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Comment by Ovah | Hacker News Reader

Anyone with hands on experience using NanoPore? I've been thinking about buying one of these to play around with. But anecdotally I've heard that they lack utility or are my concerns just myths? a) they are designed to handle many batched samples at once rather than many runs of few samples over time. So in practice they don't really last for many individual samples. b) the computational requirements are high. So while a NanoPore can be plugged into a laptop in the field it would take forever to run the data processing on said computer.

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Here's my write-up of buying one for fun: https://abarry.org/dna-sequencing-in-our-extra-bedroom/

Nice write up! How much did it cost to get a Oxford Nanopore?

$1k prices are on the website.

From your post the thing uploads the sequenced data and their service generates the report. Is the raw data available?

Also: truly remarkable phd thesis!

Yes the raw data is all there. The analysis just generates more interesting pictures!

> never arrived. Finally, I emailed them and found out I needed to join their “community.”

> Then Alice starts asking questions. I was not prepared for questions

> Then Alice started asking questions about my research

Wow, that is kinda creepy. You're seeking to pay them money for a product they sell, and they want to ask all these intrusive questions about what you do? Is there a reason for this? I mean imagine if DigiKey did this, what a nightmare that would be for electronics design.

Are they worried about you using it as part of a lab producing bioweapons? But frankly anybody with a shot at pulling that off would already know enough to lie very convincingly. I can't imagine this interview would be a hurdle to those folks.

The only other place I've come across this behavior is when seeking to buy wafers from semiconductor foundries. In that situation the foundries see themselves not as vendors to the chip designers, but rather as investors in the chip designers -- investing not money but rather production capacity, and earning not dividends but rather wafer purchases.

It wasn't creepy. It seemed like they were worried about folks who thought it was more like 23andMe "spit in a tube" than an actual lab instrument.

Oh, okay, but then why this:

> I repeat something about bacterial colonies from my card but she isn’t buying it. I manage to get out that I understand this isn’t a spit-in-the-tube-and-done thing and that’s all I’ve got. She keeps pushing

I mean you told her pretty clearly that you didn't think it was like 23andMe.

Why would she keep pushing?

Computational requirements are quite high, but OK if you have good GPUs on hand. A coronavirus sequenced sample on the fast mode without GPUs would take 3-4 hours to complete, while on the high accuracy mode days. GPU access would speed up performance considerably.

Error rate for MinIONs is still quite high (10-15%), so a human genome sequencing would be quite inaccurate in some regions.

Sequencer is quite cheap, reagents and flow cells are a little bit more expensive.

Thank you. The upfront cost of the sequencer sure makes it tempting at first sight.

My desired hobbyist use case is to key out plants, lichens and mushrooms that I find in the field. I have the bioinformatics knowhow just need the hardware. 3-4h seems lika a long time for a genome that is <30k nucleotides long. Mushrooms on average seem to have almost as many genes as coronaviruses has nucleotides. I guess partial sequences (and thus reduced comp time?) might do the trick but it's probably hard to target those partial reference sequences with a long-read method like NanoPore.

Is the error rate per base pair?

If you repeat the process many times will it reduce that error rate, or are the errors non-independent?

Last time I looked into Nanopore the cost wasn't that much better where you'd even consider this experiment.

On the other hand, when doing a genome assembly, the Nanopore reads are good for a draft sequence and then the Illumina reads can be used to polish the sequence.

Unfortunately, with nanopore the errors are biased so you tend to get errors in the same places. All sequencing techniques also have error rates but some are unbiased so running a single sample through (which will usually have many, many copies of any sequence) will average out to a good read of the sequence.

Some good info on next-gen sequencing techniques: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841808/

Still, some of the errors can be compensated for with more coverage. So if you can manage 20-30X you're left with the homopolymer problem (nanopores can't tell how long a stretch of the same repeated nucleotide is, because you can't control how long the sensed kmer stays in the pore), but lots of other types can be improved quite a lot.

A close friend of mine has worked there for many years. We've spoken a lot about the tech.

I don't know the answers to all your questions, but I do know that the emphasis is on research, not consumer (or hobbyist) use. I believe the devices are ~free, but each run requires using a consumable part that has to be either disposed of or returned for refurbishment, and I believe these are hundreds of dollars each.

The big advancement is the size and cost of the devices, the fact that a lab can have one on every desk rather than a communal machine that you have to queue your samples up for, or a device you can transport in field kit.

