The gist of between glaciers and genomes

 

This week I’ve been at the Microbiology Society Annual Conference in Birmingham and had the opportunity to present in the Microbial Diversity & Interactions in the Environment session in Tuesday. My theme was From glaciers to genomes…and back again and built around exploits with the Oxford Nanopore MinION in the last year. Cedric Laczny asked me to provide a summary of what was said and done for non-attendees – so here it is.

So far, we’ve mainly used MinION for in-field metagenomics, but I decided to only mention this as a scene setter. Our larger problem is that Earth has 70% of its freshwater stashed in glacial ice occupying 11% of its surface area and the genomic diversity microbial inhabitants of this (by volume) massive freshwater ecosystem is very poorly mapped. I believe we have fewer than 10 public bacterial genomes, less than five cyanobacterial genomes, no eukaryote or archaeal genomes and a slack handful of amplicon or shotgun datasets to cover ca. 198,000 glaciers and three ice sheets. It’s embarrassing to chat to folk in the medical microbiology community: last night I passed a poster reporting >10,000 Salmonella genomes.

As we are embarking on an unprecedented experiment in destroying glaciers and because microbes are confounding factors in that experiment it seems prudent to start discovering this genomic diversity. I have tried to engage the research community to gauge interest in a genuinely communal effort to sequence as many genomes as we can afford to, but the response has largely been muted or inclined to consolidate the project at one institution. If anyone who reads this is interested in a network based collaboration – I am all ears.

But, when starting from a low point, even incremental advances can be transformative. So for now we’ll go with me and my MinION.

The scope of my talk covered the behaviour of just a few cyanobacteria associated with cryoconite formation. My plans to show the Chris Hadfield-approved zoom shot into a Greenland cryoconite hole was scotched by IT issues – the lack of a computer mouse!

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Nevertheless, in ten minutes I needed to cover cyanobacterial sequence diversity in cryoconite on a timescale from ~12,500 years before present to Tuesday before last – literally. I need to write a separate update about the Walters Kundert “Bleakest Midwinter” project, but for the moment I’ll just say we hit our bag limit on Svalbard cryoconite samples in the “light winter” phase this March and preliminary MinION metagenomes and qPCR on our portable Mic cycler are prompting interesting hypotheses about who lives and who dies between dark and light winter. I’ll look forward to the final phase of sampling so I can then batch the samples for other analyses.

The core part of my methodology has been to resolve these genomes from shotgun metagenomes sequenced on the MinION. I’ve been multiplexing 3-6 samples per flow cell using Josh Quick’s one pot barcoding ligation protocol. In contrast to the local norms of the parish I find myself in I am not able to bask in the glory of closed, single contig genomes formed from ultra long whale reads – but then again I am working with degraded, old (ancient?) bead-beaten DNA so my expectations were adjusted downwards from the start.

Nevertheless, I have been able to learn some interesting things. As this needs to be worked up into a couple of papers, I won’t delve into too much before peer review.

In short, following error correction and assembly with Canu, and then a binning strategy based on the taxonomic classifier to select contigs with good protein-level matches to taxa of interest I do have bins corresponding to discrete bacterial genomes.  Quick annotation with prokka throws up both interesting metabolic traits and the prospect of strain resolution. Where we have non-targeted metabolomics data from the same samples, the presence/absence of pathways matching the salient metabolite fluxes  is quite gratifying. For one of my genome bins which matches a non-cyanobacterial taxon where the evolution of a particular autotrophic pathway is an unfinished business. I can tick off genes for their presence or absence in full concordance with the sequenced isolate, a bacterium with a large and complex genome which was reportedly very difficult to assemble on short reads only.

But it is with the rRNA operons that I’ve been having most fun. Agreement between the binning and rRNA operon taxonomy is excellent. Last year I co-authored a study led by Takahiro Segawa which used a retro, Sanger based long read strategy to resolve contiguous 16S-ITS environmental sequences from cyanobacteria on a global range of glaciers. I think the ITS haplotype data in that study offers the highest resolution and spatial coverage of diversity across the terrestrial cryosphere, so I have simply been looking to see where my MinION metagenome-assembled genomes have matched the Segawa haplotypes. The good news is that the 16S genes match the expected 16S OTUs and the ITS haplotypes extracted from my genomes lie within the geographic clades for the population structures of those OTUs. So – seems legit.

