Ciro Santilli

# How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it

## 1. Setting

PuntSeq is a side project led by a few Cambridge University PhDs that aims to determine which bacteria are present in the River Cam.

In July 2019, the PuntSeq team got together with the awesome Cambridge Biomakespace, an awesome biology makerspace open to all, to create a two day science outreach activity showing their procedures.

Ciro knows nothing about biology, but since he is very curious about it, in particular he jumped at this opportunity, and decided to document things as well as his limited knowledge would allow.

All participants chipped in some money to help cover the experiment’s costs. Ciro suspects that this activity was done partially to help crowdfund the experiment, but it was a worthy investment!

The impressions you get from the experiment as a software engineer will be:

• OMG, this is so labour intensive, why haven’t they automated this

• OMG, this is frightening, all the 8 hours of work I’ve just done are present in that tiny plastic tube

• Amazing! Look at that apparatus! And the bio people are like: I’ve used this a million times, it’s cheap and every lab has one, just work faster and don’t break you piece of junk!

## 2. Overview of the experiment

For those that know biology and just want to do the thing, see: Section 9, “Protocols used”.

The PuntSeq team uses an Oxford Nanopore MinION sequencer made by Oxford Nanopore Technologies to sequence the 16S region of bacterial DNA, which is about 1500 nucleotides long. This is how the MinION looks like: Figure 1, “Oxford Nanopore MinION top. Source.”.

Figure 1. Oxford Nanopore MinION top. Source.
Figure 2. Oxford Nanopore MinION side. Source.
Figure 3. Oxford Nanopore MinION top open. Source.
Figure 4. Oxford Nanopore MinION side USB. Source.

The 16S region codes for one of the RNA pieces that makes the bacterial ribosome.

Before sequencing the DNA, we will do a PCR with primers that fit just before and just after the 16S DNA, in well conserved regions expected to be present in all bacteria.

Eukaryotes also have an analogous ribosome part, the 18S region, but the PCR primers are selected for targets around the 16S region which are only present in prokaryotes.

This way, we amplify only the 16S region of bacteria, excluding other parts of bacterial genome, and excluding eukaryotes entirely.

Despite coding such a fundamental piece of RNA, there is still surprisingly variability in the 16S region across different bacteria, and it is those differences will allow us to identify which bacteria are present in the river.

The variability exists because certain base pairs are not fundamental for the function of the 16S region. This variability happens mostly on RNA loops as opposed to stems, i.e. parts of the RNA that don’t base pair with other RNA in the RNA secondary structure as shown at: Listing 1, “RNA stem-loop structure”.

Listing 1. RNA stem-loop structure
                A-U
/   \
A-U-C-G-A-U-C-G     C
| | | | | | | |     |
U-A-G-C-U-A-G-C     G
\   /
U-A
|             ||    |
+-------------++----+
stem        loop

This is how the 16S RNA secondary structure looks like in its full glory: Figure 5, “16S RNA secondary structure. Source.”.

Figure 5. 16S RNA secondary structure. Source.

Since loops don’t base pair, they are less crucial in the determination of the secondary structure of the RNA.

The variability is such that it is possible to identify individual species apart if full sequences are known with certainty.

With the experimental limitations of experiment however, we would only be able to obtain family or genus level breakdowns.

### 2.1. Why Oxford Nanopore was used instead of Illumina for the sequencing

Illumina equipment is currently cheaper per base pair and dominates the human genome sequencing market, but it requires a much higher initial investment for the equipment

The reusable Nanopore device costs just about 500 dollars, and about 500 dollars (50 unit volume) for the single usage flow cell which can decode up to 30 billion base pairs, which is about 10 human genomes 1x!

Compare that to Illumina which is currently doing about an 1000 dollar human genome at 30x, and a bit less errors per base pair (TODO how much).

Other advantages of the MinION over Illumina which didn’t really matter to this particular experiment are:

One disadvantage of the Oxford Nanopore is that its error rate per base pair read is higher than Illumina.

### 2.2. Metagenomics

This kind of "sequence some random DNA to determine what is present in the sample" analysis is called metagenomics.

Besides river sample, other types of samples where of metagenomics is used include:

One related application which most people would not consider metagenomics, is that of finding circulating tumor DNA in blood to detect tumors.

