Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures.

RNA was stored at -80 °C after extraction and thawed on ice upon use. RNA was amplified in PanEV PCR reaction using both a three primer (Arita_rev, NIBSC R3, NIBSC F4) and a pair of two primer reactions (MM_EV_F2 and MM_EV_R1, Arita_for and Arita_rev) (sequence shown in Dataset S1).

Results
For each kit and primer set, amplification was confirmed through gel electrophoresis. For the following figures, blue arrows indicate the height of the expected band.

Conclusions
Long range PCR was observed to be possible with RNA extracted using column based or magnetic bead-based method. For the column-based methods, Roche and Qiagen each gave positive bands for 8 samples using the three primer reactions, whilst Roche performed better for the two primer reactions. Zymo and NucleoSpin gave poorer amplification. Amplification of MagMax extracted RNA gave similar results to that of the Roche kit. Freshly prepared 70% ethanol in nuclease-free water

References
Nuclease-free water

References
The section "Library Preparation for the ONT MinION" is based on the Oxford Nanopore Technologies 1D PCR barcoding amplicon/cDNA (SQK-LSK109).

DNA amplification For Standard PCR-Complete steps 1 to 5 and step 13
For Nested VP1 PCR-Complete steps 1 to 13 1) Prepare a Master mix using reaction volumes as detailed below, excluding forward primer and the RNA:
• Add 15 µL (0.6 ratio) of resuspended AMPure XP beads to the reaction and mix by pipetting.
• Incubate on a rotator for 5 minutes at room temperature.
• Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant. • Keep on magnet, wash beads with 100 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard. Repeat. • Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol.
Briefly allow to dry. • Remove the tube from the magnetic rack and resuspend pellet in 25 µL nuclease-free water.
Incubate for 2 minutes at RT. • Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain 23 µL of eluate in a clean 1.5 mL Eppendorf DNA LoBind tube.
• Quantify the eluted DNA.

Library Preparation for the ONT MinION For Standard Primers-Complete all steps
For BCA primers-Complete steps 4 to 10 For Barcoded primers-Complete steps 7 to 10 1) Standardise DNA • Transfer 1 μg of DNA into a clean PCR tube.
• Adjust the volume to 23 μL with nuclease-free water.
• Incubate for 5 minutes at 20 °C and 5 minutes at 65 °C.
• Prepare the AMPure XP beads for use; resuspend by vortexing.
• Add 30 µL of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
• Incubate on a rotator for 5 minutes at room temperature.
• Spin down the sample and pellet on a magnet. Pipette off the supernatant.
• Wash beads with 100 µL of freshly prepared 70% ethanol without disturbing the pellet.
• Remove the 70% ethanol using a pipette and discard. Repeat.
• Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol.
Briefly allow to dry. • Remove the tube from the magnetic rack and resuspend pellet in 20 µL Nuclease-free water.
• Incubate for 2 minutes at room temperature.
• Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain 16 µL of eluate into a clean PCR tube.
• Quantify 1 µL of end-prepped DNA using a Qubit fluorometer -recovery aim > 700 ng. • Incubate the reaction for 10 minutes at room temperature.

3) Ligation of Barcode Adapter
• Prepare the AMPure XP beads for use; resuspend by vortexing.
• Add 20 µL of resuspended AMPure XP beads to the reaction and mix by pipetting.
• Incubate on a rotator for 5 minutes at room temperature.
• Spin down the sample and pellet on a magnet. Pipette off the supernatant.
• Keep on magnet, wash beads with 100 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard. Repeat. • Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol.
Briefly allow to dry. • Remove the tube from the magnetic rack and resuspend pellet in 25 µL nuclease-free water.
• Incubate for 2 minutes at room temperature.
• Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain 15 µL of eluate in a clean tube.

4) Standardise DNA
• Transfer 100 fmol of DNA into a clean PCR tube.
• Adjust the volume to 24 μL with nuclease-free water.

