How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Experiment background by 
Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 Updated 2025-07-16PuntSeq is a side project led by a few University of Cambridge 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.
The data collected in this experiment, together with other collection sessions done by the organizers actually led to a publication on eLife: elifesciences.org/articles/61504 "Freshwater monitoring by nanopore sequencing" by Lara Urban et al. (2021), so it is awesome to see that were are actual being part of "real science".
Ciro knows nothing about biology, but since he is very curious about it, 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!
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Overview of the experiment by Ciro Santilli 40 Updated 2026-08-21
Ciro Santilli 40 Updated 2026-08-21For those that know biology and just want to do the thing, see: Section "Protocols used".
The PuntSeq team uses an Oxford Nanopore MinION DNA sequencer made by Oxford Nanopore Technologies to sequence the 16S region of bacterial DNA, which is about 1500 nucleotides long.
This kind of "decode everything from the sample to see what species are present approach" is called "metagenomics".
This is how the MinION looks like: Figure 1. "Oxford Nanopore MinION top".
Oxford Nanopore MinION top open
. Source. 
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.
The PCR replicates only the DNA region between our two selected primers a gazillion times so that only those regions will actually get picked up by the sequencing step in practice.
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: Code 1. "RNA stem-loop structure".
This is how the 16S RNA secondary structure looks like in its full glory: Figure 5. "16S RNA secondary structure".
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.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Sample collection by Ciro Santilli 40 Updated 2026-08-21
Ciro Santilli 40 Updated 2026-08-21As 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.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it DNA extraction by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 Updated 2025-07-16 How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it PCR by Ciro Santilli 40 Updated 2026-08-21
Ciro Santilli 40 Updated 2026-08-21More generic PCR information at: Section "Polymerase chain reaction".
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 participants 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.
This process used a Marshal Scientific MJ Research PTC-200 Thermal Cycler:
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: 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 (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 Code 1. "PCR diagram".
This means that we bought a few different versions of our 27F/1492R primers, each with a different small DNA tag attached directly to them in addition to the matching sequence.
This way, we were able to:
- use a different barcode for samples collected from different locations. This means we
- did PCR separately for each one of them
- for each PCR run, used a different set of primers, each with a different tag
- the primer is still able to attach, and then the tag just gets amplified with the rest of everything!
- sequence them all in one go
- then just from the sequencing output the barcode to determine where each sequence came from!
Input: Bacterial DNA (a little bit)
... --- 27S --- 16S --- 1492R --- ...
|||
|||
vvv
Output: PCR output (a lot of)
Barcode --- 27S --- 16S --- 1492RCode 1.
PCR diagram
. Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Sequencing by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 Updated 2025-07-16 How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Bioinformatics by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 Updated 2025-07-16Because Ciro's a software engineer, and he's done enough staring in computers for a lifetime already, and he believes in the power of Git, he didn't pay much attention to this part ;-)
According to the eLife paper, the code appears to have been uploaded to: github.com/d-j-k/puntseq. TODO at least mention the key algorithms used more precisely.
Ciro can however see that it does present interesting problems!
Because it was necessary to wait for 2 days to get our data, the workshop first reused sample data from previous collections done earlier in the year to illustrate the software.
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, the data was analyzed 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 would later upload the data to well known open databases so that it will be preserved forever! When ready, a link to the data would be uploaded to: www.puntseq.co.uk/data
How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it Conclusions by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 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 External links to this page by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 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
Includes its own copy of sqlite3, you don't use the system one, which is good to ensure compatibility. The version is shown at: github.com/mapbox/node-sqlite3/blob/918052b538b0effe6c4a44c74a16b2749c08a0d2/deps/common-sqlite.gypi#L3 SQLite source is tracked compressed in-tree: github.com/mapbox/node-sqlite3/blob/918052b538b0effe6c4a44c74a16b2749c08a0d2/deps/sqlite-autoconf-3360000.tar.gz horrendous. This explains why it takes forever to clone that repository. People who don't believe in git submodules, there's even an official Git mirror at: github.com/sqlite/sqlite
It appears to spawn its own threads via its C extension (since JavaScript is single threaded and and SQLite is not server-based), which allows for parallel queries using multiple threads: github.com/mapbox/node-sqlite3/blob/v5.0.2/src/threading.h
Hello world example: nodejs/node-sqlite3/index.js.
As of 2021, this had slumped back a bit, as maintainers got tired. Unmerged pull requests started piling more, and
better-sqlite3 Node.js package started pulling ahead a little.- github.com/mapbox/node-sqlite3/issues/1381
FATAL ERROR: Error::ThrowAsJavaScriptException napi_throwwith Node.jsworker_threadsvsbetter-sqlite3Node.js package github.com/JoshuaWise/better-sqlite3/issues/237
Nick Leeson and the Fall of the House of Barings by Adam Curtis (1996) by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 Updated 2025-07-16 Oxford Master Course in Mathematical and Theoretical Physics by Ciro Santilli 40 Updated 2025-07-16
Ciro Santilli 40 Updated 2025-07-16Isomers were quite confusing for early chemists, before atomic theory was widely accepted, and people where thinking mostly in terms of proportions of equations, related: Section "Isomers suggest that atoms exist".
Microwave only found applications into the 1940s and 1950s, much later than radio, because good enough sources were harder to develop.
One notable development was the cavity magnetron in 1940, which was the basis for the original radar systems of World War II.
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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