Cyanobacterial haplotypes from pre-modern cryoconite cyanobacteria either published by Takahiro Segawa or in our possession also sit within the extant strains from those regions, hinting at the stable colonization of the cryosphere over extended timescales.

Personally I would hope this observation of congruence between pre-industrial and contemporary cryoconite ecosystem engineers helps make abundantly clear that the old chestnut “dark stuff on ice is simply pollutants” is utter dog toffee.

As always when presenting nanopore data someone is duty bound to ask the question “but isn’t the error rate terrible”?  This is based on the observation that the accuracy per base of uncorrected, raw reads is in the range 85-92%. Fair one. But we are not playing with these reads, we are playing with error-corrected assembled data.

While I can point to the efforts of folk who do multiple rounds of polishing with racon or nanopolish, and their dissatisfaction with 99.9x% accuracy for this initial effort I have only used error correction with canu. For one thing I am wary of citing CheckM statistics as remaining indels will likely undersell the estimation of completeness. For now, as the only hook I have to hang these genomes on is the ITS strain data, tracking the rRNA operons has been my goal. One of the next stages might be to look across the genome so I will be polishing and/or going hybrid as funds permit.

So what’s the error rate like in this context? Here’s a quick test: Dr Nathan Chrismas has kindly provided his BC1401 isolate of Phormidesmis priestleyi which represents the first cyanobacterial isolate genome from glacial ecosystems, sequenced with Illumina reads. For cyanobacterial genomes, one can usually read them as “metagenomes” as a number of cohabiting bacteria are difficult to get rid of, even in a unialgal culture. In his paper, Nathan devised a clever bioinformatics strategy for doing so…rather than hammering the culture with say, bleach (sorry Nathan). But, when resequencing the isolate from culture the presence of contaminants obviously recurs. So, turning the negative to a positive I can consider the ONT sequence data from BC1401 a reduced complexity, P. priestleyi dominated metagenome. This starts to sound like the real thing, covered by a similar sequencing effort, but for which there is a 213 contig genome sequenced with Illumina data to act as a benchmark. Long story short, across the 4.8kbp rRNA operon I obtain a 98.92% identity between the canu corrected and assembled ONT data and the Nathan’s Illumina assembly.  Not 99.9999% but fairly respectable for a first pass.

In summary, given the potential strain level resolution and functional insights from genomes recovered from metagenomics data generated on an USB powered device I get the feeling our MinION will be just as handy in our home lab as well as our field lab.

My thanks to collaborators Dr Joseph Cook, Dr Sara Rassner, Professor Andy Hodson and Professor Alun Hubbard for their contributions to this work in the varied form of samples, fieldwork and scripts for pulling out interesting contigs, and to the session organizers for the opportunity to speak.

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Ice Alive

On Friday night I had the pleasure of attending and presenting for the launch of Rolex Young Laureate Joseph Cook‘s documentary Ice Alive at the Royal Geographical Society.

Narrated by The Life Scientific’s Professor Jim Al-Khalili and presenting former commander of the International Space Station Colonel Chris Hadfield, UK Polar Network President Archana Dayal, Dr Jenine McCutcheon and Dr Andrew Tedstone from NERC Black & Bloom, the film explores the connections between life and ice.

Set against a backdrop of stunning imagery from Greenland and Svalbard the documentary makes an arcane research field come alive to a much broader audience than I would have ever thought possible. In Hadfield’s words: in an increasingly complex world, knowledge may be your only means of survival.  Communicating our science clearly to the world in this way helps pack our survival kit for the coming decades.

 

The evening opened with an “audiovisual exploration” of the Greenland Ice Sheet, linking Joe’s UAV imagery with Hannah Peel‘s original composition.  The exploration covered ground ice I know well as AWS S6 and RG outlet but manages to conjure the sublime and convey both the power and vulnerability of the ice. Watching it full screen and with your speakers to 11 is to be recommended.

