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Illumina by Ciro Santilli 37 Updated 2025-07-16
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The by far dominating DNA sequencing company of the late 2000's and 2010's due to having the smallest cost per base pair.
Illumina actually bought their 2010's dominating technology from a Cambridge company called Solexa.
To understand how Illumina's technology works basically, watch this video: Video 1. "Illumina Sequencing by Synthesis by Illumina (2016)".
Video 1.
Illumina Sequencing by Synthesis by Illumina (2016)
Source.
The key innovation of this method is the Bridge amplification step, which produces a large amount of identical DNA strands.
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Bridge amplification by Ciro Santilli 37 Updated 2025-07-16
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This is one of the the key innovations of the Illumina (originally Solexa) sequencing.
This step is genius because sequencing is basically a signal-to-noise problem, as you are trying to observe individual tiny nucleotides mixed with billions of other tiny nucleotides.
With bridge amplification, we group some of the nucleotides together, and multiply the signal millions of times for that part of the DNA.
Figure 1.
Illustration of the bridge amplification step of Illumina sequencing
. Source.
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Oxford Nanopore Technologies by Ciro Santilli 37 Updated 2025-07-16
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They put a lot of emphasis into base calling. E.g.:
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Oxford Nanopore Technologies product by Ciro Santilli 37 Updated 2025-07-16
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PromethION by Ciro Santilli 37 Updated 2025-07-16
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Oxford Nanopore MinION by Ciro Santilli 37 Updated 2025-07-16
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One of the sequencers made by Oxford Nanopore Technologies.
The device has had several updates since however, notably of the pore proteins which are present in the critical flow cell consumable.
Official documentation: nanoporetech.com/products/minion (archive)
Figure 1.
Top view of a closed Oxford Nanopore MinION
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Figure 2.
Side view of an Oxford Nanopore MinION
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Figure 3.
Top view of an open Oxford Nanopore MinION
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Figure 4.
Oxford Nanopore MinION side USB
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Figure 5.
Oxford nanopore MinION flow cell package.
Source.
Figure 6.
Oxford nanopore MinION flow cell front.
Source.
Figure 7.
Oxford nanopore MinION flow cell back.
Source.
Figure 8.
Oxford nanopore MinION flow cell pipette loading.
Source.
Figure 9.
Oxford Nanopore MinION connected to a Mac via USB.
Source.
Video 1.
Oxford Nanopore MinION software channels pannel on Mac.
Source.
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How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it by Ciro Santilli 37 Updated 2025-07-16
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This article gives an idea of how this kind of biological experiment feels like to a software engineer who has never done any biology like Ciro Santilli.
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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 37 Updated 2025-07-16
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PuntSeq 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!
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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 37 Updated 2025-07-16
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For 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".
Figure 1.
Oxford Nanopore MinION top
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Figure 2.
Oxford Nanopore MinION side
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Figure 3.
Oxford Nanopore MinION top open
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Figure 4.
Oxford Nanopore MinION side USB
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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.
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".
                A-U
               /   \
A-U-C-G-A-U-C-G     C
| | | | | | | |     |
U-A-G-C-U-A-G-C     G
               \   /
                U-A
|             ||    |
+-------------++----+
    stem        loop
Code 1.
RNA stem-loop structure
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This is how the 16S RNA secondary structure looks like in its full glory: Figure 5. "16S RNA secondary structure".
height=800
Figure 5.
16S RNA secondary structure
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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.
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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 37 Updated 2025-07-16
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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.
And, in the name of science, we even wore gloves to not contaminate the samples!
Figure 1.
Swans swimming in the river when during sample collection
. Source. Swam poo bacteria?
Figure 2.
Tying rope to bootle for river water sample collection
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Figure 3.
Dumping the bottle into the river to collect the water sample
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Figure 4.
Measuring the river water sample temperature with a mercury thermometer
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Figure 5.
