Loading Oxford Nanopore flow cells: a beginner’s guide

Oxford Nanopore sequencing takes place, of course, on your nanopore sequencing device. But more precisely, the sequencing itself happens thanks to the flow cell on that device.

Preparing your flow cell, adding your sample, and kicking off your sequencing experiment is fast and simple. In this Nanopore Know-How blog, we’ll walk you through the process.

What is a flow cell?

A flow cell is the consumable part in the sequencing stage of your Oxford Nanopore workflow. It contains the chemistry that allows sequencing to take place when you add your DNA, RNA, or cDNA sample, including the nanopores themselves.

The flow cell is connected electronically to your nanopore sequencing device, so that the electrical signal from DNA/RNA sequencing can be measured and used to identify the sequence of bases. To learn about this in detail, take a look at the blog: how Oxford Nanopore sequencing works.

What kinds of flow cells are available?

The MinION Flow Cell is compatible with the portable MinION, for sequencing on a single flow cell in the lab or field, as well as the flexible GridION, on which you can run up to five at once.

The high-output PromethION Flow Cell pairs with our powerful PromethION 2 Integrated and PromethION 24 devices, allowing you to run up to 2 or 24 flow cells at a time, respectively. You can place, run, and remove flow cells on your GridION or PromethION whenever you need to, without affecting any other ongoing experiments on your device.

For both MinION and PromethION Flow Cells, we offer two chemistries: one for sequencing DNA and cDNA, and another for sequencing native RNA.

What does a flow cell look like?

Within each type of flow cell, the nanopore sequencing chemistry itself is contained within the array, which sits under a layer of buffer. When your flow cell arrives, it will be filled with storage buffer, giving the array a yellow-green appearance.

The MinION Flow Cell features two different ports where you can reach this array: the priming port, which you’ll use to prepare your flow cell, and the SpotON port directly over the array, where you’ll drop your sequencing library.

The PromethION Flow Cell, meanwhile, has just the one inlet port, which you’ll use for both of these steps. Each of these ports has its own cover to prevent evaporation when they’re not in use. Both flow cells also feature a waste channel, into which excess buffer flows, and waste ports, where waste can be removed. To learn more about what’s inside a flow cell, visit the flow cells information page.

How do I prepare a flow cell for sequencing?

Here’s how to prepare and load your flow cell, step by step. When you’re ready to run your sequencing experiment, make sure to follow the protocol for your workflow at this stage, which will detail the process in full.

Insert

First, insert your flow cell into your sequencer. On a MinION or GridION, slide your MinION Flow Cell under the clip on your device, then press down over the priming port cover to ensure good contact. On a PromethION, slide your PromethION Flow Cell into the docking port on your device until the gold pins are out of sight, then press it down — you’ll hear a click when it’s in place.

Prepare

Next, slide open the priming port cover (for MinION Flow Cells) or the inlet port cover (for PromethION Flow Cells), exposing the port underneath. If you look closely, you’ll see a small amount of air in this port ahead of the storage buffer. You’ll need to remove this air pocket — a normal feature of flow cells — to make sure you don’t introduce any air to the array.

We have a trick for this. Take a P1000 pipette and, without holding on to the plunger, place the tip into the exposed port. Then, slowly twist up the wheel on the pipette plunger to increase the volume shown on the dial until a small amount of buffer is pulled into the pipette tip and the air pocket has been removed from the flow cell.

Prime

Next, it’s time to add priming mix, which will flush the storage buffer from the array and ensure conditions are perfect for sequencing your sample. This is done in two rounds.

On a MinION Flow Cell, add in your first flush of priming mix by slowly pipetting it into the priming port — which you just removed the air pocket from — and wait five minutes. Each time you pipette into your MinION or PromethION Flow Cell, make sure to stop just short of the full volume, before you reach the second stop on the plunger, to keep a little solution in your pipette tip. This prevents introducing any air into the array.

Then, lift the SpotON port cover to expose the SpotON port. Add in your second flush, not through this port but into the priming port you used the first time. As you do this, you’ll see buffer welling up a little in the SpotON port, then sinking back into the array. Once again, this ensures that there’s no air in the port, in preparation for the next step. Pause for another five minutes after this second flush.

On a PromethION Flow Cell, you’ll be using the inlet port for both flushes. Pipette your priming mix slowly into this port, wait five minutes, then repeat this process for the second flush.

Your flow cell is now primed and ready for sequencing.

Load

Last, but not least, it’s time to load your sample onto your flow cell.

On a MinION Flow Cell, add your sample via the SpotON port you opened in the previous step using a ‘dropwise’ technique. To do this, simply hover your pipette over the port without touching the tip to the surface, then slowly dispense your sample so that it enters the port one drop at a time and sinks into the array. For a quick video demonstration of this process, take a look at how to load a MinION Flow Cell.

On a PromethION Flow Cell, add your sample using the same inlet port where you added your priming mix. You don’t need to use the dropwise method for this; just pipette your sample directly into the port. For a detailed guide to PromethION Flow Cell loading, check out our masterclass video.

Finally, close the port covers, place the light shield on your flow cell that arrives with your flow cell pack, and replace the sequencer lid. Protecting flow cells from light during sequencing extends pore lifetime and improves flow cell outputs.

You’re ready to go. It’s then over to MinKNOW — the software that controls your device — for the quick process of kicking off your run. Before you know it, you’re generating nanopore sequencing data in real time.

What will you be sequencing? Buy flow cells and discover a world of applications.

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