## 3. Sample collection

As you would expect, not much secret here, we just dumped a 1 liter glass bottle with a rope attached around the neck in a few different locations of the river, and pulled it out with the rope.

The temperature of the water was measured with a mercury thermometer, and the PH with pH strips and a cell phone app to compare the color of the strip.

There were some swans in the river, so…​ swan poo bacteria I guess?

And, in the name of science, we even wore gloves to not contaminate the samples!

Who said you can’t have fun with science? Video 1, “River water sample collection with a bottle and string. Source.”

Figure 7. River water sample collection swans. Source.
Figure 8. River water sample collection tie rope to bottle. Source.
Figure 9. River water sample collection get sample. Source.
Figure 10. River water sample collection measure temperature. Source.
Figure 11. River water sample collection read PH strip. Source.
Figure 12. River water sample collection identify bottle. Source.
Video 1. River water sample collection with a bottle and string. Source.

## 4. DNA extraction

The first thing we had to do with the sample was to extract the DNA present in the water in a pure form for the PCR.

We did that with a Qiagen DNeasy PowerWater Kit.

As you would expect, this consists of a purification procedure with several steps.

In each step we take a physical or chemical action on the sample, which splits it into two parts: the one with the DNA and the one without.

We then take the part with the DNA, and throw away the one without the DNA.

The first steps are coarser, and finer and finer splits are done as we move forward.

### 4.1. Filtration with vacuum pump

The first thing we did was to filter the water samples with a membrane filter that is so fine that not even bacteria can pass through, but water can.

Therefore, after filtration, we would have all particles such as bacteria and larger dirt pieces in the filter.

From the 1 liter in each bottle, we only used 400 ml because previous experiments showed that filtering the remaining 600 ml is very time consuming because the membrane filter gets clogged up.

Therefore, the filtration step allows us to reduce those 400 ml volumes to more manageable Eppendorf tube volumes: Figure 13, “An Eppendorf tube is small and convenient. Source.”. Reagents are expensive, and centrifuges are small!

Figure 13. An Eppendorf tube is small and convenient. Source.
Figure 14. Labelled Eppendorf microcentrifuge tubes on rack. Source.

Since the filter is so fine, filtering by gravity alone would take forever, and so we used a vacuum pump to speed thing up!

For that we used:

Figure 15. Vacuum pump filter peel filter. Source.
Figure 16. Vacuum pump filter place filter. Source.
Video 2. Vacuum pump filter pour sample and turn on. Source.

### 4.2. Post filtration purification

After filtration, all DNA should present in the filter, so we cut the paper up with scissors and put the pieces into an Eppendorf: Video 3, “Vacuum pump filter cut and place in eppendorf. Source.”.

Video 3. Vacuum pump filter cut and place in eppendorf. Source.

Now that we had the DNA in Eppendorfs, we were ready to continue the purification in a simpler and more standardized lab pipeline fashion.

First we added some small specialized beads and chemicals to the water and shook them Eppendorfs hard in a Scientific Industries Inc. Vortex-Genie 2 machine to break the cells and free the DNA.

Video 5. Scientific Industries Inc Vortex-Genie 2 running. Source.

Once that was done, we added several reagents which split the solution into two phases: one containing the DNA and the other not. We would then pipette the phase with the DNA out to the next Eppendorf, and continue the process.

In one step for example, the DNA was present as a white precipitate at the bottom of the tube, and we threw away the supernatant liquid: Figure 17, “Qiagen DNeasy PowerWater Kit White Precipitate. Source.”.

Figure 17. Qiagen DNeasy PowerWater Kit White Precipitate. Source.

At various stages, centrifuging was also necessary. Much like the previous vacuum pump step, this adds extra gravity to speed up the separation of phases with different molecular masses.

In our case, we used a VWR Micro Star 17 microcentrifuge for those steps:

Figure 18. VWR Micro Star 17 microcentrifuge. Source.

Then, when we had finally finished all the purification steps, we measured the quantity of DNA with a Biochrom SimpliNano spectrophotometer to check that the purification went well:

Figure 21. Biochrom SimpliNano spectrophotometer result readout. Source.

And because the readings were good, we put it in our -20 C fridge to preserve it until the second day of the workshop and called it a day:

Figure 22. Minus 20 fridge storing samples. Source.

