This week Oxford Nanopore Technologies organized the third London Calling conference, gathering around 400 attendees (200 more than last year) in the Old Billingsgate Market directly at the Thames. This year there was not a MinION in the goodie bag (I thought because everyone already had one, but there were a lot of new users as well) instead the bag contained a voucher for a flowcell and 1D^2 sequencing kit*.
I’ll not cover each individual talk, as James Hadfield did a great job of posting a detailed writeup on enseqlopedia (day 1, day 2). Furthermore David Eccles has a very thorough transcript of Clive Browns (CTO Oxford Nanopore) talk and I’m expecting a blog from Keith Robison at OmicsOmics soon. Videos of all the talks are supposed to be online later this month.
Technology
- Read length, or more specific long reads, was an often-mentioned topic the past days. Whereas 100 kb reads were previously classified as ‘long’. These days the record is 950 kb. Long reads all hinge on the DNA extraction method. This has been described on Nick Lomans blog, as well as in the human genome seq paper. The latter paper (Fig 5a) also nicely forecasts how long reads can tremendously aid (human) genome assembly reaching a predicted N50 of> 80 Mbs (basically a full chromosome)
- Clive announced (although I don’t have the exact wording) ONT would not discontinue pore chemistries any more. Which was previously flagged by quite a few attendees as limiting the implementation of nanopore sequencing in the ‘production’ environment.
- Most of the users get stable results with R9 compared to the more variable R7.x chemistry of last year (but apparently not everyone, so ONT is trying to help individual users and also organizes hands on workshops etc.).
- Direct RNA Seq is available. Although the throughput is not as high as the cDNA version (“which is just very great”). However, direct RNA seq does allow users to map base modifications as showcased by this cool direct 16S preprint from Smith et al.
- The dCas9 enrichment looks really promising, although this is not publicly available yet. Slides presented by Andy Heron from ONT included a few old ones from last year in New York, but spiced up with more recent data. For example work on increasing the local concentration of DNA at the pore using beads. On an E. coli sample this makes a 300x target enrichment possible.
- Mick Watson showed it is possible to do complete genome assembly from a metagenomic sample.
Devices
ONT now has a whole portfolio of products at different stages of the development process. I’ll segment them by their availability
- In use
- MinION, currently R9.4, will switch later this month to R9.5 pore to support 1D^2. However the 1D kits will still run on the R9.5 pore. I assume there are just a few modifications made to the pore protein that attract/guide the tether from the 1D^2 complement strand to the pore. Currently users routinely get out between 5-10 Gbase, 15-20 Gbase is in-house possible
- First PromethION flowcells are running in the field, but the users are asked for their patience as all the hardware is new (flowcell, chips, box) compared to the MinION. (This is not the case for the flonge which is just ‘reusing’ MinION hardware, see below). A full running setup with 48 PromethION flowcells is supposed to generate far more data than Illuminas Novoseq flagship.
- First shipment later this month:
- GridION is marked as a device for users who want to be a service provider. Basically it is 5 MinIONs in one box + basecaller, so no hassle with updating 5 computers. The GridION will in the future be compatible with the high-performance PromethION flowcells.
- VolTRAX (the automated sample prep) is already deployed in the field, but not yet with the reagents to actually carry out a library prep. However the release of the reagents is imminent. It will be very exciting to see first results from this, also as a way for the community to share and standardize DNA extraction protocols. Next stage are lyophilized reagents, which are scheduled for end 2017 and will be most welcomed by users doing in-field experiments.
- Somewhere in the pipeline
- Flonge is an adapter that allows a down-scaled version of the MinION flowcell to be used, thereby lowering the flowcell costs significantly. The device is in the process for regulatory approval and thus the main entrance for ONT into the healthcare market, which Gordon Sanghera (CEO) described as much harder to get a hold on than the R&D market.
- SmidgION uses the same lower pore density flowcell as the flonge but allows direct connection to a phone.
- An unnamed-basecall-dongle. Basecalling will in the future be done on dedicated hardware, a field programmable array (FPGA), which should be able to basecall 1M bases per second. This will initially make users without access to clusters or remote use pretty happy.
What will the coming year bring?
Compared to two years ago I saw a lot of cool applications and trials. Zamin Iqbal tuberculosis sequencing, Justin O’Grady urinary tract infection sequencing, Nick Loman and Josh Quick Zika Brazil project and Richard Leggett pre-term infant microbiome sequencing. It is clear the ONT platform is starting to mature and the initial hicks up are over. From a healthcare perspective these technologies are just waiting to be tried in the clinic, as Nick also mentions “Why has nobody sequenced yet in a NHS (National Health Service) lab?” So I expect presentations to be in this clinical direction at the 2018 conference. I also believe we will see large (nanopore only) genome assemblies of plants, funky eukaryotes, phased human genomes as well as metagenome assemblies being produced by the platform due to the increased throughput and read length. Eventually I expect the base modifications (both on RNA and DNA) to receive quite some coverage because of the improvements in the basecallers and kit chemistries.
In conclusion, I’m very much look forward to the coming developments as its clear that ONT is very passionate about R&D and continues to crank out improvements.
Disclaimer: I was an invited speaker at LC17 and received travel and accommodation subsidy.
*Update 05-09: Apparently new users did receive a MinION