They do have cloud services that do much of the processing for you, but I suspect you'd want to be able to manipulate the data so you'd need your own data processing tools locally. It's not going to give you a 23andMe style report, it's more likely to say "yep, that's a human" vs "you're ecoli". I believe they do have training for how to do this data analysis, but I suspect this is targeted at customers on large contracts.

Thank you for the practical insight. I suspected that NanoPores are not just yet geared towards hobbyists. I happen to have some bioinformatics knowhow so it's mainly a matter of hardware for me. As both you, u/bioinformatics and u/searine mention it is the overhead cost of flow cells etc that worries me from a hobbyist point of view.

Yep the flow cells are expensive, but more generally saying the hardware costs $1000 is probably more of a technicality – almost all sales are bigger contracts, with training, support, flow cells, hardware, included.

Also that's great you have some bioinformatics experience, but are you sure it will apply here? I'm very unknowledgeable about this so forgive me if I'm wrong, but I believe that traditional machines do short-reads, whereas Nanopore does long-reads, which I believe invalidates many techniques for reconstructing the data, the tools, etc. This might not be something you have to be concerned about depending on the level you're working at, I don't know (maybe this is all "solved" by the time you're doing analysis?).

I think theoretical/practical background will allow me to figure stuff out as I go with relative ease. Often times in bioinformatics there are useful vignettes a Google search away. But you are right that specialized tools and libraries which I haven't used before are likely needed. At least for me the main bottleneck will be proper space for processing and the aspect of (reusable) hardware to keep costs down costs. While NanoPores appear to have hidden costs it still seems to be way cheaper than other methods if I want to have my own sequencing rig.

They are a fun tool, great for doing molecular work in the field. The error rate is still very high compared to short reads, but if you know this and plan for it going in you should be fine.

Flowcells last for one sample. The machine should last indefinitely. You can sometimes add more of the same DNA to a flowcell after one use to get a bit more out of it, but the quality degrades quickly. 500-1000 dollars each for flowcells, depending on how much you order.

My experience in field use, I was using Oxford Nanopores software which does processing remotely and was able to run the the platform on just a regular 2015-era laptop.

Is the price a question of scale? If this technology would become commonplace, would the price go down? Are there patents that would prevent cheaper chemical production?

I assume scale and more R&D on how to produce nanopores more cheaply would be the main ways to drive price down. As for patents, Oxford Nanopore has a pretty big portfolio for all things nanopore, so a direct competitor based on nanopores that would drive the price down seems unlikely (though they obviously have to compete on price with other sequencing methods to some degree).

What is a flow cell made out of and why is the cost so high?

It's made of plastic, glass, and the special protein pores which split the strand and read the DNA. Reagents and sample are applied to it to make the reaction happen.

The flowcell gets contaminated with your sample after one run so they are 'one time use'. The nanonpore protein eventually stops working also.

They are expensive because doing molecular biology is expensive. It requires expensive machines and expensive reagents at atomic scales to create. Thus money is required.

Actually, one of the main features of this tech apart from the obvious size-factor is that it's a streaming process. You can analyse data on the fly and decide when to stop the run. Wash the flowcell, and use it for another sample. Eventually the pores die, yes, how fast depends on the sample type. I think they guarantee 48 hours or something of the sort.

The expensive part is not the chemistry. Each flowcell has a very expensive piece of metal that senses the very small current variations that each kmer causes when going through each pore. They've actually come up with a device (horribly named "flongle") that has the same shape of a flowcell but no pores, and the mini flowcell it uses is ~90USD (against ~900USD for a full flowcell). Of course, yield is much lower.

I used to run a department at a biotech where ~50% of our data came from MinIONs (although, that said, I'm a bioinformatician, rather than a molecular biologist), so I can answer your questions. For (a.), you can for sure "batch" samples. The term of art you're looking for is "multiplexing". Nanopore provide prep kits that allow you to "barcode" different samples (i.e. tag all the molecules in a given sample with a unique, synthetic sequence, which allows them to be distinguished by software downstream), but note that (as with all DNA prep kits, but some more than others) you'll need access to a fair whack of lab equipment and consumables to use it (these kits aren't "all-in"). For (b.), for one anecdata point, I used to process a whole flow cell's data on an M4800 with a 4th Gen i7 and 32 GB of RAM in a few hours. Most of the "high" computational requirements you hear about relate to either assembly or variant calling (both of which are downstream of just retrieving "usable" sequencing data); and even both of those I've managed on that same laptop overnight. Actually acquiring the data (you can delay base calling if you like, although you probably wouldn't need to) is real-time and only needs very modest hardware (IMHO the Nanopore "system requirements" are very much on the "safe-side".) "In the field", your challenge would be physically preparing the samples!

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