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Dec 1, 2021 42 tweets 21 min read Read on X
Up in a few minutes, Clive G Brown @the_taybor with a plenary talk on @nanopore sequencing. We'll live tweet in this thread 1/n #nanoporeconf
@The_Taybor CB: our goal is to enable the analysis of anything, by anyone, anywhere #nanoporeconf
CB: A reminder of how nanopore works #nanoporeconf
CB: with @nanopore, any fragment length can be sequenced and accuracy is not read length dependent. #nanoporeconf
CB: @nanopore CAN sequence short fragments (<200bp) and show good results - the data is the same. We are now introducing software configurations to enable Short Fragment Mode to support as short as 20bp. You can do an F-tonne of short fragments #nanoporeconf
CB: New short read mode default to be released into MinKNOW in new year. 250M reads @~200bp possible from a single PromethION flow cell, with native human DNA, including a proportion of perfect reads #nanoporeconf
CB: We are now releasing a new kit – “kit 12”, which includes “Q20+” chemistry and is Duplex enabled. This enables >Q20 (>99%) modal raw read simplex accuracy with recently released R10.4 nanopore #nanoporeconf store.nanoporetech.com/sample-prep.ht…
Apols @the_taybor, Clive's handle is @The__Taybor
CB: We have refined the movement of the motor - while it is a bit slower we get a significant shift in the baseline accuracy with Kit 12 and R10.4 #nanoporeconf
CB: diff types of accuracy; Duplex is enabled with kit12, enabling >99% modal raw read simplex accuracy with recently released R10.4 nanopore. Consensus is at Q50 on bacterial genomes, but coverage required is lower with kit12&R10.4 #nanoporeconf
CB: We operate process of continuous improvement to algorithms, releasing features into research basecallers before moving to production. Eg Learnings from Bonito integrated into SUP basecaller in MinKNOW #nanoporeconf
CB: Duplex – reading both forward and reverse strands immediately after one another – occurs at a natural baseline rate, but we can make amendments to increase this rate. #nanoporeconf
CB: Changes to kit 12 have increased Duplex rate, with internal data seeing up to 45% pairable data for accuracy around Q30 per single molecule
Pioneered by @ianholmes lab #nanoporeconf
store.nanoporetech.com/us/sample-prep…
CB: The longest Duplex paired read to date has been 156 kb at ~Q30; there’s no inherent limit to duplex read length #nanoporeconf
CB: I wonder what ultra-long Duplex would do to assembly? Hard to believe this wouldn't have a significant effect, we hope to see more on this in the coming months #nanoporeconf
CB: Reminder: modification data is inherent in the nanopore signal, and so is available “for free”. We even showed data on this at AGBT 2012 #nanoporeconf
CB: Marcus Stoiber @stoibs11 will talk about this in more detail. However, in short we believe methylation data with R9.4.1 to be better than “gold-standard” bisulfite, and R10.4 is better than R9.4.1 #nanoporeconf
CB: Making calling base modifications more accessible. With no more need to re-basecall to see modifications – new Remora tool runs alongside basecalling to annotate modifications (as a lightweight second pass). Remora is in Bonito now and Guppy in the new year #nanoporeconf
CB: what's next? We have a hit list of modifications. This may be a bit vertebrate-centric so please speak up of you want others #nanoporeconf
CB: Accuracy improvements are not only in algorithms; there are lots of levers to pull and chemistry changes play a big part. Enzyme movement kinetics can be made more uniform, there are new versions in R&D pipeline #nanoporeconf
CB: Innovations around the length of a nanopore drive accuracy even further. R10.4 better at homopolymers than R9.4.1. Next gen pores with even longer read heads are in research #nanoporeconf
CB: The enzyme motor on DNA can also be influenced by changing temperature. Higher speeds may mean slight drop in accuracy, but more yield. Potential for on-flow cell control by users to select the data they want #nanoporeconf
CB: Everything covered so far is in progress already. Time to look at more future-focused work #nanoporeconf
CB: There are basically two ways of putting DNA or RNA through pore: “Inny” or “Outy” (like a belly button). Product is currently in “Inny” mode but assessing enzymes for “Outy” suitability. Benefit to Outy in e.g. size selection #nanoporeconf
CB: here is the original AGBT presentation from 2012 so you can compare where we are now to that vision. f1000research.com/slides/4-1206 #nanoporeconf
CB: More detail on Outy: DNA threads through pore and molecule can be sized. And then:
#nanoporeconf
CB: Outy can potentially be used to re-read a single molecule many times. Pause at end of the strand, motor releases, DNA drops back, caught again and repeat. So you could "to-and-fro" a single molecule for very high accuracy consensus on that single molecule #nanoporeconf
Oh by the way, the catflap reference from earlier: #nanoporeconf
CB: Adaptive sampling, where we can hold on to the molecule, and then going up and down that molecule until you have the accuracy you need: Adaptive Accuracy #nanoporeconf
CB: By combining Adaptive Sampling with Adaptive Accuracy we could illuminate rare single molecule mutations at ultra-high accuracy. Find your needle in a haystack, then confirm its significance #nanoporeconf
CB: This could be the essence of liquid biopsy in a range of samples..blood..river..air droplets – fish through molecules not of interest, and find the key ones to give your answer #nanoporeconf
CB: No big hardware changes needed to enable this concept - and there are potential applications in labs and environment – Anything, Anyone, Anywhere #nanoporeconf
CB: Our sample-to-answer concept is built on our existing flow cell tech, with a new sample chamber added above pores. Raw sample added there, samples prepared, DNA electrophoretically moved to the pores beneath #nanoporeconf #skunkworx
CB: Same concept opens up possibility for sensing other types of molecules, eg small molecules, metabolites. #nanoporeconf
CB: Introducing a new device. We introduced this concept introduced at LC 2019, and now PromethION 2 is available for preorder. Human WGS for less than $1000, with ~$10k starter pack. Falls between Grid and P24 #nanoporeconf nanoporetech.com/products/p2
CB: P2 Solo is a modular sequencing unit which uses separate compute. Compatible with GridION, easier to place in automation robots #nanoporeconf
CB: We have a proposed change to browser basecalling framework also. Use standard web browser for operation of basecalling, still exploit local GPU resource. #nanoporeconf
CB: Bonito proof of concept now live for browser-based basecalling. Have a go! bonito.epi2me.io #nanoporeconf
CB: Browser based operation will be at the heart of the intended Mk1C replacement – MinION Mk1D. Custom tablet case with MinION integrated. Register your interest here register.nanoporetech.com/minion-mk1d #nanoporeconf
CB: we are supporting/subsidising users who are sequencing critically endangered species – 22 draft genomes have been uploaded (open source) in a few months, we'd like to see more from scientists working with species in the field. org.one/oo #nanoporeconf
CB: we'd like to see people using eDNA to make a positive impact in biodiversity #nanoporeconf

