Setting a sequencing run with a nanopore MinION and the Rapid Sequencing gDNA kit (SQK-RAD004)

Thaw all reagents in box 1 and 2 of the RAD004 kit.

Take a flow cell from 4-8°C and leave it at room temperature (RT).

Prepare the DNA to the suggested concentration of 400ng total in 7.5µL, or a concentration of 54 ng/ul in a 0.2mL sterile thin-walled PCR tube. Adjust the volume using nuclease-free water. Mix by gently flicking the tube 5 times. DO NOT vortex. Spin down briefly in a micro-centrifuge.

Add 2.5µL of FRA to the tube containing the prepared DNA from the previous step. Mix by gently flicking the tube as above and spin down.

Incubate in Thermocycler at 30°C for 0h 1m 0s, then at 80°C for 0h 1m 0s. Let it cool at RT.

Add 1µL of RAP to the tube. Mix gently by flicking 5 times and spin down. Incubate for 0h 5m 0s. After this step your library DNA is ready for mixing with the sequencing reagents and loading to the flow cell. While this step is running you can start setting up the priming of the flow cell.

Flow cell priming:

Mix FB and FLT (independently) by vortexing and spin down. Then add 30µL of FLT to 1 tube of FB and mix by pipetting thoroughly. This mixture will be use to prime the flow cell.

Place the RT flow cell in the MinION device and proceed with the flow cell QC by clicking on flow cell check and wait till the program reports back how many live pores are in the flow cell. If live pores are below 800, do not continue sequencing (if very important samples) . Contact the vendor if the flow cell is still under warranty for a replacement.

If the flow cell passed QC then open the priming port by sliding the priming port cover clockwise.

By inserting a pipette tip on the priming port, remove approximately 30µL avoiding introducing bubbles.

Add 800µL of the FB+FLT solution prepared on step 7 to the priming port, avoiding introducing bubbles, by slowly dialing down the pipette. Wait 0h 5m 0s.

After the 0h 5m 0s, gently open the flow cell SpotON cover and expose the loading port. Add 200µL of FB+FLT to the priming port (NOT the SpotON port). Make sure that the loading port does not have any bubble.

To the tube containing the DNA library add the following:

• 34µL of the sequencing buffer (SQB) - red

• 25.5µL of loading beads (LB) - pink (Thoroughly mixed before use)

• 4.5µL of nuclease-free water

Mix thoroughly by pipetting and make sure that the solution looks homogenous. This is the sequencing reaction ready to be loaded to the flow cell.

Add the 75µL of the sequencing library to the SpotON port in a dropwise fashion. Make sure that every drop falls into the port and that it goes in, before adding the next drop.

Replace the cover over the SpotON and close the priming lid port, and close the MinION device lid

On the Minknow software select Start, then Start sequencing and follow the instructions for setting the sequencing run. Experiment name (I use the same as the flow cell serial number), then select the kit (RAD004), The run options I leave it mostly default except that I change the time to 48h 0m 0s, the basecalling to fast (or high accuracy, your choice), Leave the output default (you can point it to wherever you want the run to be save, then just click start at the end and your sequencing run will start.