TITLE: Amplicon sequencing on the MinION platform
AUTHORS: Yoshiyuki Matsuo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Amplicon sequencing on the MinION platform</div></div>

[STEPS]
?. [Materials]
KAPA2G Robust HotStart ReadyMix (2X) (KAPA BIOSYSTEMS, KK5701)Inner primer (forward): 5'-TTTCTGTTGGTGCTGATATTGC - target-specific sequence -3' (User-supplied)Inner primer (reverse): 5'-ACTTGCCTGTCGCTCTATCTTC - target-specific sequence -3' (User-supplied)PCR Barcoding Kit (Oxford Nanopore Technologies, SQK-PBK004)AMPure XP (Beckman Coulter, A63880)70% ethanolElution buffer: 10 mM Tris-HCl pH 8.0 with 50 mM NaClQuantiFluor ONE dsDNA System (Promega, E4871)Flow cell R9.4 (Oxford Nanopore Technologies, FLO-MIN106D)Flow Cell Priming Kit (Oxford Nanopore Technologies, EXP-FLP001)
?. [Equipment]
Thermal cyclerGel electrophoresis deviceMagnetic rackQuantus Fluorometer (Promega, E6150)MinION sequencer (Oxford Nanopore Technologies)
?. [Workflow]
?. [1st PCR with inner primers]
Prepare the PCR master mix.ComponentVolumeFinal conc.Template DNAx µl10 µM FW/RV primer mix0.5 µl0.2 µM each2X KAPA2G12.5 µl1XWater12 - x µlTotal25 µl
ComponentVolumeFinal conc.Template DNAx µl10 µM FW/RV primer mix0.5 µl0.2 µM each2X KAPA2G12.5 µl1XWater12 - x µlTotal25 µl
?. [1st PCR with inner primers]
Perform PCR.StepTemperatureTimeCyclesInitial denaturation95°C3 min1Denaturation95°C15 sec25-35Annealing55°C15 secExtension72°C30 secHold4°C∞1
StepTemperatureTimeCyclesInitial denaturation95°C3 min1Denaturation95°C15 sec25-35Annealing55°C15 secExtension72°C30 secHold4°C∞1
?. [1st PCR with inner primers]
Analyze the PCR product (2-5 µl) with gel electrophoresis to verify successful amplification.
?. [2nd PCR with barcoded outer primers]
Prepare the PCR master mix.ComponentVolume1st PCR products1.0 µlLWB 01-120.5 µl2X KAPA2G12.5 µlWater11 µlTotal25 µl
ComponentVolume1st PCR products1.0 µlLWB 01-120.5 µl2X KAPA2G12.5 µlWater11 µlTotal25 µl
?. [2nd PCR with barcoded outer primers]
Perform PCR.StepTemperatureTimeCyclesInitial denaturation95°C3 min1Denaturation95°C15 sec8-10Annealing62°C15 secExtension72°C30 secHold4°C∞1
StepTemperatureTimeCyclesInitial denaturation95°C3 min1Denaturation95°C15 sec8-10Annealing62°C15 secExtension72°C30 secHold4°C∞1
?. [2nd PCR with barcoded outer primers]
[Optional] Analyze the PCR product (1 µl) with gel electrophoresis.
?. [PCR cleanup]
Resuspend the AMPure XP beads by vortexing.
?. [PCR cleanup]
For selecting fragments of over 500 bp, add 10 μl of AMPure XP to 20 µl of PCR product (0.5x ratio).
?. [PCR cleanup]
Mix by pipetting and incubate for 5 min at room temperature.
?. [PCR cleanup]
Place the tube on a magnetic rack for 2 min to magnetically separate out the beads.
?. [PCR cleanup]
Pipette off the supernatant.
?. [PCR cleanup]
Keeping on the magnetic rack, add 200 μl of 70% ethanol without disturbing the bead pellet, and discard the supernatant.
?. [PCR cleanup]
Repeat step 15 (wash the beads twice in total).
?. [PCR cleanup]
Spin down and place the tube back in the magnetic rack.
?. [PCR cleanup]
Pipette off any residual ethanol.
?. [PCR cleanup]
Air-dry for 1 min.*Do not over dry the magnetic beads.
?. [PCR cleanup]
Remove the tube from the magnetic rack and resuspend the beads in 10 μl of elution buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl).
?. [PCR cleanup]
Incubate for 2 min at room temperature.
?. [PCR cleanup]
Place the tube on a magnetic rack for 2 min.
?. [PCR cleanup]
Transfer the eluate to a new tube.
?. [DNA quantification]
Warm QuantiFluor ONE dsDNA dye to room temperature.
?. [DNA quantification]
Add 1 µl of eluted sample to 200 μl of QuantiFluor ONE dsDNA dye in 0.5 ml tube.
?. [DNA quantification]
Mix thoroughly by vortexing.
?. [DNA quantification]
Incubate for 5 min at room temperature, protected from light.
?. [DNA quantification]
Measure fluorescence using the Quantus Fluorometer for quantifying DNA concentrations.
?. [DNA quantification]
[Optional] Analyze the sample with gel electrophoresis.
?. [Sequencing library preparation]
Pool all barcoded amplicons to a total of 50-100 fmoles in 10 μl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl.*For full-length 16S rRNA gene amplicons (approximately 1,500 bp), 50-100 fmoles of DNA equates to ~50-100 ng.
?. [Sequencing library preparation]
Add 1 μl of RAP and mix gently by pipetting.*RAP is supplied in the PCR Barcoding Kit (SQK-PBK004).
?. [Sequencing library preparation]
Incubate for 5 min at room temperature.
?. [Sequencing library preparation]
Store the library on ice until ready to load.
?. [Flow cell check]
Open the MinION lid and insert the flow cell under the clip.
?. [Flow cell check]
Perform flow cell QC.
?. [Flow cell check]
Check the number of active pores available for the experiment.
?. [Sample loading]
Prepare flow cell priming mix by adding 30 µl of Flush Tether (FLT) to the tube of Flush Buffer (FB).*FLT and FB are supplied in the Flow Cell Priming Kit (EXP-FLP001).
?. [Sample loading]
After opening the priming port, load 800 µl of the priming mix into the flow cell via the priming port and wait for 5 min.
?. [Sample loading]
Prepare the sequencing library for loading.Component VolumeLibrary11 µlWater4.5 µlSequencing Buffer (SQB)34 µlLoading Beads (LB)25.5 µlTotal75 µl
Component VolumeLibrary11 µlWater4.5 µlSequencing Buffer (SQB)34 µlLoading Beads (LB)25.5 µlTotal75 µl
?. [Sample loading]
Lift the spotON sample port cover and load 200 μl of the priming mix into the flow cell via the priming port (NOT the sample port).
?. [Sample loading]
Mix the prepared library gently by pipetting just prior to loading.
?. [Sample loading]
Load the library (75 µl) into the flow cell via the SpotON sample port in a dropwise fashion.
?. [Sample loading]
Replace the SpotON sample port cover and close the priming port.
?. [Sample loading]
Start the sequencing run.