TITLE: Library preparation protocol to sequence full length 16S rRNA gene in Nanopore MinION sequencer
AUTHORS: Somasundhari Shanmuganandam, Yiheng Hu, Benjamin Schwessinger, Robyn Hall

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was optimised from an existing protocol to achieve maximum pore occupancy in a MinION flow cell. This protocol was performed using Ligation sequencing kit (ONT) in combination with native barcoding kit (ONT). Using this protocol, I was able to achieve ~12 Gbp of data from one MinION run from samples with very low input concentration. </div></div>

[STEPS]
?. [PCR amplification ]
1For a 100 µl PCR reaction, use the following setup  AB1ReagentsVolume (µl) for 100 µl reaction22X Platinum SuperFi PCR Master Mix50 µl3Water, nuclease-freeupto 100 µl4Template DNA5 µl (11.5 ng)510 µM forward primer5610 µM reverse primer5 µl
AB1ReagentsVolume (µl) for 100 µl reaction22X Platinum SuperFi PCR Master Mix50 µl3Water, nuclease-freeupto 100 µl4Template DNA5 µl (11.5 ng)510 µM forward primer5610 µM reverse primer5 µl
?. [PCR amplification ]
The PCR conditions are:initial denaturation at 98°C for 30 sec28 cycles of98°C for 10 sec55°C for 15 sec72°C for 40 sec with final extension at 72°C for 5 min
?. [PCR product cleanup]
Add 1 volume (100ul) of resuspended beads into each PCR reactions and mix by flicking the tubes
?. [PCR product cleanup]
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature
?. [PCR product cleanup]
Prepare 20 ml of fresh 70% ethanol in nuclease-free water.
?. [PCR product cleanup]
Spin down the sample and pellet on a magnet. Keep the tube on the 96 well magnet rack, and pipette off the supernatant.
?. [PCR product cleanup]
Keep on magnet, wash beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet using the multichannel pipette and 70% ethanol in a trough. Remove the 70% ethanol using a pipette and discard. Repeat.
?. [PCR product cleanup]
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Briefly allow to dry for 3 -5 mins
?. [PCR product cleanup]
Remove the tube from the magnetic rack and resuspend pellet in 15 µl nuclease-free water. Incubate for 2 minutes at room temperature.
?. [PCR product cleanup]
Pellet beads on magnet until the eluate is clear and colourless.
?. [PCR product cleanup]
Run 3 µl of the cleaned up DNA product in 1% agarose gel to ensure the cleaned product is free from primers.
?. [PCR product cleanup]
Run 1 µl  of DNA in qubit using high sensitivity assay kit and 1 µl in nanodrop to ensure the cleaned up DNA is free from contamination from RNA and/or protein.
?. [PCR product cleanup]
Based on the qubit reading, take 500 ng of cleaned up DNA in 25 µl of nuclease free water.
?. [End prep]
Add the reagents below in the same order in a fresh 0.8 ml PCR strip tube  AB1ReagentsVolume2500 ng input DNA25 µl3Ultra II End-prep reaction buffer3.5 µl4Ultra II End-prep enzyme mix1.5 µl5Total30 µl2
AB1ReagentsVolume2500 ng input DNA25 µl3Ultra II End-prep reaction buffer3.5 µl4Ultra II End-prep enzyme mix1.5 µl5Total30 µl
?. [End prep]
Mix gently by flicking the tube, and spin down
?. [End prep]
Incubate for 10 minutes at 20 °C and 30 minutes at 65 °C using the thermal cycler with a lid temperature of 70°C.
?. [End prep]
Add 1 volume (30 µl) of resuspended AMPure XP beads to the end-prep reaction and mix by flicking the tube.
?. [End prep]
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature
?. [End prep]
Spin down the sample and pellet on a 96 well magnet rack. Keep the tube on the magnet, and pipette off the supernatant individually
?. [End prep]
Keep on magnet, wash beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet using the multichannel pipette and 70% ethanol in a trough. Remove the 70% ethanol using a pipette and discard. Repeat.
?. [End prep]
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Briefly allow to dry for 3 -5 mins at room temperature.
?. [End prep]
Remove the tube from the magnetic rack and resuspend pellet in 15 µl nuclease-free water by flicking the tubes. Incubate for 2 minutes at room temperature.
?. [End prep]
Pellet beads on magnet until the eluate is clear and colourless.
?. [End prep]
Quantify 1 µl of end-prepped DNA using Qubit high sensitivity assay kit
?. [End prep]
For next stage, we need 80 ng per sample and hence, dilute the sample concentration to 80 ng to make a final volume of 9 µl in a new strip tube
?. [Adding barcodes]
Adding the following reagents in the same order as below  AB1ReagentsVolume280 ng end prep DNA9 µl3Native Barcode1 µl4Blunt/TA Ligase Master Mix10 µl5Total20 µl
AB1ReagentsVolume280 ng end prep DNA9 µl3Native Barcode1 µl4Blunt/TA Ligase Master Mix10 µl5Total20 µl
?. [Adding barcodes]
Mix gently by flicking the tube, and spin down
?. [Adding barcodes]
Incubate the reaction for 10 minutes at room temperature
?. [Adding barcodes]
Incubate the reaction for 10 minutes at room temperature. Add 20 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
?. [Adding barcodes]
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature
?. [Adding barcodes]
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
?. [Adding barcodes]
Keep on magnet, wash beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet using the multichannel pipette and 70% ethanol in a trough. Remove the 70% ethanol using a pipette and discard. Repeat.
?. [Adding barcodes]
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Briefly allow to dry for 3 - 5 minutes at room temperature.
?. [Adding barcodes]
Remove the tube from the magnetic rack and resuspend pellet in 12 µl nuclease-free water. Incubate for 2 minutes at room temperature
?. [Adding barcodes]
Pellet beads on magnet until the eluate is clear and colourless.
?. [Adding barcodes]
Quantify 1 µl of barcoded DNA in Qubit high sensitivity assay.
?. [Adding barcodes]
Based on the qubit reading, add 36.4 ng of each barcoded sample in a 1.5 ml eppendorf tube to get a final concentration of more than 400 ng. If the volume exceeds 50 µl, perform 2.5 x volume AMPure XP bead cleanup with final elution volume of 52 µl of nuclease free water.
?. [Adding barcodes]
Quantify 1 µl of barcoded DNA in Qubit high sensitivity assay to concentration is greater than 400 ng.
?. [Adapter ligation]
Thaw the reagents in ice
?. [Adapter ligation]
Add the reagents in following order and flick between addition of each reagents  AB1ReagentVolume2pooled barcoded DNA ( > 400 ng)50 µl3Barcode Adapter Mix (BAM)20 µl4NEBNext Quick Ligation Reaction Buffer (5X)20 µl5Quick T4 DNA Ligase10 µl6Total100 µlaeR
AB1ReagentVolume2pooled barcoded DNA ( > 400 ng)50 µl3Barcode Adapter Mix (BAM)20 µl4NEBNext Quick Ligation Reaction Buffer (5X)20 µl5Quick T4 DNA Ligase10 µl6Total100 µl
?. [Adapter ligation]
Incubate the reaction for 10 minutes at room temperature.
?. [Adapter ligation]
Prepare the AMPure XP beads for use; resuspend by vortexing. Prepare another 1ml of fresh 70% Ethanol. Prepare all the tubes and megnetic rack ready.
?. [Adapter ligation]
Add 40 µl of resuspended AMPure XP beads to the adapter ligation reaction from the previous step and mix by flicking.
?. [Adapter ligation]
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature. Take out the flowcell from the fridge to let it recover to room temperature.
?. [Adapter ligation]
Place on magnetic rack, allow beads to pellet and pipette off supernatant.
?. [Adapter ligation]
Add 140 μl of the Adapter Bead Binding buffer 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.
?. [Adapter ligation]
Remove the tube from the magnetic rack and resuspend pellet in 15 µl Elution Buffer by flicking. Incubate for 10 minutes at room temperature.
?. [Adapter ligation]
Pellet beads on magnet until the eluate is clear and colourless, Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Store on ice.
?. [Adapter ligation]
Quantify 1 µl of adapter ligated DNA in Qubit high sensitivity assay to ensure concentration is close to 200 ng
?. [Priming flowcell]
prepare the priming mix as follows in a 2 ml Eppendorf tube:  AB1ReagentVolume2Nuclease-free water576 µl3RPF624 µl4Total1200 µl
AB1ReagentVolume2Nuclease-free water576 µl3RPF624 µl4Total1200 µl
?. [Priming flowcell]
Take out the flowcell from the box, flip back the MinION lid and slide the priming port cover clockwise so that the priming port is visible.
?. [Priming flowcell]
After opening the priming port, check for small bubble under the cover. Draw back a small volume to remove any bubble (a few µls): Set a P1000 pipette to 200 µl;Insert the tip into the priming port;Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.Visually check that there is continuous buffer from the priming port across the sensor array.
?. [Priming flowcell]
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 minutes.Pipette ~830 ul of the priming mix, before putting the tip on the priming port, slightly drop a little of the liquid to cover the port and attach the tip to the port. Slowly pipette the priming mix out but DO NOT pipette out everything. Remaining the last few (~20ul) of priming mix in the tip and take off the tip straightway. This is to make sure no air goes into the port.
?. [Loading library]
Thoroughly mix the contents of the RBF and LLB tubes by pipetting, Prepare the library for loading as follows:  AB1ReagentVolume2RBF35.0 µl3LLB25.5 µl4Nuclease-free water2.5 µl5DNA library12 µl6Total75.0 µl
AB1ReagentVolume2RBF35.0 µl3LLB25.5 µl4Nuclease-free water2.5 µl5DNA library12 µl6Total75.0 µl
?. [Loading library]
Complete the flow cell priming:     1.Gently lift the SpotON sample port cover to make the SpotON sample port accessible.    2. Load 200 µl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles. The same idea, pipette ~230ul of the priming mix and be careful as the previous priming step.
?. [Loading library]
Mix the prepared library gently by pipetting up and down just prior to loading.
?. [Loading library]
Add 75 μl of 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.
?. [Loading library]
After the last drop was absorbed, quickly replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION lid.
?. [Loading library]
Run the MinION sequencer