The unreasonable effectiveness of plasmid sequencing as a service | Hacker News Reader

The unreasonable effectiveness of plasmid sequencing as a service

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48 pointsabhishaike20 comments

Sounds great. But the focus on the competition with in-house sequencing is odd. When i was doing a PhD twenty years ago, we never even considered doing it in-house - i would just post tubes to a commercial sequencing service and get results back a few days later (or, in one case, take tubes to their office myself to save some time).

So, Plasmidsaurus are actually winning against those services. It would be more useful to focus on why that is. It sounds like the two distinguishing factors are fast turnaround, and long reads.

But there are other players offering basically the same service now, and faster or cheaper:

https://eurofinsgenomics.com/en/products/nanopore-sequencing...

https://plasmidsng.com/

So we'll see how that pans out.

My experience was similar when I was doing a lot of cloning 15 years ago: We would routinely send tubes to a commercial sequencing service. How Plasmidsaurus competes against those other services is indeed the more interesting question.

Really hard for them to compete at certain institutions. Some of these sequencing reps show up every afternoon to collect the days samples. Prices are so low that margins are probably tracing paper thin. Then some places will even have their own sequencing core facility offering all of that service at cost with even better turnaround, sometimes that day.

When I did the same, also 15 years ago, the lab on campus would only Sanger sequence my insert - and for targets larger than ~600bp I also had to include internal sequencing primers (that had to be ordered separately, shipped, etc). Plasmidsaurus will return the entire plasmid sequence so you can verify it is intact, you don't have to provide any internal sequencing primers, simple repeats will be more accurately sequenced, and you can just pop some tubes in a padded mailer in the post at room temperature.

I'm no longer doing wet bench work but Plasmidsaurus seems like a service I'd happily use.

Regarding the question why scientists don't buy the sequencing machines themselves I think a major factor is likely that sequencing is something that everyone expects to be a service and not something you do yourself. I would not even have considered doing this in-house.

At $15 per plasmid there is also no real advantage for typical labs, they're unlikely to save much money here. It could be quicker because you don't have to send the samples, but that likely isn't a big factor here. And having another device that either someone has to run as a service locally or that you have to teach everyone is a rather big effort.

I doubt it would be faster in a typical microbiology lab. Every time I've used plasmidsaurus in the recent past I've had results next day. It is a reasonable little bit of labor to prep plasmids for sequencing and then there is a bit of technical expertise required to format the data into a readable format. It's certainly doable, of course! Most lab managers / techs have more pressing things to do than to work on sequencing plasmids. Though I suppose it depends on the scale of your sequencing needs. I found plasmidsaurus to be amazing, and the full plasmid sequencing helped identify some errors that had been propogating in my old lab that never showed up on Sanger sequencing.

"In house" could mean in the institute, though. I never worked anywhere that had in-house sequencing, but better-funded places like Lincoln's Inn Fields did. Those places were big enough to make it cost-effective, and it's hard to beat the turnaround time of a sequencing lab two floors above you.

Some universities do it in house too

I work in a large biomedical research institute with in-house sequencing. It's a constant point of contention that many labs continue to send their sequencing out to external companies as they can still be cheaper and more reliable.

Running the service could involve a more efficient division of labor. Think of Adam Smith's pin factory. I don't know how many manual steps are involved in sequencing. I live in a town with a couple of big diagnostic test labs, and they are very much organized as an assembly line. They hire hundreds of technicians and scientists. There's also modern quality control, which isn't going to happen in an academic lab.

Some labs do but that is a bunch of extra labor that they need to manage. Sometimes some labs do more sequencing than others and they might have more researchers to do the regular sequencing. Many labs are smaller and can't afford to hire so many people or buy so much tertiary equipment. Grad students are already overloaded so they probably don't sequence unless its directly a part of their project

It also depends on what you are doing in the lab. The sequencers for most technologies are not cheap. The prep kits arent cheap. The assays take time. Plus for a lot of labs that sequencer might get used once a year. It just doesn’t make sense to invest to have this tech in house especially when it might end up obsolete in a few years, after you used it a whopping 10 times.

Productionizing and scaling nanopore sequencing is definitely an achievement. With that operational obstacle out of the way, you get to think about sequencing in terms of “streams” of data rather than “batches” of huge amounts of data (illumina). That confers a huge operational and commercial benefit.

Can you explain what you mean by streams? My familiarity with long read data is that you still deal with batches of huge amounts of data at the end of the day. Just the sequencing read is of course longer than 150bp paired end reads.

Nanopore spits out data as dna goes through the pores. And depending on the flowcell you are using, you can load or top up the flow cell in smaller amounts. So you can potentially have quick turn around time without having to wait for enough samples to pile up before you batch sequence on a larger illumina machine.

Illumina machines are cost efficient in terms of cost per basepair, but only at large batches. They are trying to rectify this after seeing other benchtop sequencing machines (Element Biosci) moving into the mid-throughout niche and doing well. Their solution is the miseq i100 that they just announced.

