TITLE: nCoV-2019 McGill Nanopore LibPrep Protocol, 10 ng NB
AUTHORS: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol works for 12 native barcodes, 24 native barcodes, and 96 native barcodes.</div></div>

[STEPS]
?. [Native barcoding]
Barcode the amplicon pools using native barcodes.
This is a ‘one-pot ligation’ protocol for native barcoded ligation libraries. We have seen no reduction in performance compared to standard libraries, and is 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.
?. [Native barcoding]
Set up the following reaction for each sample:Component                                            VolumeDNA amplicons                                       Nuclease-free water                               Ultra II End Prep Reaction Buffer         Ultra II End Prep Enzyme Mix               Total
5 µl
7.5 µl
1.75 µl
0.75 µl
15 µl
?. [Native barcoding]
Incubate at room temperature for  Incubate at   for Incubate on ice for
65 °C
?. [Native barcoding]
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
0.75 µl
[35.75 ul total with the 15 ul from step 19.1]
?. [Native barcoding]
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.
?. [Native barcoding]
Clean-up the native barcodes using the following protocol (before pooling):Add 0.8X of SPRI beads (28.6 ul) to the sample tube and mix gently by pipetting.Incubate for 5 min at room temperature. Pellet on magnet for 5 min. Remove supernatant.Add 200 ul of 80% ethanol to the pellet and wash twice. Elute in 20 ul elution buffer.
?. [Native barcoding]
Quantify the barcodes using a fluorimetric dsDNA assay.
?. [Native barcoding]
Normalize the barcodes to 10 ng each and pool (except neg. ctrl - which will be equal volume).
?. [Native barcoding]
Clean-up the barcode pool using the following protocol. Add 0.8X of SPRI beads to the sample tube and mix gently by pipetting.Incubate for 5 min at room temperature. Pellet on magnet for 5 min. Remove supernatant.Add 200 ul of 80% ethanol to the pellet and wash twice. Elute in 30 ul elution buffer.
?. [Native barcoding]
Set up the following AMII adapter ligation reaction:Component                                                                    VolumeBarcoded amplicon pools                                            NEBNext Quick Ligation Reaction Buffer (5X)          AMII adapter mix                                                           Quick T4 DNA Ligase                                                   Total
30 µl
10 µl
5 µl
5 µl
50 µl
?. [Native barcoding]
Incubate at room temperature for
?. [Native barcoding]
Clean-up the native barcodes using the following protocol:Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by pipetting.Incubate for 5 min at room temperature. Pellet on magnet for 5 min. Remove supernatant.Add 200 ul of SFB to the pellet and resuspend beads completely by pipette mixing. Pellet on beads, remove supernanant, and repead the wash step with another 200 ul of SFB.Elute in 15 ul EB (provided in the ONT kit).Incubate at room temperature for Place on magnetic rack.Transfer final library to a new 1.5 mL Eppendorf tube.
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. [Native barcoding]
Quantify the final library using a fluorimetric dsDNA assay.
Final library can be now be stored in 10 mM Tris pH 8 at 4°C for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [Native barcoding]
Prime the flowcell and load 20 ng to 40 ng sequencing library onto the flowcell. Dilute library in EB if required.
The original protocol says 20 ng, but this leads to only ~50% pore occupancy. Loading 40 ng leads to ~70% pore occupancy but the flow cell needs to be refueled.
?. [Native barcoding]
Thaw the following reagents at room temperature before placing on ice:  Sequencing buffer (SQB)Loading beads (LB)Flush buffer (FLB)Flush tether (FLT)
?. [Native barcoding]
Add  FLT to the FLB tube (1.16 mL) and mix well by vortexing.
30 µl
?. [Native barcoding]
If required place a new MinION flowcell onto the MinION by flipping open the lip and pushing one end of the flowcell under the clip and pushing down gently.
?. [Native barcoding]
Rotate the inlet port cover clockwise by 90° so that the priming port is visible.
?. [Native barcoding]
Take a P1000 pipette and tip and set the volume to   . Place the tip in the inlet port and holding perpendicularly to the plane of the flowell remove any air from the inlet port by turning the volume dial anti-clockwise.
800 µl
Be careful not to remove so much volume that air is introduced onto the rectangular array via the outlet.
?. [Native barcoding]
Load   of FLB (plus FLT) into the flow cell via the inlet port, dispense slowly and smoothly trying to avoid the introduction of any air bubbles.
800 µl
?. [Native barcoding]
Wait for
?. [Native barcoding]
Gently lift the SpotON cover to open the SpotON port.
?. [Native barcoding]
Load another  of FLB (plus FLT) into the flow cell via the inlet port, this will initiate a siphon at the SpotON port to allow you to load the library dilution.
200 µl
?. [Native barcoding]
In a new tube prepare the library dilution for sequencing:Component                                  VolumeSQB                                               LB                                                  Final library                                  Total
37.5 µl
25.5 µl
12 µl
75 µl
?. [Native barcoding]
Mix the prepared library gently by pipetting up and down just prior to loading.
?. [Native barcoding]
Add the   library dilution to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop siphons into the port before adding the next.
75 µl
?. [Native barcoding]
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the inlet port and close the MinION lid.
?. [Native barcoding]
Start the sequencing run using MinKNOW.
?. [Native barcoding]
If required plug the MinION into the computer and wait for the MinION and flowcell to be detected.
?. [Native barcoding]
Choose flow cell 'FLO-MIN106' from the drop-down menu.
?. [Native barcoding]
Then select the flowcell so a tick appears.
?. [Native barcoding]
Click the 'New Experiment' button in the bottom left of the screen.
?. [Native barcoding]
On the New experiment popup screen, select the running parameters for your experiment from the individual tabs:Experiment: Name the run in the experiment field, leave the sample field blank.Kit: Selection: Select LSK109 as there is no option for native barcoding (NBD104).Run Options: Set the run length to 6 hours (you can stop the run once sufficient data has been collected as determined using RAMPART).Basecalling: Leave basecalling turned but select 'fast basecalling'.Output: The number of files that MinKNOW will write to a single folder. By default this is set to 4000 but can be reduced to make RAMPART update more frequently.Click 'Start run'.
?. [Native barcoding]
Monitor the progress of the run using the MinKNOW interface.
?. [Native barcoding]
Repeat the clean-up step to avoid any unligated barcode carryover to the next ligation reaction. Add 0.8X of SPRI beads to the pooled sample tube and mix gently by pipetting.Incubate for 5 min at room temperature. Pellet on magnet for 5 min. Remove supernatant.Add 200 ul of 80% ethanol to the pellet and wash twice. Elute in 30 ul elution buffer.
?. [Native barcoding]
Optional: Run a tapestation to verify that no unligated barcodes are present in the elute.
?. [Native barcoding]
Pool the samples.