Want to build a sequencer? 454.bio opens up their plans
omicsomics.blogspot.com
omicsomics.blogspot.com
They are clearly at the early stage of tech dev. And the reward of $200k for the polymerase to allow longer reads seems pretty low to me... unless you're a grad student and the university doenst take your IP...
Honestly it's pretty hard to get lab access without somebody also getting rights to your IP, and labs are a bit more expensive than setting up an H100, so it might be tough going.
Even if no external party solves their polymerase problem I have a good feeling about this direction though. Godspeed.
On the other hand, $200k might be enough today if someone finds a way to apply AlphaFold and friends to this problem.
On the other other hand, I'm not convinced a better polymerase will solve their problems. Photobleaching might be their biggest problem, and I don't know that the polymerase itself can do much to protect against that.
But, the contest isn’t really about length — it’s about polymerase preference for fluorescently labeled dNTPs vs unlabeled.
From: https://454.bio/blog/2024/01/23/polymerase-competition/
> 454 Bio have opened a competition with a prize of $200,000.00 to evolve a DNA polymerase optimized to use Lightning Terminators™ while not incorporating their hydroxymethyl cleavage products with a minimum 1000:1 ratio.
This seems to be an issue with their chosen “One Pot” reaction mixture. When you can’t wash away byproducts after each cycle, with this method, you get a build up of non-labeled molecules. And these are the ones that the polymerase prefers. So, of course they end up with single-digit read lengths… the polymerase is doing what it does best. This seems like figuring out this chemistry should have been fleshed out before trying to build the sequencer.
So, I guess it turns out that there is a validation endpoint, but still not a very convenient way to measure it…
But this is an obvious issue. With this type of photo-reaction, you’ll get a build up of unlabeled nucleotides. There is still some potential for the technology, but it’s really hard for me to see how this works in one tube without some kind of washing (which also increases costs by a lot).
Maybe there is a way to add multiple moieties to the NTPs, such that removal of the fluorophore would cause a bigger hindrance? (Fluorophore + extra-moiety would bind, but extra-moiety alone would have more trouble binding?)
I’ll mention that your example of a protease is a good counter example — the function of the protein is to bind the larger peptide and cleave it. The evolutionary pressure was to bind the larger molecule and release the smaller (cleaved) ones. Here, the evolutionary pressure was to bind the smaller (unlabeled) nucleotide to build the DNA strand. This is a complex reaction that involves a lot of ligand binding and intermediates. Trying to add a function to preferentially bind a larger fluorophore labeled base seems like it would be overly disruptive or at least reduce the rate of polymerization.
I don’t know enough about the structure of DNA polymerase to say how feasible this really is — but the extra bulk of the fluorophore seems like it would cause issues.
But, is it possible? Sure! This is biology — there is always a way (or three)! I’d expect for there to be a way to generate a polymerase that does this via mutagenesis. But it would be very difficult and probably more expensive than the prize.
If they were able to keep the concentration of fluorescently labeled nucleotides higher, this wouldn’t be an issue. But due to the technology choices (one tube reactions, laser release), they are stuck trying to get a better polymerase.
I certainly wish them luck, I’d love for this to work for more than 5-6 bp.
Any recommendations on learning how to do this? As well as the limitations to your approach? I've been interested in engineering site-specific DNA binding proteins for over a decade now, but the tools I've looked at are specific for protein to small-molecule or protein to protein interfaces.
> We applied the protein G B1 domain (GB1) models to design a sequence that binds to immunoglobulin G with substantially higher affinity than wild-type GB1.
[1] https://www.pnas.org/doi/pdf/10.1073/pnas.2104878118?downloa...
But, anyway .. Here's another amazing open source sequencer, hard and soft, worthy of the attention of the sequencer-loving subdivision of the HN music-making aristocracy:
https://github.com/FundamentalFrequency/
Yet to build one myself, but having immense fun with the hardware emulator that is included in the project .. definitely worth the build.
When PacBio was scaling their technology, they had lots of problems with photobleaching. It turns out shining intense laser light at a polymerase-nucleotide-fluorophore complex and inducing lots of electron flux in the fluorophore eventually causes an electron to get overexcited, jump around, and damage the machinery. PacBio eventually invested a lot in builidng elaborate shields into the nucleotide-fluorophore link, able to absorb the stray electrons and save the polymerase.
Given that experience, I'd expect shining intense UV light into a TIRF volume to break the reversible terminator link will cause lots of photobleaching. It will be interesting to see if 454.bio can overcome this.