5) Barcoding PCR
• Set up a barcoding PCR reaction as follows for each sample: • Add 40 µL (0.8 ratio) of resuspended AMPure XP beads to wells and mix by pitpetting.
• Incubate on a rotator for 5 minutes at room temperature.
• Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant. • Keep on magnet, wash beads with 100 µL of 70% ethanol without disturbing the pellet.
Remove the 70% ethanol using a pipette and discard. Repeat. • Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol.
Briefly allow to dry. • Remove the tube from the magnetic rack and resuspend pellet in 25 µL nuclease-free water.
Incubate for 2 minutes at room temperature. • Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain 23 µL of eluate in a clean PCR plate.
Optional-If pool volume >47 µL concentrate with the following step: • Prepare the AMPure XP beads for use; resuspend by vortexing.
• Add the required amount of resuspended AMPure XP beads to the DNA pool and mix by pipetting. • Incubate on a rotator for 5 minutes at room temperature.
• Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant. • Keep on magnet, wash beads with 100 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard. Repeat. • Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol.
Briefly allow to dry. • Remove the tube from the magnetic rack and resuspend pellet in 50 µL nuclease-free water.
Incubate for 2 minutes at room temperature. • Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain 47 µL of the eluate in a clean 1.5 ml Eppendorf DNA LoBind tube.

8) End-prep and dA-tailing
• Add the following reagents in a 0.2 mL PCR tube. • Incubate for 10 minutes at 20 °C and 10 minutes at 65 °C.
• Place on ice for 30 seconds.
• Transfer sample to a 1.5 mL Eppendorf DNA LoBind tube.
• Prepare the AMPure XP beads for use; resuspend by vortexing.
• Add 60 µL of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
• Incubate on a rotator for 5 minutes at room temperature.
• Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant. • Keep on magnet, wash beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard. Repeat. • Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol.
Briefly allow to dry. • Remove the tube from the magnetic rack and resuspend pellet in 61 µL Nuclease-free water.
Incubate for 2 minutes at room temperature. • Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain 60 µL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube. • Mix gently by flicking the tube, and spin down.

9) Adaptor ligation
• Incubate the reaction for 10 minutes at room temperature.

10) AMPure XP bead binding
• Prepare the AMPure XP beads for use; resuspend by vortexing.
• Add 40 µL of resuspended AMPure XP beads to the adapter ligation reaction from the previous step and mix by pipetting. • Incubate on a rotator for 5 minutes at room temperature.
• Place on magnetic rack, allow beads to pellet and pipette off supernatant.
• Add 250 μL of the LFB/SFB to the beads. Close the tube lid and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant. Repeat. • Spin down the tube and place back on the magnet. Pipette off residual supernatant and briefly air dry. • Remove the tube from the magnetic rack and resuspend pellet in 15 µl Elution Buffer.
Incubate for 10 minutes at room temperature. • Pellet beads on magnet until the eluate is clear and colourless.
• Remove and retain the eluate which contains the DNA library in a clean 1.5 mL Eppendorf DNA LoBind tube • Dispose of the pelleted beads The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.

Appendix S3 -Comparison of the effect of VP1 primer modification by addition the barcode adapter or barcode sequences.
A dilution series of PV1 RNA was set up starting from a 10 -1 dilution of the RNA concentrate down to a 10 -7 dilution. RT-PCR was carried out as previously described (Appendix S2), and subsequent VP1 PCR nest was carried out with the Y7/Q8 primer set, the primers with the barcode adapter (VP1-BCA) or with ONT barcode sequence (VP1-BC70) appended to the original primer sequence (all primer sequences in Table S1). PCR products, including those from the RT-PCR, were visualised on a 1% agarose gel stained with SYBRsafe (Invitrogen) and run at 120V for 1hour with a 1kb plus DNA ladder (NEB, Figure 1).

C D
After the barcoding PCR, Y7/Q8 products were seen up to the 10 -4 dilution of original input RNA; for the VP1-BCA products, a band was only seen up to the 10 -3 dilution ( Figure 2). In the original PCR nest, the barcoded primers gave a visible product up to the 10 -4 dilution (Figure 1), with a slightly brighter band being produced in comparison to the Y7/Q8 products after the barcoding PCR. This implies that there is little difference in sensitivity of the Y7/Q8 primers -which would then have to go through the full library preparation process -and of the barcoded primers which offer a much quicker option for library preparation.