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
Ciro Santilli 37 Updated 2025-07-16One 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 PCR verification with gel electrophoresis by Ciro Santilli 37 Updated 2025-07-16
Ciro Santilli 37 Updated 2025-07-16For this reason, it is wise to verify that certain steps are correct whenever possible.
Gel electrophoresis separates molecules by their charge-to-mass ratio. It is one of those ultra common lab procedures!
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.".
Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination.
Source. 
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.
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
Ciro Santilli 37 Updated 2025-07-16After 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".
Cutting vacuum pump filter and placing it 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 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.
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:
file 5.36 however does use it to display file type as explained at: stackoverflow.com/questions/34519521/why-does-gcc-create-a-shared-object-instead-of-an-executable-binary-according-to/55704865#55704865Please refer to Video "Linus Torvalds saying "Nvidia Fuck You" (2012)".
Does not happen every time, only some times. Can't figure out why. Usually happens when has suspended for a longer time.
bugs.launchpad.net/ubuntu/+source/nvidia-graphics-drivers-470/+bug/1946303 sounds like a likely report, Nvidia driver version 470, but can't find those error messages anywhere. The last line of:once was:which is when sleep starts.
journalctl -o short-precise -k -b -1PM: suspend entry (deep)This suggests that it is not a video bug then, seems that it is not waking up at all? Gotta try to SSH into it. OK. I did SSH into it, and that was fine, so it is just the video that won't start.
PM: suspend exitbugs.launchpad.net/ubuntu/+source/linux/+bug/1949977 is another possible bug, based on kernel version. I'm running 5.13, which is one of the failing versions on the report. Can't find any interesting dmesg though.
In another crash:had the following interesting lines:and there was a corresponding
journalctl -o short-precise -k -b -1nvidia-modeset: WARNING: GPU:0: Lost display notification (0:0x00000000); continuing.
[24307.640014] NVRM: GPU at PCI:0000:01:00: GPU-18af74bb-7c72-ff70-e447-87d48378ea20
[24307.640018] NVRM: Xid (PCI:0000:01:00): 79, pid=8828, GPU has fallen off the bus.
[24307.640021] NVRM: GPU 0000:01:00.0: GPU has fallen off the bus.
[24328.054022] nvidia-modeset: ERROR: GPU:0: The requested configuration of display devices (LGD (DP-4)) is not supported on this GPU.
[repeats several more times]
[24328.056767] nvidia-modeset: ERROR: GPU:0: The requested configuration of display devices (LGD (DP-4)) is not supported on this GPU.
[24328.056951] nvidia-modeset: ERROR: GPU:0: Failed to query display engine channel state: 0x0000927c:0:0:0x0000000f
[24328.056955] nvidia-modeset: ERROR: GPU:0: Failed to query display engine channel state: 0x0000927c:1:0:0x0000000f
[24328.056959] nvidia-modeset: ERROR: GPU:0: Failed to query display engine channel state: 0x0000927c:2:0:0x0000000f
[24328.056962] nvidia-modeset: ERROR: GPU:0: Failed to query display engine channel state: 0x0000927c:3:0:0x0000000f
[24328.056983] nvidia-modeset: ERROR: GPU:0: DP-4: Failed to disable DisplayPort audio stream-0
[24328.056992] nvidia-modeset: ERROR: GPU:0: Failed to query display engine channel state: 0x0000947d:0:0:0x0000000f/var/crash/_usr_sbin_gdm3.0.crash.Related "GPU has fallen off the bus": askubuntu.com/questions/868321/gpu-has-fallen-off-the-bus-nvidia
To run examples on a specific database:All examples can be tested on all databases with:
./index.jsor./index.js l: SQLite./index.js p: PostgreSQL. You must manually create a database calledtmpand ensure that peer authentication works for it
cd sequelize
./testOverview of the examples:
- nodejs/sequelize/index.js: a bunch of basic examples
- nodejs/sequelize/update.js: This file is also where we are storing our expression-foo for now, e.g. how to do stuff like
col1 + col2. Such knowledge can however be used basically anywhere else however, e.g. inASorWHEREclauses, not just inUPDATE. - nodejs/sequelize/count.js: a simplified single-table count example. In practice, this will be usually done together with JOIN queries across multiple tables. Answers: stackoverflow.com/questions/22627258/how-does-group-by-works-in-sequelize/69896449#69896449
- nodejs/sequelize/date.js: automatic date typecasts
- nodejs/sequelize/like.js: LIKE
- nodejs/sequelize/camel_case.js: trying to get everything in the database camel cased, columns starting with lowercase, and tables starting with uppercase. The defaults documented on getting started documentation do uppercase foreign keys, and lowercase non-foreign keys. It's a mess.
