TITLE: Nanopore Influenza A Virus WGS using one step RT/PCR with RBK barcoding
AUTHORS: Tom Williams

[DESCRIPTION]
A protocol to generate Influenza A Virus sequences from RNA extracts in under 24 hours with less than two hours of hands on time.

Based on:
King, J., Harder, T., Beer, M., & Pohlmann, A. (2020). Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses.BMC Infectious Diseases,20(1). https://doi.org/10.1186/s12879-020-05367-y
Zhou, B., Donnelly, M. E., Scholes, D. T., st. George, K., Hatta, M., Kawaoka, Y., & Wentworth, D. E. (2009). Single-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses.Journal of Virology,83(19), 10309–10313. https://doi.org/10.1128/jvi.01109-09

Tested with up to 20 RNA extracts from samples per run, alongside:
Negative control of nuclease-free water
Positive control of H1
Positive control of H3
Positive control of lambda phage, introduced at barcoding stage (i.e. not amplified), as an assessment of barcode crosstalk

[STEPS]
SECTION: Combined RT/PCR
1. Prepare up to 20 RNA extracts from samples plus:
Negative control of nuclease-free water 
Positive control of H1N1
Positive control of H3N2

If samples previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on cool block at all times.
SECTION: Combined RT/PCR
2. In the template-free pre-PCR hood, prepare the following master mixes in Eppendorf DNA LoBind tubes, mix thoroughly by pipetting, and pulse spin to collect liquid at the bottom of the tube.

Pool A
 

Pool B
 

SECTION: Combined RT/PCR
3. In the template-free pre-PCR hood, aliquot 9.5 µL of Pool A master mix into in 0.2 mL 8 well PCR strip-tubes, and 9.5 µL of Pool B into a seperate 0.2 mL8 well PCR strip tubes.

SECTION: Combined RT/PCR
5. In the post-PCR lab, incubate the reaction as follows:

 

SECTION: Combined RT/PCR
4. In the template pre-PCR hood, transfer 3 µL of sample into the corresponding well of both Pool A and Pool B. Mix by pipetting each well up and down. Spin down.

SECTION: Pooling and clean-up
10. Briefly spin down the Barcode reaction product to collect the liquid at the bottom of the wells prior to opening.
SECTION: Priming and loading flow cell
28. Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII), Flush Tether (FLT) and Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down the SBII and FLT at room temperature.
SECTION: Priming and loading flow cell
29. To prepare the flow cell priming mix, add 30 µL of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

SECTION: Priming and loading flow cell
30. On the GridION Mk1, place flow cell and perform flow cell check. Proceed if more than >800 pores.
SECTION: Priming and loading flow cell
31. Slide the priming port cover clockwise to open the priming port.
SECTION: Priming and loading flow cell
32. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):
Set a P1000 pipette to 200 μl
Insert the tip into the priming port
Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, or until you can see a small volume of buffer entering the pipette tip
Note:Visually check that there is continuous buffer from the priming port across the sensor array.
SECTION: Priming and loading flow cell
33. Load 800 µL of the priming mix into the flow cell via the priming port, again by turning wheel of pipette and not by pressing on button, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.

SECTION: Priming and loading flow cell
34. Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting
SECTION: Priming and loading flow cell
35. In a new tube, prepare the library for loading as follows:

 

SECTION: Priming and loading flow cell
36. Complete the flow cell priming:
    1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
    2. Load200 µL of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

SECTION: Priming and loading flow cell
37. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Priming and loading flow cell
38. Add 75 µLof sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
SECTION: Priming and loading flow cell
39. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.
SECTION: MinKNOW settings
40. When starting the sequencing run, ensure the following settings are selected

Rapid Barcoding Kit 96 (SQK-RBK110.96)
High accuracy base calling
Enable barcoding
Mid-read barcoding filtering on
Over-ride minimum barcode score 60
Fastq size set to 250 reads

Sequencing performed on MinKNOW version 22.10.7 with guppy_basecaller 6.3.9
SECTION: Pooling and clean-up
11. Pool 10 µL from each sample into a 2ml Eppendorf

SECTION: Pooling and clean-up
12. Vortex pooled samples
SECTION: Pooling and clean-up
13. Resuspend the AMPure XP Beads (AXP, or SPRI) by vortexing
SECTION: Pooling and clean-up
14. To the pooled barcoded sample, add an 0.4 x volume of resuspended AMPure XP Beads (AXP, or SPRI) (e.g. 40 µL of beads to 100 µLof pooled barcode reaction) and mix by pipetting.

SECTION: Pooling and clean-up
15. Incubate on a Hula mixer (rotator mixer) for5 min at room temperature.

SECTION: Pooling and clean-up
16. Prepare at least3 mL of fresh 80% ethanol in nuclease-free water.

SECTION: Pooling and clean-up
17. Spin down the sample and pellet on a magnet for 3 min. Keep the tube on the magnet, and pipette off the supernatant.

SECTION: Pooling and clean-up
18. Keep the tube on the magnet and wash the beads with 1 mL of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: Pooling and clean-up
19. Repeat the previous step.
SECTION: Pooling and clean-up
20. Briefly spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 s, but do not dry the pellet to the point of cracking.

SECTION: Pooling and clean-up
21. Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 µL Nuclease Free water. Incubate for 10 min at room temperature.

SECTION: Pooling and clean-up
22. Pellet the beads on a magnet for 3 minuntil the eluate is clear and colourless.

SECTION: Pooling and clean-up
23. Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Pooling and clean-up
24. Quantify DNA concentration by using the Qubit dsDNA BS Assay Kit.

SECTION: Pooling and clean-up
25. Take forward 1000 ng of library and make up the volume to11.25 µL Nuclease Free water.

SECTION: Pooling and clean-up
26. Add0.75 µLof Rapid Adapter F (RAP F) to 11.25 µLof barcoded DNA.

SECTION: Pooling and clean-up
27. Incubate at room temperature on Hula mixer (rotator mixer) for 5 min. Then store on ice until ready to load

SECTION: Rapid Barcoding
6. Add 5 µL of pool A and 2.5 µL of pool B of each sample to a new strip tube.

SECTION: Rapid Barcoding
7. In another well of the new 0.2ml PCR strip tube, add0.1 µL of lambda phage to  7.4 µL nuclease free water

SECTION: Rapid Barcoding
8. Transfer 2.5 µL from the Rapid Barcode Plate to the corresponding well of 7.5 µL containing pooled PCR product. Mix by pipetting. Spin down.
SECTION: Rapid Barcoding
9. Incubate the plate:

30 °C for 2 min
80 °C for 2 min 
4 °Chold