On Svalbard in August I had the pleasure of meeting Leverhulme Trust Artist in Residence Naomi Hart while I was kicking off my own Leverhulme research fellowship. We formed an unlikely partnership – she needed someone with a gun to cover her while she explored the ice, I needed a field buddy while I did what she describes as “cutting edge science with teabags and Danish coins”. IMGP0951

Naomi on the ice

Naomi’s project is to document the links between ice, climate, life and coal on Svalbard from the days of Eric Rugnose Brown to Andy Hodson as heads of geography departments in Sheffield University. Our view of life on ice has changed from Rugnose Brown (“of course the ice is devoid of life”) to Andy (e.g. the seminal review: Hodson et al [2008] Glacial Ecosystems, Ecol Monographs).  Naomi presented these links in a compelling talk and exhibition.  From Naomi I’ve learned the way of the artist and the scientist are often closer than we think: both build upon experimentation to find and communicate truths.

And of course, I couldn’t let the evening pass without sticking my oar in. As the current RGS Walters Kundert Arctic Fellow, and speaking at one of the great homes of exploration, my theme focused on the exploration of Earth’s microbial frontiers. My contention is that exploration is far from a done deal, and we face not the final frontier, but rather fractal frontiers. Here’s a snapshot.

I’ve likened this to the coastline paradox: the more we discover, the more there is to explore.  For my part, these frontiers are microbial, and our current cutting edge is to access the genomes of the microbes while on the frontier. I spoke about how the public health crisis represented by the Ebola outbreak in West Africa stimulated me to think about how we are tackling the environmental health crisis of Arctic warming, leading to in field DNA sequencing using Nanopore devices. I have little doubt that as our lenses sharpen yet again there will be more to discover.

My heartfelt thanks to Joseph Cook for the invitation to attend and present, to Proudfoot Films for an awesome documentary and to the Rolex Enterprise Awards and for making the event possible.

No bucks, no Buck Rogers

A few years ago, a friend gave me a copy of The Right Stuff, following a conversation over a brew in Greenland about the philosophy of working on challenging frontiers, and the kind of people it attracts. Joseph Cook has also written about the parallels between astronauts and Arctic scientists so I’ll not explore that here. Moreover, I’m several inches too tall for a ride on Soyuz, and my personality has far too many rough edges to take this kind of thing on breakfast TV with such flawless grace. Let’s not even talk motion sickness. It’s fair to say I therefore have no aspirations to be an astronaut!

Instead, I’m going to write about something even harder than becoming an astronaut. Funding a career in science.

A more recent read in the genre of astrobiography recalled lines from two Mercury 7 astronauts in the film of The Right Stuff:

Gordon Cooper: You know what makes this bird go up? FUNDING makes this bird go up.

Gus Grissom: He’s right. No bucks, no Buck Rogers

The relevance to Arctic science could not be clearer.  Any future in research has to be fundable. No bucks, no Buck Rogers.

In December I spent some time helping a talented younger scientist debug a fellowship application. I could see some great potential in the science and the applicant which wasn’t coming through clearly on paper in the drafts I looked at, early-on. You may have The Right Stuff, but no bucks…no Buck Rogers.

It occurred to me that in this era of enlightenment such topics as the basics of grant-craft make it on to graduate school curricula. It seems not. Things may not have changed since my own graduate training.

I have a chequered history with grant capture. Sure, there are many projects where I have been proud to secure funding from challenging sources (e.g. NERC, Royal Society and Leverhulme) as a PI in my late twenties/early thirties but this has often felt like more luck than judgement.  My boss made clear that while there was room for academic freedom, there was no room for failure: I should expect to bring in grants and 4* REF papers, building a group from the bench and pipettors I used for my PhD. The sooner the better. A kind colleague gave me photocopies of the case for support for two highly rated NERC grants, describing them as following the formula for success. The rest is the product of tolerant coinvestigators (ca. 2010: “Arwyn, have you fecked [Full Economic Costed] this grant yet up yet?” “Mind your language!”), tenaciously supportive research administrators and (usually) constructive comments from peer reviewers. It still feels like I’m breaking in to academia, with the sirens wailing in the distance.