Measuring the river water sample pH with a pH strip
. Source. The strip is compared with the color of a mobile app that gives the pH for a given strip color.
Figure 6.
Noting sample collection location on the water bottle
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Video 1.
Dumping the bottle into the river to collect the water sample
. Source. That was fun.
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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 37 Updated 2025-07-16
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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.
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.
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Latin letter by Ciro Santilli 37 Updated 2025-07-16
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Google Cloud Platform by Ciro Santilli 37 Updated 2025-07-16
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How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Post filtration purification by Ciro Santilli 37 Updated 2025-07-16
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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".
Video 1.
Cutting vacuum pump filter and placing it in Eppendorf
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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.
Video 2. Source.
Video 3. 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 1. "White precipitate formed with Qiagen DNeasy PowerWater Kit".
Figure 1.
White precipitate formed with Qiagen DNeasy PowerWater Kit
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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 2.
VWR Micro Star 17 microcentrifuge.
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Figure 3.
VWR Micro Star 17 microcentrifuge loading.
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 4.
Biochrom SimpliNano spectrophotometer loading sample.
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Figure 5.
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 6.
Minus 20 fridge storing samples.
Source.
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How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / PCR by Ciro Santilli 37 Updated 2025-07-16
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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.
We did 35 cycles of:
Figure 1.
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: 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 --- 1492R
Code 1.
PCR diagram
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Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.
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How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / PCR verification with gel electrophoresis by Ciro Santilli 37 Updated 2025-07-16
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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 1.
Gel electrophoresis insert comb.
Source.
Figure 2.
Gel electrophoresis top view with wells visible.
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Figure 3.
Gel electrophoresis pipette sample into wells.
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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.".
Figure 4.
Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination.
Source.
Figure 5.
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 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.
Figure 6.
Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator
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Figure 7.
Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator loading gel.
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Figure 8.
Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel.
Source.
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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
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Once we had the amplified 16S DNA, we were almost ready to start sequencing!
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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
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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.".
Figure 1.
Oxford nanopore MinION flow cell package.
Source.
Figure 2.
Oxford nanopore MinION flow cell front.
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Figure 3.
Oxford nanopore MinION flow cell back.
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Figure 4.
Oxford nanopore MinION flow cell pipette loading.
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 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.
Figure 5.
Oxford Nanopore MinION connected to a Mac via USB.
Source.
Video 1.
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.
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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
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How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Protocols used by Ciro Santilli 37 Updated 2025-07-16
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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.
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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!
Video 1.
Intro to OurBigBook
. Source.
We have two killer features:
  1. topics: topics group articles by different users with the same title, e.g. here is the topic for the "Fundamental Theorem of Calculus" ourbigbook.com/go/topic/fundamental-theorem-of-calculus
    Articles of different users are sorted by upvote within each article page. This feature is a bit like:
    • a Wikipedia where each user can have their own version of each article
    • a Q&A website like Stack Overflow, where multiple people can give their views on a given topic, and the best ones are sorted by upvote. Except you don't need to wait for someone to ask first, and any topic goes, no matter how narrow or broad
    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/derivative
    Video 2.
    OurBigBook Web topics demo
    . Source.
  2. 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.
    Figure 2.
    You can publish local OurBigBook lightweight markup files to either https://OurBigBook.com or as a static website
    .
    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.
  3. https://raw.githubusercontent.com/ourbigbook/ourbigbook-media/master/feature/x/hilbert-space-arrow.png
  4. Infinitely deep tables of contents:
    Figure 6.
    Dynamic article tree with infinitely deep table of contents
    .
    Descendant pages can also show up as toplevel e.g.: ourbigbook.com/cirosantilli/chordate-subclade
All our software is open source and hosted at: github.com/ourbigbook/ourbigbook
Further documentation can be found at: docs.ourbigbook.com
Feel free to reach our to us for any help or suggestions: docs.ourbigbook.com/#contact
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