TITLE: Nested VP1 PCR and Nanopore Sequencing from Stool and ES Samples
AUTHORS: Alex Shaw, Manasi Majumdar, Catherine Troman, Javier Martin, Nick Grassly

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is published in the paper "Rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing" by Shaw </span><span style = "font-style:italic;">et al</span><span>  in the Journal of Clinical Microbiology (2020), DOI: 10.1128/JCM.00920-20. </span></div><div class = "text-block">The protocol aims to amplify the VP1 region of poliovirus through a nested PCR using panEV primers followed by amplification of the VP1 sequence using the Q8/Y7 primer set. We advise the use of barcoded primers where possible as this greatly simplifies the subsequent library preparation process. Additional steps have however been included in the case that the second PCR step is performed either with standard Q8/Y7 primers or using Q8/Y7 with the barcode adaptors (BCA) attached. Primer sequences for the panEV primers, Q8/Y7 primers and modified Q8/Y7 primers are found in Dataset S1 of the publication. </div><div class = "text-block">Sequencing of the panEV product is also possible through the removal of the VP1 nested PCR steps and the preparation of the panEV product for sequencing. </div></div>

[STEPS]
?. [Nested PCR First Round (PanEV)]
Nested PCR First Round (panEV primers):Prepare a Master mix using reaction volumes as detailed below, excluding forward primer and the RNA:Forward Primer:   TGGCGGAACCGACTACTTTGGGTG (Arita et al. 2015)Reverse Primer:   TCAATACGGTGTTTGCTCTTGAACTG (Arita et al. 2015)  AB11 Reaction (µL)22x Master Mix12.53SS III Platinum Taq mix0.54Reverse Primer (10 µM)15Nuclease Free Water56Forward Primer (10 µM)17RNA58Total volume25
AB11 Reaction (µL)22x Master Mix12.53SS III Platinum Taq mix0.54Reverse Primer (10 µM)15Nuclease Free Water56Forward Primer (10 µM)17RNA58Total volume25
?. [Nested PCR First Round (PanEV)]
Briefly vortex and centrifuge down. Add 19 µL of master mix to each PCR tube and 5 µL of eluted RNA.
?. [Nested PCR First Round (PanEV)]
Incubate at 50 °C for 30 minutes.
?. [Nested PCR First Round (PanEV)]
Add 1 µL of the forward primer to the tubes.
?. [Nested PCR First Round (PanEV)]
Amplify using the following cycling conditions:    ABCD1CYCLESTEPTEMP (°C)TIME21Initial Denaturation942 minutes342Denaturation9415 seconds4Annealing5530 seconds5Extension684 minutes 30 seconds*61Final Extension685 minutes 7-Hold10-
ABCD1CYCLESTEPTEMP (°C)TIME21Initial Denaturation942 minutes342Denaturation9415 seconds4Annealing5530 seconds5Extension684 minutes 30 seconds*61Final Extension685 minutes 7-Hold10-
?. [Nested PCR Round 2 (VP1)]
VP1 amplification is performed using the Q8/Y7 primers. These can be modified to remove library preperation steps as shown in Shaw et al  2020, DOI: 10.1128/JCM.00920-20. The basic PCR uses the primersForward Primer (Y7): GGGTTTGTGTCAGCCTGTAATGA (Kilpatrick et al. 2011)Reverse Primer (Q8): AAGAGGTCTCTRTTCCACAT (Yang et al. 1992)These can be replaced with either Q8/Y7 primers with the barcode adaptor attached (BCA primers) or with barcodes attached (sequences in Dataset_S1 of Shaw et al  2020)Prepare a Master mix using reaction volumes as detailed below, excluding the diluted PCR product (and primers if these are barcoded):   AB11 Reaction (µL)2DreamTaq 2x master mix12.53Water9.54Forward primer (10 µM)15Reverse primer (10 µM)16PCR product1*7Total volume25* Previous protocol editions have used a 5 µL of a 1 in 20 dilution of the PCR product (5 µL in 100 µL nuclease free water); we find that 1 of the neat product works as well, if not better, and reduces the potential for contamination.
AB11 Reaction (µL)2DreamTaq 2x master mix12.53Water9.54Forward primer (10 µM)15Reverse primer (10 µM)16PCR product1*7Total volume25
?. [Nested PCR Round 2 (VP1)]
Briefly vortex and centrifuge down the master mix and aliquot 20 µL into each PCR tube (or 18 µL if using barcoded primers).
?. [Nested PCR Round 2 (VP1)]
Add 1 µL of barcoded forward primer and 1 µL of barcoded reverse primer if necessary.
?. [Nested PCR Round 2 (VP1)]
Add 1 µL of PCR product.
?. [PCR Steps]
PCR confirmation: Check a representative set of samples to confirm that the PCR has been successful.