So today I was pleased to abuse my position as the Director of an Interdisciplinary Research Centre to convene a workshop titled Your Fundable Future with its affiliated graduate research students. I reached out to the students past the middle of their PhDs. Their futures may well depend on funding sooner, rather than later. Hopefully I could pass on just some of the stuff I wished I knew at their career stage but have learned from hard knocks since.

As we handled real grant proposals (I’ve hitherto failed to gain funding for!) as part of some of  the exercises, I won’t detail the process further, but here are some of the resources which I’ve found helpful in trying to figure out the funding game.

It helps to start from a strong position with your science.

Mick Watson’s tips for early career researchers (less about grants, more about being able to submit them from a position of strength)

You will need to write clearly – your audience comprises clever people with no time or special interest in your pet project

Tim Clutton Brock – Perspective on grant writing (some technicalities dated in detail, but principles highly relevant)

Grantsmanship – Mark Pallen (a good use of 1h30+)

Writing Science (Ronseal!)

You will need to learn the rules of the game.  Ensuring perfect adherence to eligibility is important (including every detail of submission format).

Ten simple rules

Humans decide who gets funded on the basis of subjective interpretations of seemingly objective reviews, panels, criteria.

Unconscious bias – your reviewers will likely have it

Athene Donald and the ABCs of Panels

There’s other advice too. For example, it’s important to sell your project to the reader within the first few lines of a proposal. If you’ve read this far, that matters less though.

 

 

 

 

 

 

No Dramas

My recent grump-piece about field safety has returned to the foreground of my thinking as National Geographic chose to further publicize an incident from 2014 in which a scientist self-rescued from a crevasse in a snow-covered glacier in the Himalaya. By the scientist’s own admission he was alone, therefore unroped and visibly without the typical personal protective equipment (e.g. helmet).

To his credit, the scientist was able to climb out of the crevasse. Others in his situation are not so lucky, and the consequences don’t end in the crevasse: criminal charges are being brought against the employer of an Antarctic helicopter pilot who died in a crevasse fall. Some may have the savvy to extricate themselves from a crevasse, but all is for none if you’re jammed in hard, too deep or too severely injured.

I may be criticizing from an armchair, but I am not entirely an armchair critic. Crevasses are a fact of life and death for people working on glaciers. Glaciers are slot machines, and like slot machines, the odds are in the house’s favour and not yours. I’ve spent entire field seasons end-running or stepping over  slots commuting to field sites, and many trips roped up above the snowline to collect essential samples or measurements.

Here are some snaps from work which will be unlikely to be tagged with “heroic scientists risk life & limb” on any time soon on social media.

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“Chris Flynn” preparing a corer on crevassed terrain in the Swedish Arctic

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Sampling on bare ice, South Georgia. At this point in the day, the risks from crevasses were nil. But when working on unfamiliar glaciers it pays to wear a helmet and harness racked up for two reasons: firstly, it speeds up a transition to roped work, but also should a fall occur, it offers a much better point of attachment for rescuers than shell clothing. (Photo courtesy Dr Anne Jungblut).

 

 

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A team of three moving up to the snowline on a Svalbard glacier. The site is well known to us for >10 years, but the exact position of deeply incised melt channels varies, meaning a cautious approach is merited from the start of a new season while the glacier is wet. Recreational mountaineers are unlikely to work in threes, carry the full range of kit or (potentially) rope up on terrain such as this. Different ball game.

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Yours truly. Given my dainty proportions I am best placed at the tail end of a team of three. In this position, if I do not hold the fall, build decent anchors and transfer safely to them the consequences are likely to be serious for all.

For each of those trips there is a period of refresher, rehearsal and training. When working with scientists new to glaciers this is essential, but even after a decade I still practice basic skills of tying in with chest coils, moving while roped, self-rescue and building pulley systems before the start of any period of fieldwork which could require travel on snow covered glaciers.

None of these photos illustrate a scenario comparable to 127 Hours or Touching The Void – for a good reason. As such they are unlikely to have a fraction of the “reach” of a drama in the media.  Yes, we all have bad days at the office from time to time, and sometimes photos or video from such days play an useful role as “teachable moments” – be it for training or inquests. However, their promotion on social media to the general public distorts the reality that these are issues of workplace safety, not fallout from adventure for its own sake.