I can see that the complex microfluidics and reagent cycling can be a big added complexity/cost. The continuous process is definitely more elegant, but also seems really difficult to get right for longer read lengths and I wonder if it is really worth it.
What would be the potential benefits of the continuous process? I guess cheaper reagents since it is a one-pot process and faster sequencing speed since you don't need to cycle reagents?
Unfortunately pretty much biological equipment is so expensive and relatively hard to use. Case in point: my son is looking to measure amylase activity in the presence of various inhibitors for diabetes research. The cheapest spectrophotometer devices are $1k-$2k. Surely there must be a way to lose some accuracy but bring the price to $100-$200.
Ideally you could draft off of the work done for openbuilds, which is mainly focused on printers, lasers, and CNC machines.
(there are several projects that attempt this; openflexure is an example, but every time I work with their design, I go back to leadscrews with NEMA steppers and linear rail.)
There's a lot more biology than just sequencing DNA. transmitted light microscopy of cells is remarkably useful if you can do it cheap.
i'm working on reversing climate change at http://airminers.com
Why? craft software to grow the organism in a biologically plausible manner in simulation from the digital DNA... then expose evolutionary pressure to this growth to evolve the DNA in simulation into a more value added organism
real organism --> sequence it's DNA into digital DNA --> evolve the DNA in simulation software --> output real DNA from digital DNA --> insert this evolved real DNA into a real cell to clone an organism now with enhanced attributes
Yeah it's been unfortunate that the electronic sensors have not been able to keep up with the optical readouts. Ion Torrent and Genapsys are barely used due to accuracy problems, but Oxford Nanopore's considerable logistical benefits of tolerating a wide range of input DNA concentration, quick time to data, low capital cost, and long reads has kept them alive. Maybe they will grow more with higher accuracy.
Illumina's patents ending recently has prompted a few new startups beyond 454.bio, including Element and Singluar, but with the stiff headwinds of FDA regulatory uncertainty and the long capital life of existing sequencers, it's hard for them to make progress even when they are far cheaper than Illumina. And then we have Ultima and MGI. The me of 10 years ago would not believe both 1) how many strong entrants we have to DNA sequencers these days, and 2) how much Illumina continues to dominate in sequencing. At some point us biologists only have ourselves to blame for our purchasing decisions and their consequences.
https://41j.com/blog/2021/05/the-next-few-years-in-dna-seque...
> At some point us biologists only have ourselves to blame for our purchasing decisions and their consequences
Yep...
Accurancy didn't have very much to do with Ion Torrent's demise. As we're reminded in the OP, Illumina pushed the MiSeq platform, and there wasn't much reason to transition to Ion Torrent. Now, with 250 bp+ PE reads on Illumina, there really isn't much special about Ion Torrent besides its somewhat superior homopolymer performance.
> but Oxford Nanopore's considerable logistical benefits of tolerating a wide range of input DNA concentration, quick time to data, low capital cost, and long reads has kept them alive. Maybe they will grow more with higher accuracy.
This statement is reasonable in today's era, but ONT was nowhere close to being competitive with Ion Torrent and other platforms during the time where labs were considering the Ion Torrent devices vs continued spend on Illumina platforms.
The accuracy, tolerance to unusual DNA samples, and read length with clinically-relevant base qualities were unusable until 2018/2019.
Is the device able to sequence DNA with minimal errors?
Is that true? And if so, is it all open source or free?
It's something that's kind of interesting to me. I really enjoyed biotech and chemistry in HS but ended up going in a different direction. If this is something that you can just get into now as a hobby maybe I'll jump down the rabbithole
Last I checked 23andMe didn't perform whole genome sequencing but looked at some number of point mutations. It depends on what information you want. Eg for the sake of interest of actional genomics data.
And the sequencing machines and chemicals also don't come cheap, as the market has long been dominated by one vendor.
However, preparing a DNA sample for input into the machine is not trivial and requires training. As does the analysis to assemble all the chunks into your own genome.
A further challenge is even buying the equipment. Oxford Nanopore, and most biotech companies, keep very tight control of their customer lists, and if it looks like you are trying to order but don't have a proper molecular biology lab to perform sample preparation and to dispose of the waste products correctly, they almost certainly won't sell you anything. There are also strict legal agreements to return the flow cells when done, etc.
So to truly do this, add in the cost of LLC formation and signing up for a biotech startup lab to actually perform the experiments.