?. [PCR Steps]
AMPure bead purification using 15 µL (1 : 0.6 ratio) of resuspended AMPure XP beads and elute in 25 µL nuclease-free water, retaining  23 µL of eluate.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Standardise DNA:Quantify 1 µL of purified PCR product using a Qubit fluorometer Transfer 1 μg of DNA into a clean PCR tube.Adjust the volume to 23 μL with nuclease-free water.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
End-prep & dA-tailing:Prepare the following reaction mix:  AB123 µL2Ultra II End-prep reaction buffer3.5 µL3Ultra II End-prep enzyme mix1.5 µL4Nuclease-free water2.5 µL
AB123 µL2Ultra II End-prep reaction buffer3.5 µL3Ultra II End-prep enzyme mix1.5 µL4Nuclease-free water2.5 µL
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Mix gently by flicking, and spin down.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Ligation of Barcode Adapter Add the reagents in the order given below:15 µL End prep DNA10 µL Barcode Adapter25 µL Blunt/TA Ligase Master Mix
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Mix gently by flicking the tube, and spin down.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Incubate the reaction for 10 minutes at room temperature.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Standardise the DNA: Quantify 1 µL of the adapted DNA using a Qubit fluorometer Transfer 100 fmol of DNA into a clean PCR tubeAdjust the volume to 24 μL with nuclease-free water.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Barcoding PCR :Set up a barcoding PCR reaction as follows for each sample:1 µL PCR Barcode24 µL 100 fmol PCR Product25 µL LongAmp Taq 2x master mix
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Mix gently by flicking the tube, and spin down.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Amplify using the following cycling conditions:    ABCD1CYCLESTEPTEMP (°C)TIME21Initial Denaturation953 minutes312Denaturation9515 seconds4 Annealing6215 seconds5 Extension652 minutes*61Final Extension652 minutes7-Hold4-* Extension time for VP1 sequencing
ABCD1CYCLESTEPTEMP (°C)TIME21Initial Denaturation953 minutes312Denaturation9515 seconds4 Annealing6215 seconds5 Extension652 minutes*61Final Extension652 minutes7-Hold4-
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
AMPure bead purification using 40 µL (1 : 0.8 ratio) of resuspended AMPure XP beads and elute in 25 µL nuclease-free water, retaining  23 µL of eluate.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Sample Pooling:Quantify 1 µL of the barcoded DNA using a Qubit fluorometerPrepare 1 µg of pooled barcoded DNA in 47 µl Nuclease-free water.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Optional- If pool volume is >47 µL, concentrate an AMPure bead purification using a 1:1 ratio of resuspended AMPure XP beads and elute in 50 µL nuclease-free water, retaining  47 µL of eluate.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
End-prep and dA-tailing: Add the following reagents in a 0.2 mL PCR tube.47 µL 1 µg DNA3.5 µL NEBNext FFPE DNA Repair Buffer2 µL NEBNext FFPE DNA Repair Mix3.5 µL Ultra II End-prep reaction buffer3 µL Ultra II End-prep enzyme mix1 µL DNA CS
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Mix gently by flicking the tube, and spin down
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Incubate for 10 minutes at 20 °C and 10 minutes at 65 °C.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Place on ice for 30 seconds.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Adaptor ligation:Thaw and prepare the kit reagents as follows:Spin down and thaw Adapter Mix (AMX) on ice Spin down T4 Ligase from NEBNext Quick Ligation Module (E6056) on iceThaw Ligation Buffer (LNB) at room temperature, spin down, mix by pipetting. Place on ice. Thaw Elution Buffer (EB) at room temperature, mix by vortexing, spin down. Place on ice. Thaw one tube of S Fragment Buffer (SFB)* for VP1 at room temperature, mix by vortexing, spin down and place on ice.*For panEV sequencing, use L Fragment Buffer (LFB)
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Prepare the following reaction mix in a 1.5 mL Eppendorf DNA LoBind tube:60 µL DNA25 µL Ligation Buffer (LNB)10 µL NEBNext Quick T4 DNA Ligase5 µL Adapter Mix (AMX)
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Mix gently by flicking the tube, and spin down.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Incubate the reaction for 10 minutes at room temperature.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
AMPure XP cleanup: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.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.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
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Store the library on ice until ready to load into a nanopore flow cell.
?. [Nested PCR Round 2 (VP1)]
Amplify using the following cycling conditions:    ABCD1CYCLESTEPTEMP (°C)TIME21Initial Denaturation952 minutes335Denaturation9530 seconds4 Annealing5530 seconds5 Extension721 minutes61Final Extension7210 minutes 7-Hold10-
ABCD1CYCLESTEPTEMP (°C)TIME21Initial Denaturation952 minutes335Denaturation9530 seconds4 Annealing5530 seconds5 Extension721 minutes61Final Extension7210 minutes 7-Hold10-
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
For Standard Primers- Complete all steps For BCA primers- Complete steps 24 to 28For Barcoded primers- Skip to Library Preperation for the ONT MinION: Pooling and adapting
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Incubate for 5 minutes at 20 °C and 5 minutes at 65 °C
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
AMPure bead purification using 30 µL (1 : 1 ratio) of resuspended AMPure XP beads and elute in 20 µL nuclease-free water, retaining  16 µL of eluate.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
Quantify 1 µL of end-prepped DNA using a Qubit fluorometer - recovery aim > 700 ng.
?. [Library Preperation for the ONT MinION: Tailing and barcoding]
AMPure bead purification using 20 µL (1 : 0.4 ratio) of resuspended AMPure XP beads and elute in 25 µL nuclease-free water, retaining  15 µL of eluate.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
Transfer sample to a 1.5 mL Eppendorf DNA LoBind tube.
?. [Library Preperation for the ONT MinION: Pooling and adapting]
AMPure bead purification using 60 µL (1 : 1 ratio) of resuspended AMPure XP beads and elute in 61 µL nuclease-free water, retaining  60 µL of eluate in a clean 1.5 mL Eppendorf DNA LoBind tube.