One cool thing we did in this procedure was to use magnetic separation with magnetic beads to further concentrate the DNA: Figure 1. "GE MagRack 6 pipetting.".

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!

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:

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!

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 4. "Oxford nanopore MinION flow cell pipette loading.".

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 Ciro of: 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 Video 1. "Oxford Nanopore MinION software channels pannel on Mac." the software tells us which pores are still working.

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.

www.qiagen.com/gb/products/discovery-and-translational-research/dna-rna-purification/dna-purification/microbial-dna/dneasy-powerwater-kit (archive) Here is its documentation: www.qiagen.com/gb/resources/download.aspx?id=bb731482-874b-4241-8cf4-c15054e3a4bf&lang=en (archive).

Manual archive: web.archive.org/web/20190911111136/https://www.qiagen.com/gb/resources/download.aspx?id=bb731482-874b-4241-8cf4-c15054e3a4bf&lang=en

Kit to extract clean DNA from water.

www.qiagen.com/us/products/discovery-translational-research/dna-rn-a-purification/dna-purification/dna-clean-up/qiaquick-pcr-purification-kit/#orderinginformation (archive)

Manual archive: web.archive.org/web/20190911100243/https://www.qiagen.com/us/resources/download.aspx?id=e0fab087-ea52-4c16-b79f-c224bf760c39&lang=en

Removes PCR byproducts from purified DNA.

store.nanoporetech.com/ligation-sequencing-kit.html (archive)

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.

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:

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 4. "Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination.".

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

As barely seen at Figure 8. "Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel." 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.

At the time of the experiment, Illumina equipment was cheaper per base pair and dominates the human genome sequencing market, but it required a much higher initial investment for the equipment (TODO how much).

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! Note that 1x is basically useless for one of the most important of all applications of sequencing: detection of single-nucleotide polymorphisms, since the error rate would be too high to base clinical decisions on.

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:

www.fishersci.no/shop/products/nalgene-polysulfone-reusable-bottle%20-top-filters/10465781 (archive)

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.

www.knfusa.com/en/laboport/ (archive).

www.scientificindustries.com/vortex-genie-2.html (archive)

uk.vwr.com/store/product/8306728/microcentrifuges-ventilated-refrigerated-micro-star-17-17r (archive).

www.velp.com/en/products/lines/3/family/44/vortex_mixers/65/wizard_ir_infrared_vortex_mixer (archive).

www.marshallscientific.com/MJ-Research-PTC-200-Thermal-Cycler-p/mj-200.htm (archive).

www.gelifesciences.com/en/us/shop/protein-analysis/protein-sample-preparation/protein-enrichment/magrack-6-p-05761 (archive).

www.btlabsystems.com/Centrifuges/Mini_Centrifuge_Fixed_7K (archive).

Manual: web.archive.org/web/20190908094334/https://www.btlabsystems.com/downloads/BT602_Mini_Centrifuge_7K_Fixed.pdf

www.bidspotter.com/en-us/auction-catalogues/bscsur/catalogue-id-bscsur10011/lot-c6605b41-1a14-40e5-a255-a5c5000866e0 (archive) Cannot exact same product on official website, but here is a similar one: www.fishersci.co.uk/shop/products/lm-26-2uv-transilluminator/12382038 (archive).

biochromspectros.com/spectrophotometers/simplinano-cat/simplinano-spectrophotometer.html (archive)

Manual: biochromspectros.com/media/wysiwyg/support_page/support-simplinano/Simplinano-UM.pdf (archive)

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 1. "An Eppendorf tube". Reagents are expensive, and lab bench centrifuges are small!

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:

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 1. "Cutting vacuum pump filter and placing it in Eppendorf".

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 cell and free the DNA.

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 1. "White precipitate formed with Qiagen DNeasy PowerWater Kit".

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:

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:

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: