[HN Gopher] The unreasonable effectiveness of plasmid sequencing...
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The unreasonable effectiveness of plasmid sequencing as a service
Author : abhishaike
Score : 38 points
Date : 2024-10-09 19:49 UTC (4 days ago)
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| fabian2k wrote:
| 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.
| dillydogg wrote:
| 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.
| twic wrote:
| "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.
| kjkjadksj wrote:
| Some universities do it in house too
| analog31 wrote:
| 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.
| alan-hn wrote:
| 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
| kjkjadksj wrote:
| 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.
| twic wrote:
| 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.
| divbzero wrote:
| 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.
| kjkjadksj wrote:
| 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.
| koeng wrote:
| 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!)
| highfrequency wrote:
| 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?
| koeng wrote:
| 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.
| daemonk wrote:
| 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.
| kjkjadksj wrote:
| 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.
| daemonk wrote:
| 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.
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