But at the end of the day, these are all still constrained by having to think in terms of multiplexed batches which has a lot of operational complexity involved (equi-molar pooling, barcoding, etc).

Ultimately, for commercial sequencing labs, one of the more difficult problem to solve is the operational complexity of how to optimally load the sequencer for lowest cost while balancing failure rates/low coverage rates rather than the technicalities of dna prep/lib prep. Given unlimited and consistent intake samples, the problem gets easier. But most labs have some kind of seasonality or project cycle built in which means it’s not about maximizing a yearly capacity, it’s more about how many samples max you can pump through within a few days.

I do a lot of plasmid sequencing in-house with nanopore, so I have some insights here!

For years, the problem was mainly with the rates of mutations in nanopore sequencing, in particular, non-random mutations. The stuff you couldn't just sort out by sequencing more (well, theoretically at least). This was because of the pore structure. Around 2021, R10 flow cells came out, which were a major improvement on R9 cells. In the protein pore for nanopore sequencing, they basically just stacked two proteins on top of each other, so there are more contact points to get data out of.

With the improvement in nanopore sequencing fundamentals, you now have far fewer non-random mutations. This enables plasmid sequencing. Plasmidsaurus just hit it out the park with convenience of the service.

On cost: most of the cost is in their reliability. Each customer wants their particular plasmid to be actually sequenced - so $15 price tag (and probably much less in-house for them). I currently sequence plasmids for approximately $0.25 in-house, BUT only probabilistically (900/1500 plasmids get sequenced). And to my best approximation, this is a non-random distribution, so just doing it more doesn't fix it. Also you run out of material.

There is an aspect of library prep that is cheaper. Turns out, with illumina sequencing at least, most of the cost is in the indexing step, not the actual sequencing, for plasmids. This is the step of putting barcodes onto the material to figure out which well a particular sequence came from.

Finally, you still do need software! People don't realize how much software you actually have to make to be able to tell plasmid sequence from nanopore sequencing. Plasmidsaurus makes it easy for people. Here is an example from my repo of me figuring out how the hell to interpret some weird data I've gotten:

https://github.com/Koeng101/dnadesign/pull/98

If you want a real example, take a look at this pileup file:

https://github.com/Koeng101/dnadesign/blob/sequencingPileup/...

How to read: https://en.wikipedia.org/wiki/Pileup_format

At position 165, you see a non-random nanopore error that only affects a single side of the DNA, and not the other (there would be lowercase g if the complement was also "mutated"). You need to sort through these to get good data to customers. And that's not as easy (not to mention how often plasmids are mixed!)

Awesome, thanks for sharing. What is the turnaround time on your in-house sequencing? Is it purely for cost savings that you do it yourself?

Would be keen to hear more about what you are working on. Separately - what is the current state of technology (price, accuracy, accessibility etc.) for synthesizing custom RNA rather than just sequencing it?

Turnaround time is about a day, so as fast as plasmidsaurus would get back to me (I'm also using nanopore, so we're gonna essentially be the same speed, with my library prep taking time but their shipments also taking time). It is mostly cost savings.

I'm working on building a plasmid synthesis company. The price of sequence verified DNA has increased, not decreased, since 2018, even when you account for inflation. On that metric alone, we are regressing in our ability to modify the biological world. I'll be launching in a couple months with a service that costs less than half the incumbent provider, while maintaining their margin. Being able to sequence verify DNA for dirt cheap is a big part of that. Took over a year and a half to get there - it's way harder than it sounds, for a myriad of reasons (things at scale are hard!)

You could mean two things by synthesizing custom RNA - synthesizing oligos (ie, chemical synthesis) or synthesizing mRNA, which is typically from a DNA template (you can have short mRNAs be synthesized as oligos, though). The fundamental problem with oligos is the mutation rates - hence why I sequence verify so much stuff - and you're not really gonna get around that. For other mRNA from a DNA template, well, you need the DNA template in the first place (that's where I come in), and then you usually just transcribe it into RNA using an RNA polymerase. Then, there is essentially an endless rabbit-hole of optimizations for everything under the sun. But fundamentally mRNA only costs the DNA template + polymerase, which you can get for like $75 https://www.neb.com/en-us/products/m0251-t7-rna-polymerase . DNA prices are about $0.09 per base plus $35 right now, I'm aiming for $0.045 per base plus $16.

Ah, this is insightful - esp your point that sequencing more did not help in the earlier version (that was an assumption I had in my mind).

I am curious if people have played around with trying to recreate the nanopore itself in-house or at least a prototype? I wanted to fiddle with the pore and need access to the trans compartment for testing my idea, any leads would be really helpful!

They've tried and there are some folks who have made nanopores, but honestly they're SO many years away from Oxford it's almost incomparable.

To just play with a device, I think you can buy the MR1 molecular reality device for like $300, but I don't know that much about it other than it is a solid state nanopore.

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