Thread by @nanopore

Coming up in a few minutes, Clive Brown @the_taybor, with an update entitled “Nobody Expects the Strandish Exposition”. Follow the talk live in this thread #nanoporeconf

CB: Our goal is to enable the analysis of anything by anyone, anywhere. Here are some examples of global use: nanoporetech.com/portable-seque…

CB: As a reminder, this is how nanopore sequencing works. #nanoporeconf

CB: @nanopore has an information-rich signal which can be deconvoluted in real time. Basecalling updates have driven considerable improvements over the past two years.

We have moved onto self supervised methods of data analysis, and have bulked out our machine learning and data analysis teams #nanoporeconf

CB: the Bonito basecaller, introduced as a research basecaller in late 2020, is now in MinKNOW is reaching modal single read accuracy of 98.3% #nanoporeconf

https://twitter.com/kirk3gaard/status/1328087574951432192?lang=en-gb

CB: The Q20+ chemistry is delivering raw read accuracies of 99%+ in developers hands

https://twitter.com/AlbertVilella/status/1384846947446624257

#nanoporeconf

CB: Q20+ chemistry is now moving from developers into early access – register your interest here register.nanoporetech.com/q20plus-chemis…

CB: the Q20+ kit is compatible with MinION and GridION now #nanoporeconf

CB: we like to come back again and again to ideas and changing them to make them work. introducing Duplex #nanoporeconf

CB: new method, “Duplex”: following on. Enables nanopore devices to sequence a template and complement strand of a single molecule of DNA, in succession, in order to achieve very high accuracy sequencing results. #nanoporeconf

CB: terminology: Simplex= single strand. Both strands = duplex #nanoporeconf

CB: goal of Duplex experiments is to achieve a high proportion of instances where the complement follows the template strand through the nanopore and gives the system ‘two looks at the same sequence pairs’. #nanoporeconf

CB: Current runs are routinely achieving 40% of data from Duplex pairs, with 50% the current highest and 75% is targeted after further optimisation. #nanoporeconf

CB: upgraded enzyme to E8.1, improvement the movement quality on the complement strand. Complement quality is almost the same as the template strand #nanoporeconf

CB: Duplex is compatible with the ultra-long kit capable of N50s greater than 100kb. Further optimisation required. Longest duplex pair to date is 442kb
#nanoporeconf

CB: Duplex can currently achieve modal accuracy of ~Q29 for a single double stranded molecule of DNA #nanoporeconf

CB: the intrinsic data quality on this platform is extremely high, and we are now achieving that because of the software #nanoporeconf

CB: a key differentiator is the length of the duplex reads. The longest Duplex molecule sequenced to Q30 to date is 156kb. #nanoporeconf

CB: we can't see any substantial difference between our Duplex reads and another leading platform's high accuracy mode #nanoporeconf

CB: revision. Without a motor, single stranded DNA translocates the pore very quickly ~thousands of bases per second and thought to be “slip-stick” hard to control
― (blunt double stranded DNA won’t strip through) #nanoporeconf

CB: controlled movement is needed through the pore, for high accuracy sequencing. => motors #nanoporeconf

CB: Current nanopore chemistries sequence DNA as it is driven from the ‘top’ of the flow cell through the nanopore to the area underneath; this is known as ‘Inny’. #nanoporeconf

CB: many potential benefits of deploying the opposite ‘Outy’ direction, one format being where DNA threads from the 3’ end first, still moving 5’ to 3’. different DNA orientation may produce a different error profile #nanoporeconf

CB: we decided to start looking at 'outy' again. Here's an Outy mode: #nanoporeconf

Spot the difference. Nanopore duplex vs another platform in high accuracy mode

CB: …potential to enable approximate fragment sizing before sequencing – and therefore enable pre-selection of the desired read lengths. The method can also be used to perform controlled re-reading of native strands. #nanoporeconf

CB: ‘Adaptive accuracy’ possible with ‘outy’ mode #nanoporeconf

CB: we can re-read a fragment on an indefinite loop. The signal from the first read is the same as the 4th/5th.. you can basecall as you go, to a point where you are happy with the accuracy and then you move on to the next fragment #nanoporeconf

CB: a reminder of Adaptive Sampling – real-time acceptance or rejection of molecules from a nanopore based on sequence or signal – to enrich/deplete the sample for specific targets of interest
nanoporetech.com/about-us/news/… #nanoporeconf

CB: could size the fragment before sequencing it using "inny", with current Q20 chemistry #nanoporeconf

CB: this is what that looks like #nanoporeconf

CB: could combine Adaptive sampling & ultra-long sequencing & Q20+. “Adaptive finishing” for faster & even coverage and finishing, by proactive selection of ultra-long fragments that covered regions of low, or no coverage. #nanoporeconf

CB: if the input fragment size distribution is long enough, you should be able to adaptively select length of sample molecules, and adaptively sample haplotypes. I believe any genome can be assembled and closed with nanopore only.#nanoporeconf

CB: Next Generation Flongle. Developing new flow cells for the Flongle adapter, based on plastic not silicon for low cost. Plastic & silver chloride => aim for better signal/noise, cheaper/scalable manufacturing and stable currents for long run times. #nanoporeconf

CB: part of getting rid of the lab is getting rid of the pipette, and we want to do that with Flongle. We've been developing companion sample preps - should just need a swab. Prep too early to show today but big ambition #nanoporeconf

CB: CB: Custom ASIC collects data from individual nanopore channels; Low power ASICs in development to support pipeline eg SmidgION, Plongle

CB: there is no point having a SmidgION mobile phone sequencer, if you need a bunch of lab kit. We're working on it #nanoporetech

CB: PromethION 48 generating very high outputs: 10Tb run record recently. Composed of 48 flow cells so modular design…. Users are scaling up with PromethION to very high throughput facilities with automation #nanoporeconf

CB: we have dramatically improved the electronics inside the box for PromethION. Now. Here's a new product #nanoporeconf

CB: we are developing PromethION 2. P2 will run up to two PromethION flow cells at once, includes compute. Starter pack $60k includes 48 flow cells. #nanoporeconf

CB: pricing is provisional! But will be competitive #nanoporeconf

CB: why do P2? Two PromethION flow cells in small format keeps nanopore a step ahead of other devices in this range. P2 would be ideal for studying large genomes/ transcriptomes, easy with automation. You can register your interest here register.nanoporetech.com/p2-device

CB: P2 solo: Without integrated compute, to connect two flow cells to your own computer or GridION #nanoporeconf

CB: if you make the investment in @nanopore, we will keep you ahead by upgrading your technology with all the latest improvements #nanoporetech

CB: a summary of the latest performance stats - for full details nanoporetech.com/accuracy

CB: We are doing a lot of work on the idea for writing DNA, and protein sequencing. We will say more later #nanoporeconf

CB: We have started a pilot programme called org.one, where we support free flow cells for sequencing of critically endangered species, to help conservationists get to grips with the current biodiversity crisis org.one/oo #nanoporeconf

CB: we would like to support a ground-up movement to enable local sequencing of these critically endangered species, rather than conservationists sending their samples to wealthier countries for sequencing #nanoporeconf org.one/oo

CB: And that's the end of my update. #nanoporeconf

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