How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Sequencing by
Ciro Santilli 37 Updated 2025-07-16
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Pre-sequencing preparation by
Ciro Santilli 37 Updated 2025-07-16
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.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Using the Oxford Nanopore by
Ciro Santilli 37 Updated 2025-07-16
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!
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.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Conclusions by
Ciro Santilli 37 Updated 2025-07-16
- 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. The lessons learnt in this article were then an important motivation for Ciro's OurBigBook Markup, to which this article was later migrated.
- Nomacs to give Google Photos photos meaningful names and to edit people's faces out of pictures ;-)
- some scientific Wikipedia pages may or may not have been edited with better pictures during the course of writing this article
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Qiagen DNeasy PowerWater Kit by
Ciro Santilli 37 Updated 2025-07-16
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).
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Qiagen QIAquick PCR Purification Kit by
Ciro Santilli 37 Updated 2025-07-16
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.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit by
Ciro Santilli 37 Updated 2025-07-16
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.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Thermo Scientific Nalgene Polysulfone Reusable Bottle Top Filters by
Ciro Santilli 37 Updated 2025-07-16
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Fisher Scientific UVP LM-26E Benchtop 2UV Transilluminator by
Ciro Santilli 37 Updated 2025-07-16
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).
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it External links to this page by
Ciro Santilli 37 Updated 2025-07-16
- 2021-03-25: Oxford Nanopore Technologies retweeted this article, that's awesome!
- 2021: hackaday.com/author/wd5gnr1/ "SEQUENCING DNA FOR METAGENOMICS" by Al Williams (2021). This came after Ciro Santilli self promoted at: stackoverflow.blog/2021/02/03/sequencing-your-dna-with-a-usb-dongle-and-open-source-code/#comment-1411921
"De novo" means "starting from scratch", that is: you type the desired sequence into a computer, and the synthesize it.
The "de novo" part is important, because it distinguishes this from the already well solved problem of duplicating DNA from an existing DNA template, which is what all our cells do daily, and which can already be done very efficiently in vitro with polymerase chain reaction.
Notably, the dream of most of those companies is to have a machine that sits on a lab bench, which synthesises whatever you want.
The initial main applications are likely going to be:but the real pipe dream is building and bootstraping entire artificial chromosomes
- polymerase chain reaction primers (determine which region will be amplified
- creating a custom sequence to be inserted in a plasmid, i.e. artificial gene synthesis
News coverage:
Nuclera eDNA enzymatic de novo DNA synthesis explanatory animation (2021)
Source. The video shows nicely how Nuclera's enzymatic DNA synthesis works:- they provide blocked nucleotides of a single type
- add them with the enzyme. They use a werid DNA polymerase called terminal deoxynucleotidyl transferase that adds a base at a time to a single stranded DNA strand rather than copying from a template
- wash everything
- do deblocking reaction
- and then repeat until done
Pinned article: Introduction to the OurBigBook Project
Welcome to the OurBigBook Project! Our goal is to create the perfect publishing platform for STEM subjects, and get university-level students to write the best free STEM tutorials ever.
Everyone is welcome to create an account and play with the site: ourbigbook.com/go/register. We belive that students themselves can write amazing tutorials, but teachers are welcome too. You can write about anything you want, it doesn't have to be STEM or even educational. Silly test content is very welcome and you won't be penalized in any way. Just keep it legal!
Intro to OurBigBook
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- a Wikipedia where each user can have their own version of each article
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This feature makes it possible for readers to find better explanations of any topic created by other writers. And it allows writers to create an explanation in a place that readers might actually find it.Figure 1. Screenshot of the "Derivative" topic page. View it live at: ourbigbook.com/go/topic/derivativeVideo 2. OurBigBook Web topics demo. Source. - local editing: you can store all your personal knowledge base content locally in a plaintext markup format that can be edited locally and published either:This way you can be sure that even if OurBigBook.com were to go down one day (which we have no plans to do as it is quite cheap to host!), your content will still be perfectly readable as a static site.
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Figure 3. Visual Studio Code extension installation.Figure 4. Visual Studio Code extension tree navigation.Figure 5. Web editor. You can also edit articles on the Web editor without installing anything locally.Video 3. Edit locally and publish demo. Source. This shows editing OurBigBook Markup and publishing it using the Visual Studio Code extension.Video 4. OurBigBook Visual Studio Code extension editing and navigation demo. Source. - Infinitely deep tables of contents:
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