DOH Workshop Protocol Part 3: Library preparation for Rapid Sequencing DNA V14 Barcoding kit (SQK-RBK114.24) with Pronex modification

Prepare one 0.2 ml thin-walled PCR tube for each sample from the previous step. Label the top of the tube with the barcode number.

In each 0.2 ml thin-walled PCR tubes:

Pipette an appropriate amount of your sample (1µL-10µL) and add NFW (1µL-10µL), if necessary, to get 200ng-300ng of DNA in a 10µLtotal volume.

1.5µL of your chosen Rapid Barcode (RB01-24). (1 barcode per sample).

Mix by gently by stirring or pipetting until thoroughly mixed. If there are any bubbles present, spin down briefly (0h 0m 2s-0h 0m 3s) to ensure all liquid is at the bottom.

Note

Barcodes will be a thicker liquid, visually check they have been mixed in.

Incubate the tubes in the thermal cycler (PCR machine) at 30°C for 0h 2m 0s then 80°C for another 0h 2m 0s.

Briefly place the tubes On ice to cool.

Pool all your samples into a 1.5ml clean eppendorf DNA LoBind tube. Note down the total volume after pooling. It should be 11.5µLmultiplied by the number of samples.

Note

Only 1 person is required to carry out the following steps.

Resuspend the Pronex beads by vortexing for 0h 0m 10s or longer.

Use a 1:1 ratio of sample to Pronex beads and mix into the sample by slowly pipetting 10 times.

Note

If sticky clumps of bead-bound DNA form, be careful not to take any beads either in the pipette tip or on the outside of the pipette tip.

Incubate the sample at Room temperature for 0h 10m 0s.

To do while waiting

Take out Elution Buffer (EB) to thawOn ice.

Place the sample on a magnetic stand for 0h 2m 0suntil the solution becomes clear and the beads form a pellet on one side of the tube.

While leaving the tube on the magnet, carefully remove and discardsupernatant without disturbing the beads.

Wash 1: While still on the magnetic stand, carefully add 200µL of Pronex wash buffer without flushing directly onto the pellet. If 200µL is not enough to submerge the pellet, use more wash buffer.

Allow to incubate for 0h 0m 30s-0h 1m 0s .

While leaving the tube on the magnet, carefully remove and discard supernatant without disturbing the beads.

Wash 2: While still on the magnetic stand, carefully add 200µLof Pronex wash buffer without flushing directly onto the pellet. If 200µL is not enough to submerge the pellet, use more wash buffer.

Allow to incubate for 0h 0m 30s-0h 1m 0s .

While leaving the tube on the magnet, carefully remove and discard supernatant without disturbing the beads.

Allow the sample to air dry with lids open for 0h 2m 0s-0h 5m 0s watching it until the pellet is no longer shiny.

Note

Remove the tube from the magnetic rack and add 15µL of elution buffer (included in the Nanopore Rapid Barcoding kit). Resuspend the beads by slowly pipetting or stirring with the pipette tip.

Note

Be as gentle as possible while ensuring that pellet is resuspended.

Incubate the samples at Room temperature for 0h 10m 0s to elute the DNA.

Return the tube to the magnetic stand for 0h 1m 0suntil the solution becomes clear and the beads form a pellet.

Store the Pronex beads in the fridge.

Transfer 11µL of the eluate into a clean 1.5ml Eppendorf DNA LoBind tube.

Note

Take another 1µLof the elute from the tube on the magnetic stand for quantification on a fluorometer (Qubit or Quantus). The remaining beads can be kept in a closed tube On ice, for re-elution, if necessary.

In a new 1.5mL Eppendorf DNA LoBind tube, mix the following:

• 1.5µLRapid Adapter (RA)
• 3.5µL Adapter Buffer (ADB)

Add 1µL of this RA + ADB mixture to the DNA.

Mix gently by flicking and spin down briefly (0h 0m 2s-0h 0m 3s).

Incubate this for0h 30m 0s atRoom temperature.

Preparing the flowcell

Remove the following Nanopore Rapid Kit (RBK-114.24) items from the -20°Cfreezer, spin down and store On ice.

• SB (Sequencing Buffer)
• LIB (Library Beads)
• FCT (Flow Cell Tether)
• FCF (Flow Cell Flush)
• Bovine Serum Albumin (BSA) at 50mg/ml

Prepare the flow cell Priming Mix in a fresh 1.5mL Eppendorf DNA LoBind. Mix by inverting the tube.

• 1170µL FCF
• 5µL BSA (Bovine Serum Albumin)
• 30µL FCT

Remove the flow cell you want to use and slide it under the metal clip in the Mk1B or Mk1C MinION. Press down firmly to ensure correct contact with the thermal and electrical connections.

• Mk1B: Plug in the MinION Mk1B to a laptop with Minknow software
• Mk1C: Turn on the MinION Mk1C.

Complete a flow cell check to assess the number of pores available on the flow cell.

Rotate the flow cell priming port cover clockwise to open the priming port.

After opening the priming port there will be a small air bubble under the cover that needs to be removed.

• Set a P1000 pipette to 200µL
• Insert the tip into the priming port
• Turn the adjustment wheel slowly, pausing every few μls,until the pipette shows 220µL - 230µLto draw a total of 20µL-30µL out of the priming port, or until you can see a small volume of liquid entering the pipette tip.

Note

There may be a small delay before the liquid comes out of the port into the pipette tip. Do not draw out more than 30µL.Check that there is a continuous flow of buffer from the priming port to the nanopore sensor array, and that no bubbles are present.

Load slowly 800µLof the Priming Mix from Step 30 into the priming port, without introducing any bubbles. Leave for 0h 5m 0s and while waiting, proceed to the next step.

Note

Mix the LIB (Library Beads) thoroughly by pipetting.

In a new 1.5 ml LoBind tube, mix the following to prepare your library:

• 37.5µL SB (Sequencing Buffer)
• 25.5µL LIB (Library Beads), mixed immediately before use
• 12µL of DNA library (your sample)

Gently open the SpotON sample port cover

Load slowly 200µL of the Priming Mix into the priming port ( not the SpotON sample port ). Again, avoid introducing any air bubbles.

Note

Mix the prepared library by gently pipetting just prior to loading drop by drop 75µLof the prepared library from Step 37 onto the SpotON sample port. Let each drop flow into the port before adding the next drop.

Replace the SpotON sample port cover and gently press down to ensure the bung is in the port.

Rotate the Priming port cover back to close the Priming port.

Quickly cover the lid of the MinION to protect from light.

Start the run on the Minknow software interface.