TITLE: Field-adapted PPRV whole genome MinION library build
AUTHORS: Emeli Torsson, Emeli Torsson, Oskar Karlsson Lindsjö

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Peste-des-petit-ruminants virus is currently the focus of a control and eradication program launched by the FAO and OIE. One of the important steps in the control strategy is strengthening the laboratory capacity and improve molecular methods to characterize field isolates. This protocol describes the preparation of samples for sequencing using the MinION sequencer, from cDNA synthesis, PCR amplification using multiplex primers to loading off the sample on the flow cell. The miniPCR is used as the thermo cycler, making this protocol fully field-adapted and ready to use in the field or in a less well-equipped lab.</div></div>

[STEPS]
?. [Amplicon preparation]
Set up the following reaction  AB1Random hexamers (50 ng/µl)1 µl2dNTP mix (10 mM)1 µl&nbsp;3RNA1 - 11 µl4DEPCto reach 13 µl5Total&nbsp;13 µlMix and centrifuge, then heat to anneal primers
AB1Random hexamers (50 ng/µl)1 µl2dNTP mix (10 mM)1 µl&nbsp;3RNA1 - 11 µl4DEPCto reach 13 µl5Total&nbsp;13 µl
65 °C
?. [End Repair / dA-tailing]
For each sample, set up the following end-repair /dA-tailing reaction

  AB1Ultra II End Prep Reaction Buffer
3.5 μl
2Ultra II End Prep Enzyme Mix
1.5 μl
3DNA from previus step25 μl4Total
30 μl
Gently mix reaction by pipetting up and down at least 10 times, then quickly centrifuge to collect all liquid.
AB1Ultra II End Prep Reaction Buffer
3.5 μl
2Ultra II End Prep Enzyme Mix
1.5 μl
3DNA from previus step25 μl4Total
30 μl
?. [End Repair / dA-tailing]
In thermocycler (heated lid at >75 °C) sett up the following protocol  AB120 °C5 minutes265 °C (inactivate enzyme)5 minutes
AB120 °C5 minutes265 °C (inactivate enzyme)5 minutes
?. [Magnetic bead purification]
Add 30 µl of AMPure/HighPrep beads to reaction and mix by flicking tube
?. [End Repair / dA-tailing]
Put directly on ice for at least 30 seconds
?. [Adaptor ligation]
AB1Barcoded and pooled DNA from previous step20 µl2Barcode Adapter Mix II (AMII)5 μl3Blunt/TA Ligase master mix254Total
50 µl
AB1Barcoded and pooled DNA from previous step20 µl2Barcode Adapter Mix II (AMII)5 μl3Blunt/TA Ligase master mix254Total
50 µl
?. [Loading samples to flow cell]
Prepare the following reaction

  ABC1Sequencing Buffer (SQB)37.5 µl&nbsp;2Loading Beads (LB)25.5 µl&nbsp;3DNA Library&nbsp;12 µl&nbsp;4Total
&nbsp;75 µl&nbsp;
ABC1Sequencing Buffer (SQB)37.5 µl&nbsp;2Loading Beads (LB)25.5 µl&nbsp;3DNA Library&nbsp;12 µl&nbsp;4Total
&nbsp;75 µl&nbsp;
?. [Barcoding]
Pepare the following barcoding reaction.  AB1Native barcode1 µl2Blunt/TA Ligase master mix10 µl3End-prepped DNA*9 µl4Total20 µl
AB1Native barcode1 µl2Blunt/TA Ligase master mix10 µl3End-prepped DNA*9 µl4Total20 µl
?. [Adaptor ligation]
Pool samples in equimolar concentration in DNA LoBind 1,5 ml Eppendorf tubes to get a total of 0.15 pmol.Dilute pooled sample to 20 μl  (or as much as needed if volume needed exceeds 20 µl) in Nuclease-free water.
?. [RNA extraction]
Extract RNA using prefered method, spin column or magnetic beads.
