TITLE: One-pot native barcoding of amplicons (Ultra II AMII ligation)
AUTHORS: Josh Quick, Kirstyn Brunker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a ‘one-pot ligation’ protocol for Oxford Nanopore native barcoded ligation libraries using shearing. </div></div>

[STEPS]
?. Set up the following reaction for each sample:Component                                          VolumeDNA amplicons (5ng)                         Ultra II End Prep Reaction Buffer     Ultra II End Prep Enzyme Mix           Total
12.5 µl
1.75 µl
0.75 µl
15 µl
?. Incubate at room temperature for  Incubate at  for Incubate on ice for
65 °C
?. Add the following directly to the previous reactions:Component                               VolumeNBXX barcode                          Ultra II Ligation Master Mix    Ligation Enhancer                    Total
2.5 µl
17.5 µl
0.5 µl
35.5 µl
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 Incubate at  for Incubate on ice for
70 °C
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.
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                                                                    VolumeBarcoded amplicon pools                                           AMII adaptor                                                                  Ultra II Ligation Master Mix                                         Ligation Enhancer                                                        Total
44 µl
5 µl
50 µl
1 µl
100 µl
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
?. Add (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
100 µl
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   for .
37 °C
?. Place on magnetic rack and incubate for  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  SFB and resuspend beads completely by pipette mixing.
200 µl
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.
You do not need to allow to air dry with SFB washes.
?. Add EB and resuspend beads by pipette mixing.
13 µl
?. Incubate at   for .
37 °C
?. Place on magnetic rack.
?. Transfer final library to a new 1.5mL Eppendorf tube.