Fast, easy and robust DNA extraction for on-site bacterial identification using MinION

My name is Peter Rendbæk, and I’m currently a master student in the Albertsen lab. The overarching aim of my master project, is as a pre-test for several of the new big projects in the group, which focus on applying the on-line bacterial identification for process control at wastewater treatment plants. Hence, last couple of months I have been working on the project “Developing methods for on-site DNA sequencing using the Oxford Nanopore MinION platform”. The MinION has improved a lot since its release three years ago, and it can now be used to make rapid determination of bacterial compositions.

The potential for this fast and mobile DNA-sequencing is mind-blowing. However, given that the technology is here now (!), there has been relatively little focus on portable, fast, easy and robust DNA extraction. Hence, I’ve spent the last months on trying to develop a fast, cheap, mobile, robust and easy to use DNA extraction method.

There is a significant amount of bias connected with DNA extraction, but the bias associated with wastewater treatment samples has been investigated in depth. However, the “optimized method” is not suited for on-site DNA-extraction. There are 3 principle steps in DNA extraction, cell lysis, debris removal and DNA isolation, which I will cover below and discuss how I simplified each step.

In general, complex samples require bead beating for cell lysis and homogenization. The problem is that our in-house bead-beating is done by a big table top tool weighing 17 kg, which makes it hard to transport. However, I came across a blog post from loman labs about sample preparation and DNA extraction in the field for Nanopore sequencing. In the blog post, the possibilities of a portable bead beater outlined, by the use of a remodeled power-tool. I thought this was interesting, so I went out and bought an Oscillating Multi-Tool cutter and tried this with lots of duct tape…

The amazing part was that it worked! But the problem was that the samples would get “beaten” differently depend on how you taped the sample to the power-tool, which could give rise to variation large variations in the observed microbial community.

I solved this by 3D printing an adapter to the power-tool that fits the bead-beater tube (Finally, a good excuse to use a 3D printer!). I used Solidworks to design the adapter and collaborated with our local department of mechanical and manufacturing engineering (m-tech) in 3D printing it. You can make your own by simply downloading my design from Thingiverse (It did take a few iterations to make it durable enough, and I still use a little duct tape..).

 

After the bead beating, the cell debris removal is done by centrifugation. Our “standard” protocol recommends centrifugation at 14000 x G for 10 minutes at 4 C. However, in our minds that seemed a little extensive and requires a huge non-transportable centrifuge… Alternatively, there are a lot of possibilities to use small, easy to transport and easy to use centrifuges if we do not have to centrifuge at 14.000 xG at 4 C. There is even the possibility to 3D print a hand-powered centrifuge. However, I did not follow this path, as it seems a bit dangerous… After several tests, we discovered that a simple table top centrifuge could do the job perfectly well, using 2000 xG for 1 min at room temperature if we combined it with the DNA isolation described below.

The last step is DNA isolation, I tried several different methods, but we got the idea to simply use Agencourt AMPure XP that is routinely used in e.g. PCR purification (we 10 diluted the AMPure XP beads 1:10 to save some money and it seems to work just as good). And… It works..

So, now you have an overview of the method I developed. The most amazing part is that it works! It takes 10-15 minutes from the sample is taken until you’ve got ready DNA for use, compared to 60+ minutes for our “standard” protocol. Furthermore, it requires inexpensive equipment that can be carried in a small suitcase. So, just to prove that this approach is fast, I filmed myself doing the DNA extraction with a GO-PRO camera, as you can see below.

The next part is to test the MinION in the lab. How, fast can we identify bacteria and is the extracted DNA compatible with the downstream library preparation, which we hope to do on the our new and shiny Voltrax (which is now moving liquids!).

Small fry – master student at MEWE16

As a newly started master student it can be a bit intimidating to attend a conference. Nevertheless, I said yes when I was offered to attend MEWE16 in Copenhagen. I had just ended a one-year maternity leave and was eager to start my thesis and talk to adult people.

I quickly realized that I was surrounded by experts. I recognized names from articles, got a bit starstruck and started thinking: How in the … am I supposed to learn anything from this? They are so much smarter than me.

However, as the days past I realized there is a lot to take home from such an event – even for a small fry master student. So, I compiled a list:

  • Watch and learn. If you can follow the science – great! If you can’t, look for other things. In what way, did he/she present data? What worked? What did not? Did someone use other means to get their message through? One thing that I found could really keep my attention was humor. Not like comedy – a little goes a long way – but when my mind started to wander, humor redirected my attention back to the talk.
  • Poster sessions. I was surprised that the poster session was so crowded and it quickly became awkward because I was unprepared. So, take some time to browse the posters and note if anyone is of particular interest. Should there be no one of interest, use it as practice in “talking science” to people you don’t know. A suggestion could be: 1) go to a poster 2) ask: “Can you tell me about your poster?” At least for me, it is very good practice.
  • Networking can be many things. Don’t feel like you have to network with all the big guys, just because you’re in the same room. I am lucky enough to belong to a group that treasures social gatherings and I spent most of my time building my “local network”.
  • Be nice to yourself. I think you should be fair to yourself and realize that very few talks are directed toward you. It is a crowd of experts and if you want to have a relevant debate on your topic, maybe you have to narrow your talk and that’s not always for small fry. This is an opportunity to get some fresh air, see the city or browse the posters as mentioned earlier.