3 ms·

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 nanopor

by koeng 2y ago

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 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/lib/bio/pileup/data/sequencing/2eZeuUEOMFgF9zjssVEnEvASo4E.pileup https://github.com/Koeng101/dnadesign/blob/sequencingPileup/... How to read: https://en.wikipedia.org/wiki/Pileup_format 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 2y ago
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 2y ago
  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 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.
akankshay58 2y ago
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!

by koeng 2y ago

highfrequency 2y ago

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 2y ago
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 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.

akankshay58 2y ago

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!