To sum up, I’ll borrow some boldface:

The underlying principle behind all Antarctic travel is the need to apply conservative and reasoned judgements to all decisions. Accidents often result from a chain of events caused by a number of small errors and bad decisions that eventually snowball into a serious situation.

From the BAS Field Operations Manual. One can substitute Antarctic for fieldwork in any number of snow and ice environments without losing the message. In crevasse country, a gram of preparation will beat a kilogram of badass.  The Field Ops Manual is publicly available at the user’s risk (and due to be updated) – but is a good source of reference material on field safety in polar environments, along with the Royal Geographic Society Polar Expeditions Manual. Hardly clickbait, but a starting point for being safer.

 

A nocturnal upon St Lucy’s Day

At the moment I’m escaping the cold in the UK by working up on Svalbard in the High Arctic. Half way between the top of Norway and the North pole, the current temperatures are hovering around -3*C, in spite of the depths of polar night. The next sunrise here in Longyearbyen will be on the 15th of February, 2018.

My work here is part of a project funded by the Royal Geographical Society’s Walters Kundert Arctic Fellowship which is looking at microbial life on glaciers during polar night, and how changes in the Arctic’s winter climate might affect microbial activities. This is the second trip as part of the project. We currently assume microbial activities on glaciers are limited to active melting conditions in summer, but there is little in the literature to validate this assumption.

A bleak future is also being painted in the media for Longyearbyen’s human inhabitants. I consider the notion of a warm, wet, winter as a “bleakest midwinter” for the region’s microbes, disturbed from their likely hibernation.  Today marks the publication of NOAA’s 2017 Arctic Report Card (Headline: Arctic shows no sign of returning to reliably frozen region of recent past decades) and it is also St Lucy’s day – the year’s midnight, according to John Donne. So, in this post, metagenomics meets metaphysics.

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AugustAs shadow, a light, and body must be here. Svalbard glacier surface showing some algal biomass, dispersed cryoconite and cryoconite holes. All active microbial habitats.

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December: Whither, as to the bed’s-feet, life is shrunk/ Dead and interr’d. Or not: Same glacier, excavating microbial habitats from under the snowpack.

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The general balm th’ hydroptic earth hath drunk: Soaking excavated cryoconite in RNA later for return to the UK.

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new alchemy. Meanwhile, using some some spare sample material, I prepared a bucketload of DNA for shotgun metagenomics on the Oxford Nanopore Technology’s new field sequencing kit: a freeze-dried, use anywhere library preparation kit.

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A quintessence even from nothingness. Using a flow cell stored for 3+ weeks and flown to Svalbard at ambient temperatures (with >1200 active pores) I had a go at sequencing the DNA. Compared to out UK trials of the same kit, the results were poor, providing only hints at the microbial community structure.

My thanks are owed to the Royal Geographic Society for funding, and Professor Andy Hodson of UNIS for hosting.

Dead scientists don’t write papers

This is an important article on fieldwork safety written by Elizabeth Orr. It opens with a vignette from her PhD fieldwork in the Himalaya, describing trouble on slippery ice and then a river crossing gone wrong. I’m always very leery of river crossings: from another lifetime I recall a statistic that UK Special Forces have lost more people in water than in anything else they do – so not a trifling risk, but often part of the commute to the office for researchers working in remote environments.

The author notes a disconnect between geoscience fieldwork in principle and in practice. This troubles me.

So is it acceptable for graduate students to be sent into the cold as academic cannon fodder?

Thoughts? I’ve had a few.

JAFA syndrome

Rightly, much of the responsibility to train safe researchers falls to the shoulders of PIs to lead by example and make effective arrangements, but again, in my experience, the skills, qualifications and experience of PIs can vary. Can we expect (busy) academics without any formal training themselves and varying levels of skill and experience to set out an adequate scheme of training and operation? In my field I’ve encountered several principal investigators with respectable publication records which would indicate experience in the field but (as an example) are unable to use crampons to access a dry glacier. When working in a glacierized environment is such a person competent to evaluate the risks, own and mitigate them for their trainees? No.  Equally, palming the responsibility for their students off to other researchers or field stations presents complications. All fun while the Nature Geoscience papers are writing themselves, but makes for a messy inquest when it all goes wrong in the worst way.

