Reflections on #nanoporeconf

Reflections on #nanoporeconf

Another departure lounge, another blog. It has been a fun but also draining couple of weeks, skipping between the EU ITN MicroArctic kick off meeting plus some polar night fieldwork and duty as an (un)hired gunman for field parties on Svalbard, and then directly to New York City for the Oxford Nanopore Technologies (ONT) Community Meeting, where I had the honour of an invited talk about our metagenomics misdemeanours. While I kill some time at the gate, I thought I would share some thoughts on the New York side of the trip.

Disclosure – ONT kindly paid my travel expenses to present at the meeting, and I have had access to the MinION kit from MAP. That said, if I had something less positive to say, I would say it just the same.

Hardware from #nanoporeconf

Lots of exciting stuff is coming out from ONT. While high throughput, benchtop kit like PromethION would readily excite some of my colleagues at IBERS who are in to crop genomics, I was far more piqued by the lower throughput but higher portability kit. Specifically the SmidgION and the Flongle, which respectively are a mobile phone powered sequencer and a “flow cell dongle” to allow MinION users to run a smaller, SmidgION like flow cell. The potential for these platforms to do quicker and cheaper sequencing for focused applications is impressive, and I understand ONT will pursue diagnostics approval for them. For environmental applications, the potential to do cheeky on site, real time 16S profiling for ca. 20 USD a run will be a game changer. There are glaciers on Svalbard and certain spots on the Greenland Ice Sheet with good GSM signal and 3G so I would love to try some supraglacial SmidgIONing someday soon. (I am of course assuming that the app would require connection to the cloud in some way, given the reliance on a phone. I could be wrong about that.)

Disruptive technology…literally

All this said, the bit of hardware that impressed me the most is a flow cell dongle prototype which integrates sample prep with an on-MinION beadbeater. I will wait to see if this actually performs, and on what range of samples (insect bits were mentioned, but soil like matrices would be top of my list) but the impressive thing in my mind is that ONT “gets it”. Sample prep is the bottleneck. I have been saying and thinking this for a long time, but for the microbial ecologist there has been no real innovation in sample prep approaches for well over a decade. Yet it remains pivotal to good sequence quality and obtaining a “representative” view of the community of interest. Simply put the hard work invested elsewhere in developing an array of bioinformatics tools for microbiota research has not been mirrored upstream of the sequencer. So it is great to see a sequencing company explicitly considering this aspect.

Wetware from #nanoporeconf

So far my interest has been drawn to shotgun metagenomics based approaches for characterizing microbiota. As I stated in my talk, the direct sequencing of ca. 200 ng of community DNA avoids PCR amplification which incurs potential primer biases and risks coamplifying contaminants which are likely to be abundant in more rustic field settings. However ribosomal RNA has a foundational significance in microbial diversity exploration, so it was great to see Andy Herron, Andrew Smith and Lee Kerkhof present different strategies targeting rRNA in various forms, as the rRNA full operon, as 16S rRNA directly sequenced and by enrichment of 16S rRNA. My one note of alarm was the finding that data from 16S rRNA genes and 16S rRNA overlapped considerably in a trial environmental sample. I would not expect this as these compare apples and oranges.

 

Science from #nanoporeconf

It would be hard to provide a comprehensive list here, so I will just cherry pick some personal highlights

Jane Carlton sequencing malaria genomes from clinical samples in remote Indian field labs. Knowing how much attention is needed to cold chain flow cells and reagents to Arctic locations, this work by Prof Carlton and her parasitologists was all the more impressive.

Lee Kerkhof with 16S to 23S rRNA operon amplicon sequencing. Something that ONTs long reads readily lends itself to. Nice to see it is being done well. Kudos to Prof Kerkhof for unashamedly stating that the ensuing bioinformatics had to be point and click, GUI, Excel spreadsheet based. As an unapologetic mudfetcher I feel this is exactly what is required if one has the primary goal of understanding a microbial community, rather than simply processing the data. Until that write_paper.py script produces a functional stacked bar chart, PCoA-or-nMDS, ANOSIM and network with a passable cover letter for ISMEJ I feel it is a distraction.

Julie Hachey on the potential for MinION to sequence extraterrestrial nucleotides for life detection on Mars. Enough said.

Johanna Rhodes on real time fungal genomic epidemiology in a hospital outbreak of an emerging Candida species associated with a high case fatality rate. By now my sole interaction with medical mycology is giving a couple of second year lectures on key fungal pathogens, and I emphasize to the class just how ferocious fungi can be, especially for patients with immune system dysfunction. I always detect some scepticism about mycoses from folk in the class who have been enticed by HIV, Ebola, Zika et al. as the “big killers” so it was nice to see UK medical mycology being put firmly on the map in this context.

The Talk

It is fair to say I was severely bricking it. The list of speakers was impressive, let alone what they had to say. I very rarely speak at conferences, given the cost of attendance, and I would not really count myself as one of the “nanoporati” or “illumina-ti” to say the least.

Hopefully some of what I had to say about the importance of generating a genomics based perspective of environmental change in the cryosphere got through. These are very rapidly changing environments where microbes are both sensitive to the change in play and consequently drive landscape scale feedbacks which make the situation worse (e.g. methane production, biological darkening of ice). But we lack baseline data as genomic variation in space, time and phylogenomic dimensions of cold environments remains severely undersampled. We can use MinION to help change that.

Introducing the MetagenNomad

MetageNomadToothbrush sawn in half style kit and philosophy required to let metagenomics break free from the lab. May involve roughing it, cuffing it and duct tape. c.f. Patagucci.

What seemed to create most buzz (if my twitter timeline is any indication) is that we have assembled all the kit and consumables to go from environmental DNA extraction, QC, shotgun library or PCR prep, through to nanopore sequencing and offline bioinformatics in a 45 litre by 7 kilogram package. Even the choice of rucksack got some attention. A UK military issue daysack was not the first choice. I really wanted a yellow Tespack solar backpack, as it would visually resemble a Minion cartoon character, or failing that just a Minion daysack, but the requirements analysis for a resilient bit of kit scotched the minion bag, and the Tespack seemed less important as the first first few missions with the kit are in dark environments. So I went with a rucksack we used on Greenland last year which has the advantage of detachable side pouches to allow users to carry field safety gear (bullets, chocolate) or more plasticware or powerpacks. While others are pursuing hardcase based systems, I guess as a rucksack this becomes the first “wearable” sequencing lab. I know what I would prefer to use abseiling into a crevasse.

The key feature of the kit is that it all runs from 12V DC, is comprised of off the shelf kit and our preliminary bioinformatics tools do not require cloud based basecalling or taxonomic analysis. It should have sufficient endurance for a few sequencing runs. As I am continuing to fine tune the kit I will write in a little more detail about the exact content and rationale after our next field based MinION mission in a few weeks’ time.

Time for more coffee.

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About Aber Cryoconite

Senior Lecturer in Microbiology at Aberystwyth University, Wales, UK. Research interest: polar and alpine microbiology. Views my own.
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