## 5. PCR

Because it is considered the less interesting step, and because it takes quite some time, this step was done by the event organizers between the two event days, so I did not get to take many photos.

PCR protocols are very standard it seems, all that biologists need to know to reproduce is the time and temperature of each step.

We did 35 cycles of:

• 94˚C for 30 seconds

• 60˚C for 30 seconds

• 72˚C for 45 seconds

Figure 23. Marshal Scientific MJ Research PTC-200 Thermal Cycler. Source.

We added PCR primers for regions that surround the 16S DNA. The primers are just bought from a vendor, and we used well known regions are called 27F and 1492R. Here is a paper that analyzes other choices: https://academic.oup.com/femsle/article/221/2/299/630719 (archive) "Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment" by "Yuichi Hongoh, Hiroe Yuzawa, Moriya Ohkuma, Toshiaki Kudo Published" published 01 April 2003.

One cool thing about the PCR is that we can also add a known barcode at the end of each primer as shown at Listing 2, “PCR diagram”. This way, we were able to:

• add a different barcode for samples collected from different locations

• sequence them all in one go

• then just from the sequencing output the barcode to determine where each sequence came from!

Listing 2. PCR diagram
Bacterial DNA (a little bit)
... --- 27S --- 16S --- 1492R --- ...

|
|
v

PCR output (a lot of)
Barcode --- 27S --- 16S --- 1492R

Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.

### 5.1. PCR verification with gel electrophoresis

Biology experiments are hard, and so they go wrong, a lot.

For this reason, it is wise to verify that certain steps are correct whenever possible.

And so this is the first thing we did on the second day!

Gel electrophoresis separates molecules by their charge-to-mass ratio. It is one of those ultra common lab procedures!

This allows us to determine how long are the DNA fragments present in our solution.

Since we know that we amplified the 16S regions which we know the rough size of (there might be a bit of variability across species, but not that much), we were expecting to see a big band at that size.

And that is exactly what we saw!

First we had to prepare the gel, put the gel comb, and pipette the samples into wells present in the gel:

Figure 24. Gel electrophoresis insert comb. Source.
Figure 25. Gel electrophoresis top view with wells visible. Source.
Figure 26. Gel electrophoresis pipette sample into wells. Source.

To see the DNA, we added ethidium bromide to the samples, which is a substance that that both binds to DNA and is fluorescent.

Because it interacts heavily with DNA, ethidium bromide is a mutagen, and the biology people sure did treat the dedicated electrophoresis bench area with respect! Figure 27, “Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination. Source.”.

Figure 27. Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination. Source.
Figure 28. Gel electrophoresis dedicated waste bin for centrifuge tubes and pipette tips contaminated with ethidium bromide. Source.

The UV transilluminator we used to shoot UV light into the gel was the Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator. The fluorescent substance then emitted a light we can see.

As barely seen at Figure 31, “Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel. Source.” due to bad photo quality due to lack of light, there is one strong green line, which compared to the ladder matches our expected 16S length. What we saw it with the naked eyes was very clear however.

Figure 29. Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator. Source.
Figure 30. Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator loading gel. Source.
Figure 31. Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel. Source.

## 6. Sequencing

Once we had the amplified 16S DNA, we were almost ready to start sequencing!

### 6.1. Pre-sequencing preparation

One cool thing we did in this procedure was to use magnetic separation with magnetic beads to further concentrate the DNA: Figure 32, “GE MagRack 6 pipetting. Source.”.

The beads are coated to stick to the DNA, which allows us to easily extract the DNA from the rest of the solution. This is cool, but bio people are borderline obsessed by those beads! Go figure!

Figure 32. GE MagRack 6 pipetting. Source.
Figure 33. GE MagRack 6 eppendorf with magnetic beads mounted. Source.

Then we prepared the DNA for sequencing with the Oxford Nanopore specific part: Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit.

Here some of the steps required a bit more of vortexing for mixing the reagents, and for this we used the VELP Scientifica WIZARD IR Infrared Vortex Mixer which appears to be quicker to use and not as strong as the Vortex Genie 2 previously used to break up the cells:

Figure 34. VELP Scientifica WIZARD IR Infrared Vortex Mixer running. Source.