Hardly a season in a remote field station is complete without some entirely predictable disaster befalling one research team or other: bad weather, bad logistics, bad company. Under the pressure of watching a costly field season circle the drain, bad decisions are made, pushing the window on weather, sleep/food or logistics. This is when Dr Murphy pays a visit. Better to have Plans B-Z and stick to them – factoring in weather from the start. This advice comes from someone who cut short his honeymoon to deliver a field project, only to spend three weeks waiting for conditions that never improved. It’s part of the package of fieldwork: best learn early.

So, to the pushy PI – I’d argue unsafe data is unethical data and thus unusable data. For the hard-headed, here’s a very pragmatic point of view: if you’re sending students who are borderline hypothermic and nurturing frostnip to collect the next n=2000 sample set, is the execution of the protocol you drafted in an air-conditioned office going to be of the same rigour and attention to detail as you would demand? I doubt it. PIs need to factor in safety at the heart of a fieldwork programme for their students.

Learn and live beats live and learn

Almost any institution will offer courses to grad students on everything from R to referencing (useful, no doubt) but the topic of stayin’ alive and working efficiently in the field is rarely on the environmental research curriculum of any institution I’ve heard of*.  Certainly in my own graduate education there were no opportunities for such training. It fell to my own initiative to upskill. In the UK, NERC has sponsored an advanced training short course for polar science students, but its availability is limited and the content highly condensed.

It’s a good start: but it only targets early career researchers, perhaps on the imperfect assumption their seniors have evaded Darwinism long enough to learn.

When my opinion has been sought, I have made the case that specific training for Arctic scientists should be mandatory in the way it is for Antarctic researchers upon deployment. Considering the many pathways and venues for Arctic science make it a relative free-for-all, this is more sustainable than insisting upon field guide “minders”.  Submitting a grant with >xx% polar north fieldwork? Enter your training certificate number in the box on Je-S (other grant submission interfaces are available) and prove your competence against a set standard.

Funders should care about this issue at this level: well trained people reduce the risk of fieldwork failure. In accepting a Royal Geographical Society Arctic & Mountain Research Fellowship recently I was pleased to note their insistence on detailed vetting of fieldwork plans and risk assessments.

It’s the day job

Predictably, people who build careers on fieldwork often love the outdoors and may be very accomplished in the realm of outdoors sports. This can be extremely positive, bringing a lifetime skills and experience to the table. But for new fieldworkers who live for the outdoors, it can also present problems adapting from playing hard to working hard.

Acceptable risks and concessions to safety in your own time may take on a very different legal and practical complexion in the workplace. Now, the goal is not adventure, but to deliver on (often publicly funded) science safely and effectively. If having an adventure is your primary goal in seeking a career in field science, get out and then get out there. You will not be satisfied by using science as a vehicle to quench your thirst for adventure, nor will you necessarily approach decisions in the field from a professional perspective.

Human factors

Notable by its omission is any discussion of fieldwork harassment. Sure, this is a crucial topic to address in its own right, but a working environment where harassment occurs is not a safe environment by definition. I do not seek to diminish the importance of preventing and confronting sexual harassment by stating that unacceptable behaviour in field settings is a problem which occurs between many demographics. Even from my level of privilege as a white European male, a harmful experience as a student still deeply affects how  I interact with anyone I meet in the field a decade later. I can only imagine the impact of behaviour as extreme as recent allegations of misconduct in Antarctica on an early career researcher.

Perhaps we could learn from history. The history of Antarctic exploration is not a shining example of promoting equality and diversity (e.g. the ice ceiling) but I recall a “Golden Era” Swedish expedition forced to overwinter as its ship sank. The standing order from the expedition’s commander was that everyone’s first duty was to be kind to each other. Simple, but powerful in achieving harmony within a cramped, dangerous environment.

Be the change

While the article’s fifteen point set of recommendation contains very good advice, I think we need a smarter approach at all levels.

We all had a good laugh at #fieldworkfail (well I did, right up until my employer’s PR officer got in touch to ask why my stumbling into a cryoconite hole on Greenland was trending in Germany) but for every minor embarrassment and tale of derring do there is a deeply unfunny tale. Light hearted books have been written about #fieldworkfail, but Elizabeth Orr’s article is one of the few contemporary attempts to address the topic to reach my radar.