?. [Amplicon preparation]
Prepare the following reaction  AB15x SuperScript IV buffer4 µl2DTT (100 mM)1 µl3Ribonuclease Inhibitor1 µl4SuperScript IV Reverse Transcriptase1 µlMix and centrifuge, then add to the annealed RNA (13 µl)Set up the following thermocycling settings  AB123 °C10 min255 °C15 min380 °C10 min
AB15x SuperScript IV buffer4 µl2DTT (100 mM)1 µl3Ribonuclease Inhibitor1 µl4SuperScript IV Reverse Transcriptase1 µl
AB123 °C10 min255 °C15 min380 °C10 min
?. [Amplicon preparation]
Put directly on ice for at least 1 minute
?. [Amplicon preparation]
Add 1 µl RNase H to each tube to degarde left over RNA
37 °C
?. [Primer design]
Primers designed using the Zibra project: http://primal.zibraproject.org/For PPRV we used an amplicon lenght of 800 bp and 100 bp overlap.Prepare two primers pools (10µM), one with even numbered primer pairs and on with odd number primer pairs (https://www.nature.com/articles/nprot.2017.066)
?. [Amplicon preparation]
Prepare the following reaction  AB1Q5 reaction buffer (5x)5 µl2dNTP mix (10 mM)0.5 µl3Q5 DNA polymerase0.25 µl4Primer pool 1 or 2 (10 µM)variable*5DEPCUp to 22.5 µl6Total22.5 µlAdd 2.5 µl of cDNA from step 4
AB1Q5 reaction buffer (5x)5 µl2dNTP mix (10 mM)0.5 µl3Q5 DNA polymerase0.25 µl4Primer pool 1 or 2 (10 µM)variable*5DEPCUp to 22.5 µl6Total22.5 µl
?. [Amplicon preparation]
Set up the following thermocycling settings  ABC1198 °C30 sec22-4098 °C15 sec32-4065 °C5 min
ABC1198 °C30 sec22-4098 °C15 sec32-4065 °C5 min
?. [Amplicon purification]
Add AMPure/HighPrep magnetic beads to PCR reaction tube according to the following equationVolume of beads per reaction = 1.8 x Reaction VolumeFor example: PCR reaction volume 50 µl + 90 µl AMPure beads
?. [Amplicon purification]
Mix the beads and PCR reaction thoroghly by pipetting up and down 10 times.Incubate in RT for 3-5 minutes
[Incubate at RT]
?. [Amplicon purification]
Place tube in magnetic rack for 5-10 minutes to seperate beadsAspirate the now clear solution from the reaction and discard
?. [Amplicon purification]
Add 200 µl newly prepared 70% ethanol to reaction tube when still on magnetic rack. Incubate for 30 sec at room temperature (RT).Aspirate the etahnol and discard. Repeat wash once more.
[Incubate at RT]
[Incubate at RT]
?. [Amplicon purification]
Allow reaction tubes to air-dry in RT until completly dry and total evaporation of residual ethanol. 10-20 min
?. [Amplicon purification]
Add 40 µl of elution buffer (TE buffer, 10 mM Tris-Acetate pH 8.0, or reagent grade water) to each reaction tube. Mix thoroghly by vortex or pipetting up and down.Replace tube back to magnetic rack. When solution is cleared, remove and save for downstream reactions.
?. [End Repair / dA-tailing]
Dilute 0.12 pmol PCR products in nuclease free water to 25 µlFor our amplicons of 800 bp this means 60 ng of PCR product in 25 µl water. To convert mass/moles: https://nebiocalculator.neb.com/#!/dsdnaamt
?. [Magnetic bead purification]
Incubate on Hula mixer for 5 min at RT
?. [Magnetic bead purification]
Centrifuge tube and place on magnetic rack to pellet the magnetic beads. When solution is clear, pipet of supernatant and discard
?. [Magnetic bead purification]
While on magnatic rack, add 200 µl of freshly prepared 70 % ethanol to the tube. Remove ethanol without disturbing the bead pellet. Repeat wash one more time.
?. [Magnetic bead purification]
Spin down beads and then place again on magnetic rack. Pipette of any residual ethanol. Allow to air dry for 3-5 min at RT
?. [Magnetic bead purification]
Remove tube from magnetic rack and resuspend pellet in 15 µl nuclease-free water and mix by flicking the tube.Incubate for 2 min at RT.
?. [Magnetic bead purification]
Place tube on magnatic rack and allow beads to pellet.
?. [Quantification]
Quantify 1 µl of end-prepped DNA using Qubit flourometer.Recovery aim: 50-70 % of input
?. [Barcoding]
Thaw Native Barcodes at RT, individually mix by pipetting, and then place on ice.
