I watched an updated version of the “Hot to load a PromethION Flow Cell” training tonight. Tomek Dobrzycki, a Field Applications Scientist from Oxford Nanopore Technologies, was the presenter. This is part of the masterclass series with the tagline “from sample to answer.” Dobrzycki began by explaining how one nanopore per well can be arranged. These differences can be seen in the flow cells: Flongles have 126 channels and can be used for library QC, plasmid, viral, and bacterial sequencing. MinION flow cells have 512 pores, can be used to generate 10-20 Gbases of data and multiplex small genomes. PromethION flow cells can generate hundreds of gigabases of data. On any of these flow cells, reads as short as twenty bases can be sequenced. Different flow cells are compatible with different devices: Flongle and MinION flow cells can be used with MinION (single position) or GridION (five positions) devices. The PromethION flow cells can be run on the P2 solo, P2 Integrated, or the PromethION 24 or 48. This is the first time I have seen a demo with the P2 with a compute unit. Dobrzycki explained that they recommend taking out ~20 minutes before using to equilibrate to room temperature. Each flow cell has a single priming/loading port and a printed ID number. The port is opened by rotating clockwise 90 degrees. The flow cells are shipped with an air pocket that is removed using a pipette to remove a little liquid. Using a P1000 set to 200, the liquid is slowly removed by turning the volume to 230. The priming mix (0.5 ml) is inserted into the inlet port. A tip shared was to leave a little bit of liquid in the tip to avoid introducing air bubbles. Now, you wait five minutes before repeating the step and adding 0.5 ml of priming mix. The waste channel fills as liquid goes over the array. The loading beads settle, and inverting them several times is essential. I have been vortexing them and wonder if I shouldn’t do that! The inlet channel is filled with 200 ul of the library. After loading the library, the next step is to close the inlet port to prevent evaporation. Then, the flow cell light shield is placed. A recorded demo describing the loading of MinION flow cells was shared. The loading is very similar, with 800 ul of priming mix followed by an additional 200 ul. This last step is done with the SpotON port open to remove any air bubbles. The library is loaded by hovering above the SpotON port and adding the library dropwise. Another recording was played describing how Flongle flow cells are loaded. Flongle flow cells use a piece of tape to cover the port, and the loading is done by directly making contact between the tip with the library and the Flongle port. The comparison of loading requirements was useful.