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More from @nanopore

Dec 31, 2022
As 2022 draws to a close, we’ve been reflecting on some of the highlights (in no particular order) — you can read the full article here: bit.ly/3IfaKYc, or digest the thread below. Wishing you a happy new year and all the best for 2023! 1/23
The #cancer research community has made huge strides leveraging the unique attributes of #nanopore sequencing to characterise cancer at unprecedented resolution, unlocking previously hidden variation and accelerating research to support human health. nanoporetech.com/cancer-research 2/23
In May, @mdelledon took a group of scientists into the Gobi Desert to capture small mammals, including bats, to study their microbiome and they set up a genomics lab in a tent. They demonstrated the utility of the MinION in the harsh, off grid and sandy desert environment! 3/23
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May 19, 2022
Time for the latest @nanopore technology updates. Up next in the auditorium: an Update from the Oxford Nanopore team. #nanoporeconf Image
Please note, we invite you to read the disclaimer in the slides
CB: More information on Clive’s previous technology updates:
Read 71 tweets
May 19, 2022
Please take your seats for @gordon_sanghera's opening plenary of London Calling 2022. #nanoporeconf
GS: Lord Kelvin believed in meaningful measurement to see the whole picture. With nanopore sequencing you gain more comprehensive insights than ever before. #nanoporeconf
GS: In the 3 years since the last time we were at Old Billingsgate, @nanopore has released 200+ products, increased our single-molecule raw read accuracy from 95% to 99.6% & hugely improved our scalability & output. #nanoporeconf
Read 13 tweets
Dec 1, 2021
Opening the Nanopore Community Meeting Tech Update is James Clarke, valiantly speaking through a bad, bad cold. We'll be live tweeting in this thread, with Stu Reid and Rosemary Dokos up next #nanoporeconf
JC: @nanopore accuracy has come a long way. Right now, in just 3-4 seconds, I can pass 1,000 bases of a **single molecule** of DNA through a hole, and know its sequence with over 99% accuracy. That’s better than Q20, by looking at a single molecule - once #nanoporeconf
JC: Beyond single molecule, you can of course use multiple strands in a pile-up. We’ve achieved Q50 consensus accuracy on bacterial genomes, from 20X coverage. #nanoporeconf
Read 74 tweets
Dec 1, 2021
For those using @ArticNetwork Midnight for #SARSCoV2 sequencing: analysis of the available data (55 genomes) showed that 100% were correctly identified as Omicron. We’ve observed 1 amplicon dropout out of 29. This is outside of the S gene & does not impact correct lineage ID 1/4
Whilst the amplicon dropout does not affect identification of the Omicron variant, the Midnight kit will be revised to include an additional primer to improve amplification efficiency of Omicron Amplicon 28. We expect this to be available in 2-4 weeks 2/4
We are again thankful to @freed_nikki and the entire @ArticNetwork community for quick and open data sharing so that we can continue to provide high quality solutions for #SARSCoV2 surveillance around the globe 3/4
Read 4 tweets
May 20, 2021
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
Read 51 tweets

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