After all that was done, the DNA of the several 400 ml water bottles and hours of hard purification labour were contained in one single Eppendorf!

### 6.2. Using the Oxford Nanopore

With all this ready, we opened the Nanopore flow cell, which is the 500 dollar consumable piece that goes in the sequencer.

We then had to pipette the final golden Eppendorf into the flow cell. My anxiety levels were going through the roof: Figure 38, “Oxford nanopore MinION flow cell pipette loading. Source.”.

Figure 35. Oxford nanopore MinION flow cell package. Source.
Figure 36. Oxford nanopore MinION flow cell front. Source.
Figure 37. Oxford nanopore MinION flow cell back. Source.

At this point bio people start telling lab horror stories of expensive solutions being spilled and people having to recover them from fridge walls, or of how people threw away golden Eppendorfs and had to pick them out of trash bins with hundreds of others looking exactly the same etc. (but also how some discoveries were made like this) This reminded me of: https://youtu.be/89UNPdNtOoE?t=919 Alfred Maddock’s plutonium spill horror story.

Luckily this time, it worked out!

We then just had to connect the MinION to the computer, and wait for 2 days.

During this time, the DNA would be sucked through the pores.

As can be seen from Oxford Nanopore MinION software channels pannel on Mac. Source. the software tells us which pores are still working.

Figure 39. Oxford Nanopore MinION connected to a Mac via USB. Source.
Video 6. Oxford Nanopore MinION software channels pannel on Mac. Source.

Pores go bad sooner or later randomly, until there are none left, at which point we can stop the process and throw the flow cell away.

48 hours was expected to be a reasonable time until all pores went bad, and so we called it a day, and waited for an email from the PuntSeq team telling us how things went.

We reached a yield of 16 billion base pairs out of the 30Gbp nominal maximum, which the bio people said was not bad.

## 7. Bioinformatics

To be honest, because I’m a software engineer, and I’ve done enough staring in computers for a lifetime already, and I believe in the power of Git, I didn’t pay much attention to this ;-)

TODO at least mention the key algorithms and link to GitHub software.

I can however see that it does however present interesting problems!

Because we had to wait for 2 days to get our data, the workshop first reused sample data from previous collections done earlier in the year.

First there is some signal processing / machine learning required to do the base calling, which is not trivial in the Oxford Nanopore, since neighbouring bases can affect the signal of each other. This is mostly handled by Oxford Nanopore itself, or by hardcore programmers in the field however.

After the base calling was done, we analyzed the data using computer programs that match the sequenced 16S sequences to a database of known sequenced species.

This is of course not just a simple direct string matching problem, since like any in experiment, the DNA reads have some errors, so the program has to find the best match even though it is not exact.

The PuntSeq team uploaded / will upload the data to well known open databases so that it will be preserved forever! When ready, a link to the data will be uploaded to: https://www.puntseq.co.uk/data

## 8. Conclusions

• against all odds, the experiment worked and we got DNA out of the water, despite a bunch of non-bio newbs actively messing with random parts of the experiment

• PuntSeq and Biomakespace people, and all those tho do scientific outreach, are awesome!

• biology is hard

• creating insanely media rich articles like this is also hard, but the following helped enormously:

• Wikimedia Commons to store large media files out of Git

• Asciidoctor extensions to easily include those media files

• Nomacs to give Google Photos photos meaningful names and to edit people’s faces out of pictures ;-)

## 9. Protocols used

Protocols are the biologist term for "recipe".

I found that a lot of biology comes down to this: get the right recipe, follow it well even though you don’t understand all the proprietary details, and pray.

### 9.1. Qiagen DNeasy PowerWater Kit

Kit to extract clean DNA from water.

Figure 40. Qiagen DNeasy PowerWater Kit open box. Source.

### 9.2. Qiagen QIAquick PCR Purification Kit

Removes PCR byproducts from purified DNA.

### 9.3. Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit

Repairs the ends of DNA, and also attaches an adapter protein to the DNA that makes them go through the pores of e.g. an Oxford Nanopore MinION.

## 10. Equipment used

### 10.1. Thermo Scientific Nalgene Polysulfone Reusable Bottle Top Filters

This is where we poured the water. It was not large enough for the entire 1L sample, so we had to do it a few times.