?. [Magnetic bead purification]
Add 20 µl of AMPure/HighPrep beads to reaction and mix by flicking tube
?. [Magnetic bead purification]
Incubate on Hula mixer for 5 min at RT
?. [Magnetic bead purification]
Centrifuge tube and place on magnetic rack to pellet the magnetic beads. When solution is clear, pipet of supernatant and discard
?. [Magnetic bead purification]
While on magnatic rack, add 200 µl of freshly prepared 70 % ethanol to the tube. Remove ethanol without disturbing the bead pellet. Repeat wash one more time.
?. [Magnetic bead purification]
Spin down beads and then place again on magnetic rack. Pipette of any residual ethanol. Allow to air dry for 3-5 min at RT
?. [Magnetic bead purification]
Remove tube from magnetic rack and resuspend pellet in 10 µl nuclease-free water and mix by flicking the tube.Incubate for 2 min at RT.
?. [Adaptor ligation]
Incubate for 10 min at RT
?. [Loading samples to flow cell]
Mix the prepared library gently by pipetting up and down just before loading.Add 75 µl of sample to the flow cell via the SpotON sample port dropwise, making sure that each drop flows into the port before adding the next.Gently replace the SpotON sample port cover, close priming port and replace the MinION lid.
?. [Purification and wash]
Add 20 µl of AMPure/HighPrep beads to reaction and mix by flicking tube.If you needed to increase smaple volume in prevoius step, calculate 0.4X beads per reaction volume.
?. [Purification and wash]
Centrifuge tube and place on magnetic rack to pellet the magnetic beads. When solution is clear, pipet of supernatant and discard
?. [Purification and wash]
Add 250 µl of S Fragment Buffer (SFB). Flick the tube to resuspend beads, then return tube to magnetic rack to pellet the beads. Pipet of supernatant. Repeat this step once more. Make sure to get all of the SFB out. Spin down and pipet out again if needed.
?. [Purification and wash]
Remove tube from magnetic rack and resuspend pellet in 15 µl nuclease-free water and mix by flicking the tube.Incubate for 10 min at RT.
?. [Purification and wash]
Place tube on magnatic rack and allow beads to pellet. Remove the now clear solution and add to new 1.5 ml DNA LoBind eppendorf tube. Store on ice.
?. [Purification and wash]
Incubate on Hula mixer for 5 min at RT
?. Start sequence run
?. [Quantification]
Quantify 1 µl of end-prepped DNA using Qubit flourometer.Recovery aim: > 0.011 pmol /sample (equal to ~5.5 ng per sample for 800 bp amplicons).
?. [Quantification]
Quantify 1 µl purified DNA using Qubit flourometer.
?. [Loading samples to flow cell]
Thaw Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT), and one tube of Flush Buffer (FLB) at RT, then place on ice as soon as thawed.
?. [Loading samples to flow cell]
Take out flow cell. Open lid and slide the priming port cover clockwise to make the priming port visiable.
?. [Loading samples to flow cell]
Check for small bubbles under the cover. Draw back a small volume of buffer to remove possible bubbles (Set P1000 pipette to 200 µl, insert tip in port, turn until pipette set to 220-230 µl)
?. [Loading samples to flow cell]
Load 800 µl of priming mix into flow cell via priming port, avoid introducing air. Wait for 5 minutes.
?. [Loading samples to flow cell]
Prepare the flow cell priming mix.Add 46 µl of thawed FLT directly to tube of thawed and mixed FLB. Mix by pipetting up and down.
?. [Barcoding]
Mix gently by flicking the tube, spin down.Incubate the reaction at RT for 10 min.
?. [Magnetic bead purification]
Place tube on magnatic rack and allow beads to pellet.
?. [Loading samples to flow cell]
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.Load 200 µl of priming mix into the flow cell via the priming port (not SpotOn port), do not introduce air.
?. [Amplicon preparation]
Pool each sample, so both primer pools per sample are in the same tube
?. [Optional: Gel eletrophoresis]
(Optional step to check PCR reaction)Run 3-5 µl of sample on a 1 % agarose gel, 6-7 V/cm, 50-60 min.Expected outcome: A clear band at amplicon size (e.g. 800 bp) and possibly a second band at x3 amplicon size (e.g.  ~2400 bp)