A few years ago in pre-deployment training for an Antarctic project, an instructor asked what the participants considered to be an acceptable fatality rate for such work. Estimates from the class reached as high as 3%. The same organisation was achieving nearly those rates annually until the mid 1980s. Since then they have avoided all but one fatal accident. A remarkable change. How? By making everyone responsible for their actions, and in particular the leadership. Simply, leaders lead. Everyone else follows.

We may never achieve 0%, but the onus is on all to put safe work at the heart of their field agenda.

 

 

(*Correction: Archana Dayal kindly reminded me UNIS puts staff and students through fieldwork safety training. As a general point “Stayin’ Alive 101” isn’t on the graduate curriculum of many larger institutions as far as I am aware)

#nanoporeconf

It’s hard to believe a year has passed since I last wrote up some personal highlights from the Nanopore Community Meeting in New York. Nevertheless, it’s been a year which has marked considerable progress within the world of nanopore sequencing – and in the ways we use nanopore sequencing in our lab.

Summaries of day 1 and day 2 are available here. This year’s meeting was packed with many, many talks from the user community and relatively few announcements of dramatic new tech directions. It seems right now is the time to polish many of the emerging strengths of nanopore sequencing.

To be honest, what is already being achieved is extremely impressive. Consider antimicrobial resistance as a case in point – a major societal challenge. Existing approaches take time. Time can cost lives, as bluntly demonstrated by Charles Chiu’s slide on time to effective antibiosis in sepsis. Several speakers showed how nanopore sequencing changes the dynamic: Patricia Simner reported detecting carbapenem antibiotic resistance genotypes within minutes of sequencing. I expect the next frontier is cost: where the tests are more expensive than many commonly prescribed antibiotics, the impact will be diluted. Low cost, application specific nanopore tools may well change this economic balance.

I think the highlight of the conference for me was that during an interactive plenary, discussion moved from technology itself to its societal implications of everyday genomics. If technologies such as nanopore sequencing are to be transformative for society, in what ways do we all wish society be transformed? Would we opt for our children to be sequenced at birth, and what would that mean for insurance? Or a future where your genome could be sequenced from a handshake and resynthesized in a genome foundry. Hypothetical for now, but the convergence of simplified sequencing, powerful computing and synthetic biology in a biohacker’s lab is one to watch.

This year I had the privilege of presenting in the microbial breakout session once more, reporting on our underground sequencing work. In two experiments in a coal mine, we demonstrated microbial identification using our lightweight metagenomad kit without benefit of power or internet. This experience has been useful in mounting our Arctic campaigns this summer, using rapid library preparations to characterize the microbial communities of the Greenland Ice Sheet while camped at the ice margin, achieving species level ID of cyanobacterial ecosystem engineers, and 16S rRNA gene sequencing on Svalbard to look at community responses to habitat changes on the glacier surface. Having shown in field metagenomics from sample-to-preprint publication within the likely doubling time of a glacier microbial community in 2016, it’s high time for me to consolidate a body of work in this area.

Cramming a little bit too much into the talk, I also presented our results from the lyophilized field kit on metagenomic sequencing. Until now the cold chain has been a significant constraint on deep field sequencing: essentially, in-field sequencing has moved the cold chain from sample return to lab to the deployment phase. It’s not going to be a problem for long. Using flow cells stored at +20*C for six days and freeze dried library reagents I was able to sequence a metagenome within two hours of Oxford Nanopore Technologies releasing the protocol. Although the “field site” was my kitchen table rather than the glacier – I’m very happy with the concordance between the field kit, nanopore rapid libraries and Illumina data from the same microbial community. I’ll post more about the lyophilized kit when I return to the UK, and release some data from a second experiment.

#nanoporeconf

Looking forward immensely to the Nanopore Community Meeting and hearing about all the exciting developments since last year. For my part I’ll be presenting in Friday’s breakouts session on microbes and metagenomics. I’ll mainly be talking about our work on sequencing in inappropriate places: Deep, Cold and Dry…

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