Mar 07, 2020
One-pot native barcoding of amplicons (Ultra II AMII ligation)
Forked from One-pot native barcoding of amplicons
- 1University of Birmingham;
- 2University of Glasgow
External link: http://lab.loman.net/protocols/
Protocol Citation: Josh Quick, Kirstyn Brunker 2020. One-pot native barcoding of amplicons (Ultra II AMII ligation). protocols.io https://dx.doi.org/10.17504/protocols.io.bdaqi2dw
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: Working
We use this protocol and it's working
Created: March 05, 2020
Last Modified: October 28, 2024
Protocol Integer ID: 33840
Abstract
This is a ‘one-pot ligation’ protocol for Oxford Nanopore native barcoded ligation libraries using shearing.
Note
This protocol has been slightly modified from Josh Quick's original to use the Ultra II ligation for barcoding and adaptor ligation
Attachments
Guidelines
Scope:
There has been no evidence of a reduction of performance compared to standard libraries, yet it can be made faster by using the Ultra II ligation module which is compatible with the Ultra II end repair/dA-tailing module removing a clean-up step.
The FFPE DNA repair step is optional. If you have the time, we recommend using the double incubation times in bold. If you are in a hurry, the times in italic are a good compromise between speed and efficiency.
Required:
g-TUBEs (optional)
SQK-LSK108 1D ligation kit
Native barcoding kit
Ultra II End Repair/dA-Tailing Module
Ultra II Ligation Module
FFPE DNA Repair Mix (optional)
Ampure XP beads
80% ethanol
EB (10 mM Tris-HCl pH 8)
Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.
Set up the following reaction for each sample:
Component Volume
DNA amplicons (5ng) 12.5 µL
Ultra II End Prep Reaction Buffer 1.75 µL
Ultra II End Prep Enzyme Mix 0.75 µL
Total 15 µL
Incubate at room temperature for 00:10:00
Incubate at 65 °C f for 00:05:00
Incubate on ice for 00:01:00
Add the following directly to the previous reactions:
Component Volume
NBXX barcode 2.5 µL
Ultra II Ligation Master Mix 17.5 µL
Ligation Enhancer 0.5 µL
Total 35.5 µL
Note
Use one native barcode from the EXP-NBD104 (1-12) or EXP-NBD114 (13-24) per sample. Use from 6 to 24 barcodes in a library, any fewer and there will be insufficient total material to achieve good yields.
Incubate at room temperature for 00:15:00
Incubate at 70 °C for 00:10:00
Incubate on ice for 00:01:00
Note
The 70°C incubation is to inactivate the DNA ligase to prevent barcode cross-ligation when reactions are pooled in the next step.
Pool all barcoded fragments together into a new 1.5 ml Eppendorf tube and perform a SPRI bead clean-up. Elute in 45ul.
Note
PAUSE POINT: As long as you have not yet ligated the sequencing adapter, the library can be stored at 4 °C and continue with the prep at a later point. It is better to store at 4°C, as freezing and thawing can introduce nicks or breaks in the DNA. Several days to weeks in the fridge are possible. For longer-term storage, the library can be placed at -20 °C, though unnecessary freeze-thaw cycles should be avoided for best results.
Quantify the barcoded amplicon pools using the Quantus Fluorometer using the ONE dsDNA assay.
Set up the following AMII adapter ligation reaction:
Component Volume
Barcoded amplicon pools 44 µL
AMII adaptor 5 µL
Ultra II Ligation Master Mix 50 µL
Ligation Enhancer 1 µL
Total 100 µL
Note
The input of barcoded amplicon pools will depend on the number of barcoded pools and should be between 40 ng (8 barcodes) and 120 ng (24 barcodes).
Incubate at room temperature for 00:20:00
Add 100 µL (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
Note
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate at 37 °C for 00:10:00 .
Place on magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear.
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add 200 µL SFB and resuspend beads completely by pipette mixing.
Note
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Remove supernatant and discard.
Repeat steps 14-16 to perform a second SFB wash.
Pulse centrifuge and remove any residual SFB.
Note
You do not need to allow to air dry with SFB washes.
Add 13 µL EB and resuspend beads by pipette mixing.
Incubate at 37 °C for 00:10:00 .
Place on magnetic rack.
Transfer final library to a new 1.5mL Eppendorf tube.