Ever since we first heard about MinION genetic sequencers we’ve been excited to see how we can use this technology at Science Practice. We recently managed to try it out for the first time.
We first received our MinION last year but have had to go through several steps to get started. Firstly, hunting down a computer with very specific requirements (Windows 7 is becoming increasingly hard to find!), and then verifying our hardware and software setup by running a data exchange with a dummy (“configuration”) flow cell. The following screenshot shows uploading, processing and downloading 20 test data files with Metrichor, which is Nanopore’s cloud-based analysis tool.
Only then could we order our flow cells and their accompanying reagents. These reagents need to be stored at -20 °C so we bought a household freezer hopeful that -18 °C would do the trick.
Inspired by stories and pictures of using MinION in exotic places, we were raring to go with our MinION and new laptop. However we soon realised that we would need a table-full of other lab equipment. Besides specialist equipment for quantifying DNA in a sample, even standard equipment like pipettes and vortexers add up to a basic equipment bill of around £5000, and facilities for storage and disposal are also necessary. This equipment list made the MinION significantly less portable than it first appeared.
To get started with MinION we decided to first trial the protocol in an established lab. Lauren Cowley from Public Health England had previously given a talk about her work with MinION at one of our Mobile Health Meetups, and she kindly agreed to show us the ropes.
When you first start using a MinION, Oxford Nanopore recommends running a standard control ‘burn-in’ experiment to familiarise yourself with the protocol, the equipment and what the data looks like. The burn-in involves sequencing a small viral genome called lambda phage, and comparing it against a reference to check that everything is working properly.
Over in Lauren’s lab at Public Health England, our first step in running the burn-in was to set up all the equipment and reagents we would need for the experiment. Cue a line up of pipettes, tubes for mixing, a magnetic rack, reagents on ice, and heating, mixing and measuring equipment.
A genetic sample needs a few processing steps before it can be loaded onto the MinION for sequencing. This is known as ‘library preparation’ because a long DNA strand is broken up into a ‘library’ of DNA fragments with special sequences on their ends.
Fragmented DNA of around 8kb is recommended for the burn-in, as this allows important enzymes to be bound to the ends of each fragment, and also allows the DNA to efficiently pass through the pores of the flow cell. Fragmentation can be done in a number of ways, but for the purpose of this simple burn-in we relied on DNA’s natural fragmentation during processing.
The next step is to bind enzymes to each end of the DNA which facilitate the sequencing reactions. One enzyme is responsible for recognising the pore on a flow cell and directing the DNA fragment into it, while the other creates a ‘hairpin loop’ between the double strands of DNA. This means that after a single strand of DNA has been through a pore, its complement is pulled through afterwards, creating two reads of each fragment.
After each sample-prep step it is important to clean up the DNA and get rid of lingering chemicals from previous steps. The clean up step relies on tiny magnetic beads with enzymes that bind and unbind DNA under certain chemical conditions, which allows the separation of DNA from non-genetic material. In the clean up step, Eppendorfs containing the DNA sample mixed with magnetic beads are placed on a magnetic tube rack. The strong magnet concentrates and holds onto the magnetic beads and DNA, so that the rest of the fluid can be carefully pipetted off.
Because it was our first experiment, sample preparation took us about 2-3 hours, but it could be done in an hour.
With the library prepped, we were ready to connect our MinION to our laptop, open up the sequencing program called MinKNOW on our laptop, and load our sample into the MinION for sequencing.
The day wouldn’t have felt complete without some drama, so naturally the MinION and our laptop stubbornly refused to talk to each other at first. Once that was resolved, we found that MinKNOW needed an internet connection to start sequencing the lambda phage, but we were offline. Luckily the lab wasn’t too far underground and we were able to get just enough signal by leaning a mobile phone on the windowsill to create a wifi hotspot for our laptop.
The MinION itself is a chassis which houses (semi-) disposable cartridges called flow cells:
Prepared DNA is loaded into a flow cell, where it flows over a membrane speckled with nanopores. Probably the trickiest part of the whole experiment is preventing any bubbles from flowing across the membrane. The flow cell also contains the electronics to read the electrical signatures of DNA bases as they pass through the pores.
A nice feature of the MinKNOW software is that it displays the status of how well each pore is functioning. The high number of pores labelled green on screen told us that our flow cell was in great shape.
As soon as we started the sequencing run in MinKNOW we could see the data coming in because the sequencing is in real-time. Our experiment ran for several hours overnight, but we did manage to disconnect our phone without interrupting it. The burn-in produced about 16,000 .fast5 files which amounts to 4GB. This is a pretty amazing amount of data considering that the 60kb genome we sequenced is considered tiny. This gives an idea of the amount of data that can be generated by genetic sequencing, and the special processing requirements that are necessary for handling it.
We are really excited to investigate how and where we can use MinION, especially as part of our focus on citizen science, resourceful engineering and bringing scientific analysis outside of the lab. Now that we’ve worked through the protocol in a fully equipped lab, we can see how it could be adapted for the field. Our excellent guide Lauren recently published a paper in Nature (Go Lauren!), describing how they used Minion to track Ebola in Guinea by packing all the required equipment into a flight suitcase. This gives us plenty of hope and inspiration to use the sequencer in difficult, and unusual contexts. For our next adventures in sequencing, watch this space!
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