- nodejs/sequelize/ignore_duplicates.js: ignore query on unique violation with
ignoreDuplicates: truewhich does SQLiteINSERT OR IGNORE INTOor PostgreSQLON CONFLICT DO NOTHING. Closely related Upsert versions:- Upsert
- nodejs/sequelize/upsert.js:
.upsertselects the conflict column automatically by unique columns. Both SQLite and PostgreSQL doINSERT INTO ON CONFLICT. PostgreSQL usesRETURNING, which was added too recently to SQLite: www.sqlite.org/lang_returning.html - nodejs/sequelize/update_on_duplicate.js:
.bulkCreate({}, { updateOnDuplicate: ['col1', col2'] }. Produces queries analogous to.upsert. This method is cool because it can upsert multiple columns at once. But it is annoying that you have to specify all fields to be updated one by one manually. - stackoverflow.com/questions/29063232/how-to-get-the-id-of-an-inserted-or-updated-record-in-sequelize-upsert/72092277#72092277
- stackoverflow.com/questions/55531860/sequelize-bulkcreate-updateonduplicate-for-postgresql
- Upsert
- nodejs/sequelize/inc.js: demonstrate the
incrementmethod. In SQLite, it produces a statement of type:UPDATE `IntegerNames` SET `value`=`value`+ 1,`updatedAt`='2021-11-03 10:23:45.409 +00:00' WHERE `id` = 3 - nodejs/sequelize/sync_alter.js: illustrates
Model.sync({alter: true})to modify a table definition, answers: stackoverflow.com/questions/54898994/bulkupdate-in-sequelize-orm/69044138#69044138 - nodejs/sequelize/truncate_key.js
- nodejs/sequelize/validation.js: is handled by a third-party library: github.com/validatorjs/validator.js. They then add a few extra validators on top of that.The
args: truething is explained at: stackoverflow.com/questions/58522387/unhandled-rejection-sequelizevalidationerror-validation-error-cannot-create-pr/70263032#70263032 - nodejs/sequelize/composite_index.js: stackoverflow.com/questions/34664853/sequelize-composite-unique-constraint
- nodejs/sequelize/indent_log.js: stackoverflow.com/questions/34664853/sequelize-composite-unique-constraint
- association examples:
- nodejs/sequelize/one_to_many.js: basic one-to-many examples.
- nodejs/sequelize/many_to_many.js: basic many-to-many examples, each user can like multiple posts. Answers: stackoverflow.com/questions/22958683/how-to-implement-many-to-many-association-in-sequelize/67973948#67973948
- ORDER BY include:
- nodejs/sequelize/many_to_many_custom_table.js: many-to-many example, but where we craft our own table which can hold extra data. In our case, users can like posts, but likes have a integer weight associated with them. Related threads:
- nodejs/sequelize/many_to_many_same_model.js: association between a model and itself: users can follow other users. Related:
- nodejs/sequelize/many_to_many_same_model_super.js
- nodejs/sequelize/many_to_many_super.js: "Super many to many": sequelize.org/master/manual/advanced-many-to-many.html This should not exist and shows how bad this library is for associations, you need all that boilerplate in order to expose certain relationships that aren't otherwise exposed by a direct
hasManywith implicit join table.
- nested includes to produce queries with multiple JOIN:
- nodejs/sequelize/nested_include.js: find all posts by users that a given user follows. Answers: stackoverflow.com/questions/42632943/sequelize-multiple-where-clause/68018083#68018083
- nodejs/sequelize/nested_include_super.js: like nodejs/sequelize/nested_include.js but with a super many to many. We should move this to nodejs/sequelize/many_to_many_super.js.
- two relationships between two specific tables: we need to use
as:to disambiguate them- nodejs/sequelize/many_to_many_double.js: users can both follow and like posts
- nodejs/sequelize/one_to_many_double.js: posts have the author and a mandatory reviewer
- hooks
- internals:
- nodejs/sequelize/common.js: common utilities used across examples, most notably:
- to easily setup different DBRM
- nodejs/sequelize/min_nocommon.js: to copy paste to Stack Overflow
- nodejs/sequelize/min.js: template for new exapmles in the folder
- nodejs/sequelize/common.js: common utilities used across examples, most notably:
You can connect form an Ubuntu 22.04 host as:When in but be aware of: Raspberry Pi Pico W freezes a few seconds after after screen disconnects from UART.
screen /dev/ttyACM0 115200screen, you can Ctrl + C to kill main.py, and then execution stops and you are left in a Python shell. From there:- Ctrl + D: reboots
- Ctrl + A K: kills the GNU screen window. Execution continues normally
Other options:
- ampy
runcommand, which solves How to run a MicroPython script from a file on the Raspberry Pi Pico W from the command line?
Superconducting qubits are good because superconductivity is macroscopic by Ciro Santilli 37 Updated 2025-07-16
Ciro Santilli 37 Updated 2025-07-16Superconducting qubits are regarded as promising because superconductivity is a macroscopic quantum phenomena of Bose Einstein condensation, and so as a macroscopic phenomena, it is easier to control and observe.
This is mentioned e.g. in this relatively early: physicsworld.com/a/superconducting-quantum-bits/. While most quantum phenomena is observed at the atomic scale, superconducting qubits are micrometer scale, which is huge!
Physicists are comfortable with the use of quantum mechanics to describe atomic and subatomic particles. However, in recent years we have discovered that micron-sized objects that have been produced using standard semiconductor-fabrication techniques – objects that are small on everyday scales but large compared with atoms – can also behave as quantum particles.
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
Ciro Santilli 37 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!
There are unlisted articles, also show them or only show them.