Oxford Nanopore flow cell priming and loading tutorial

This video is a recording from a training session for one of our studies funded by BSAC (Twitter @AmrCovid) - link below:
The tutorial shows you how to prime and load a flow cell, ready for sequencing and gives extra tips on how to make sure that you are not introducing any air to your flow cell. Please note that this is a practice session and (as mentioned in the video) we are practicing on a USED flow cell for training purposes. The steps are the same as priming and loading a new flow cell for sequencing. Note that the camera cut out just before the end, so the Zoom recording was used for the final few minutes. ONT have a KZbin video showing you how to prime and load a flow cell that you should watch first!
All the documentation and links mentioned in the video can be found on our The Global Health Network's hub page pandora.tghn.o...

[@user-qe8uj2tw9j]

Could you please help me to understand the tether story, what is it, why do i add it? is it added to the sample in the library preparation step? does it bind to the flow cell or DNA or both? Many thanks in advance!

[@fatimaruna9855]

While you prime the flow cell or before you load your sample, if you notice a bubble in the flow cell channel, but hasn't reached to the sensor array yet, how would you try getting rid of the bubble? What if there is a big bubble already before priming, how do you get rid of it, we are not meant to pipette out more than 20-30 ul, what if the bubble is not completely tackled by taking 30 ul out? Thanks.

[@pandora-id-netconsortium1846]

The best cure for bubbles is to carefully avoid adding them in the first place. But if there is a bubble before the ASIC panel, you could test to see if it is stuck in it's position, or whether it will move. If it is stuck in place, you might be able to add your buffer without disturbing it, if you are slow and careful. If it does move, you could try and suck it back up through the priming port, but be careful to make sure not to suck up so much that the pores are exposed to air from the other end of the channel.

[@ariomstudios7366]

When you place the spot-on cap back on (especially when reusing flow cells, is there a risk of introducing contaminants from previous experiments?

[@pandora-id-netconsortium1846]

There is no chance of contaminating the run if it is a new flow cell, because samples need barcodes, adaptors etc. to be sequenced and recognised. When washing a flow cell for re-use, you should flush the wash buffer through with the SpotON port closed. That should ensure that any DNA from previous samples will be broken down. The longer you leave it, the less the chance of cross-contamination, but that is of course not completely guaranteed. If reusing a flow cell, it's recommended the samples are very different (e.g. tuberculosis and then SARS-CoV-2), so that if contamination has occurred, the contaminating reads can be removed post-sequencing. I hope that's helpful.

[@melanieshields6816]

Thank you! A very useful video. Every video I have watched refers to a priming or sequencing buffer, but I can’t find what this buffer is. Can I make this buffer, or is there a reagent I can buy?

[@pandora-id-netconsortium1846]

Hi Melanie, what we mean when we (and others) refer to the 'priming' or 'sequencing' buffer, is the combination of Flush Buffer (FB) and Flush Tether (FLT) that you then apply to the priming port, prior to loading your sample into the SpotON port. With most sequencing kits, you will also receive the Flow Cell Priming Kit (EXP-FLP002), which is made up of these components. The preparing and addition of the priming/sequencing buffer is shown in another of our tutorial videos: kzbin.info/www/bejne/hmbcZZ-cpaiIqtk which you might find useful. I hope that helps!

[@melanieshields6816]

@@pandora-id-netconsortium1846 Many thanks for the information and additional video!

[@katerinapa8652]

That's very informative, thank you. However, it seems to me that you did create an air bubble there, no?

[@pandora-id-netconsortium1846]

Nope, no air bubbles here! We've found that doing each of the steps (sucking up a bit first, then creating a small blob before you add buffer in) really eliminates air bubble risks. Also worth keeping an eye on the channel between the priming port and the sensor array (where the pores are), so you can check for any potential air movement and stop before you let air hit the pores (if there's air in the channel, you'll see that it's pale grey in colour, rather than black)