 30 seconds.
00:00:30
Add the following directly to the previous reaction:
| AMX 1D | 20 μl | |
| Ultra II Ligation Master Mix | 40 μl | |
| Ligation Enhancer | 1 μl | |
| Total | 91 μl |
Incubate at RT for 20 minutes.
00:20:00
20 °C
Add 45.5 μl Ampure XP beads.
45.5 µL Ampure XP beads
Incubate at room temperature for 10 minutes.
00:10:00
20 °C
Spin down briefly and place on a magnetic rack until solution clears.
Taking care to avoid the pellet remove the supernatant.
Add 150 μl ABB and resuspend by gently flicking (wash 1/2).
150 µL ABB
Spin down briefly and place on a magnetic rack until solution clears (wash 1/2).
Taking care to avoid the pellet remove the supernatant (wash 1/2).
Add 150 μl ABB and resuspend by flicking (wash 2/2).
150 µL ABB
Spin down briefly and place on a magnetic rack until solution clears (wash 2/2).
Taking care to avoid the pellet remove the supernatant (wash 2/2).
Spin down again and remove all residual ABB with a P10 pipette.
Add 12 μl ELB and resuspend beads by flicking.
12 µL ELB
Incubate at RT for 10 minutes.
00:10:00
Spin down briefly and place on a magnetic rack until solution clears.
In a new tube prepare library dilution for sequencing:
| With LLB | Without LLB | ||
| RBF | 35 µl | 35 µl | |
| Nuclease-free water | 2.5 µl | 28 µl | |
| LLB | 25.5 µl | - | |
| Library | 12 µl | 12 µl | |
| Total | 75 µl | 75 µl |
Note
N.B. I would generally include LLB unless working with HMW DNA, which can cause them to clump, or if I plan to flush/reload the flowcell, in which case they would get flushed out anyway.
Mix by gently flicking before removing 1 µl to assess concentration by Qubit (wait until beads have settled before measuring).
1 µL Library
Expected result
Expected recovery is 50-80% of starting material, lower recovery is indicative of presence of short fragments or inaccurate quantification of the input material. Loading insufficient library into a flowcell will significantly impact yield.