Thread by @ewanbirney

Here is the slightly cheesy montage for the great #nanoporeconf for 2023 - and, with a reminder of my conflict of interest - I am a longestablished paid consultant for Oxford Nanopore and a shareholder - here are my thoughts on the conference.

https://twitter.com/nanopore/status/1659872069004271618

For long time nanopore scientists -and I am definitely one of those- one can definitely both plot progress London Calling conference (on the Thames in London) both in terms of what the company presents as near and long horizon+how the plenary speakers use and talk about nanopore

From the company side, much of this was giving a roadmap of key software and flow cells; the R10 flow cells (which is a distinct step up in quality) are now routine; what is not yet is high yield duplex which has being moving from Oxford to Alpha to broader Beta testers.

As well as the high yield duplex flow cells (some magic in keep the second strand in the right place to follow the first) as important is making the duplex calling one-button push; currently one needs a rather complex workflow to find and recall the duplex.

The high yield duplex revives an old (2018? 2019?) idea to have a synthetic second strand protocol with spiked in modified bases of the company's own design.

This should solve the homopolymer issue as the modified bases from a DNA polymerase perspective are incorporated against a homopolymer randomly, but they will give changes in nanopore signal. With R10's long read head one only needs 1 in 9 - say 1 in 5 in practice to get length

Talking to some of the "nanopore wizards" (JC and Stuart) afterwards they are bullish about this, as is obviously Clive (otherwise he wouldn't have shown so much). Clearly it is fiddly (what bit of single molecule sequencing is not) but clearly do able.

This means, with exisiting nanopore tech (no need for outie chemistry for example) one can see a path to Q20+ single read, Q30+ duplex *including* homopolymers, and frankly it should start getting hard to assess consensus quality as it goes through Q40 onwards

(past Q40 - 1 in 10,000 error rate, goint to Q50 - 1 in 100,000 it gets ever so hard to have big enought truth sets with enough accuracy and consistency- you have to know the right answer crudely at least 10 fold better - so 1 in 100,000 / million errors on your truth set)

Another big thing are the new chips (ASICs) which I can see the nanopore team are excited about. It is the first ground up redesign of the chips, and uses less silicon per channel (no more of these 4 muxes - they have gotten better at putting in one pore per membrane)

There are many other tweaks, most of which I could not follow but sensed the collective excitment. The end results is more channels per silicon, far, far less energy (really bring in mobile applications) and integrated (so cheaper to make all in)

I'm not sure quite when we will see products based on these, but they already committed that some of them will be compatible with MinIONs (so these will be "just" a new type of flowcell). Low power rating means true mobile, hands off applications are conceptually doable

(one still have to sort out the "front end" - molecular biology - or "molly-bolly" in internal slang. One option is the revamped VolTrax - now named something else. I wonder if for blood and water/or simple inputs there could be just a bead/column/simple attachment thing

This was a 2019 idea - ubiquity or something - and clearly they can get some of this working. I think there will be multiple ways to get the front end to work, ideally "technician hands free", but this is an active area of research)

The final theme for me was modifications, both DNA and RNA. As long known and expected (I remember I think back in 2015 or 2016 saying that CpGs can be read directly) nanopore can sense many modifications; what has changed is a robust training mechanism

So now, already, R10 nanopore called native DNA can have CpG methylation and CpG hydroxymethylation (it is going to drive us all up the wall, but the most common way to get methylation read out - bisulphite conversion - actually converts both of these. Sort of union CpG methyl)

Here the hidden world of RNA I think will open up - RNA is not some one shot, passive molecule either structurally doing things (rRNA) or passing information (mRNA); rather it is an actively managed molecule in cells, being marshalled, sorted, translocated and degraded.

We've known for a long time mRNA is super dynamic; we've known for a long time mRNA is modified; we've not had the technology to look at how modifications either are deliberate marks of their management or "just" reliably bystanders on their journey.

On a personal note, it is great to see one of my ex-postdocs, @AdrienLeger2, leading this area in Oxford Nanopore and a good example of academia to industry transition and fun.

Turning away from the company to the presentations, unsurprisingly it was more on the production schemes available over the last year, ie, R9 and R10 simplex (with some R10 duplex), but the innovation of how to use nanopore was going up

Some of this is obvious - long assemblies (are long reads - and really long reads - useful for assemblies - of course they are!). Here the "nanopore only" assemblies closing in on the "best blend" and being good enough for many applications was interesting

(I thought the NIH Alzheimer talk by Kimberley Billingsley of 200 human genomes from brains, being able to get good diploid assemblies with methylation and some somatic mutations was an excellent talk).

The other obvious use of nanopore is in cancer, where the combination of long reads for structural variation, methylation+hydroxymethylation (and more in the future) "for free" and fast turn around is going to be great

I am biased as I know the work from @hbelrick on this from @emblebi and Helen Webb's talk from @GenomicsEngland (also an area I know) was good to see getting out of R&D and closer to production

(someone moaned at me that it wasn't yet there for GeL, which is... naive about the difference between "works for clinical research" - we've seen plenty of this - to "is a routine offering in a healthcare system". They are related things - but not the same!)

One surprise to me was the utility of long read single cell transcriptomics with multiple talks on this. I had stupidly thought that the only real benefit of long read transcriptomics was splicing - and the bulk of the information was in the expression.

However, both Rachel Thijssen and Hanlee Ji made huge use of linking the mutation (somatic cancer, or engineered) to the transcript-cell level bar code to immediately be able to track specific mutation to cell barcode. this is ... big.

I can see some really interesting use cases of this, and it removes the need for one layer of index tracking potentially in engineering (when one is dealing with transcript mutations, but those are some of the most important ones).

In general the long reads being able to link information - whether it is SNPs to Methylation, or mutations to bar codes, and I think in the future things like artificial modifications (think... chip adaptations) to SNPs, CRISPR sites or between types it is going to be... great

Finally adaptive sampling - controlling how the chip accepts or rejects nanopore reads- is coming good. Close to my heart is @weilgunyl's BOSS-RUNs (dynamic adaptive sampling) and in the future dynamic adaptive assembly.

But adaptive sampling is both empowering for routine uses (eg, "just the exome") through to this more sophisticated lets update the sampling strategy as we see the data, which I think will be useful in broader and broader applications.

So, as ever the future is bright for @nanopore (when has it not been - steady progress each year) with the endless utility of measuring DNA (and RNA) - both natural and manipulated/engineered in a variety of ways.

There is plenty of headroom in the key features - such as error rate (nanopore is nowhere near its maximal), speed (silicon density really) and sensing (eg, modifications, already realised, "just" needs training) and plenty of future in its use.

A final reminder - I have a conflict of interest as a long established nanopore consultant and a shareholder. No company vets my tweets.