Opportunities multiply as they are seized – Sun Tzu
We have just published a paper in Environmental Research Letters’ Focus on Cryospheric Ecosystems detailing a metagenome we sequenced from cryoconite in the Alps. The paper is open access, and I cobbled together an insight piece about its implications for ERL (“cryoconite: not to be confused with kryptonite”) riddled with cliché (“genetic blueprints”, for starters). This week, the paper and some of our other recent work was discussed on the Society for General Microbiology’s Microbe Post.
In a sentence, we sequenced a lot of DNA ripped from Alpine cryoconite and found most of it to be from devious bacteria forced to steal carbon and nutrients in order to survive. Don’t take our word for it: the metagenome should be publically available on MG-RAST (#4491734.3) for you to re-analyse for your own purposes. We haven’t quite finished with it yet, though, but don’t let that stop you.
So, rather than précis the paper’s scientific context, what I wanted to write about here was a little about the paper’s backstory. A defining theme is encapsulated in the pretentious Sun Tzu quote at the start: seizing opportunities.
The paper has its roots in an email I sent the awesome International Woman of Science (who has an award to prove it), Professor Birgit Sattler in March of 2010. I had finished my PhD a few months earlier, and despite being jobless, grantless and paperless I wasn’t quite done with science. Having worked with Birgit on Svalbard, I suggested an Alpine reunion might be fun. Opportunity #1 presented itself: the Society for General Microbiology’s Presidential Fund for Research Visits. I applied for, and duly won my first grant. An interview with the SGM about my experience was published here. In a nutshell, if you are an UK based early career microbiologist, apply for it. The perspective, experience and CV points of getting a grant to go do science elsewhere you will gain are worth it.
Six months later, I found myself in the Austrian Alps padding around a glacier called Rotmoosferner, collecting cryoconite as I went, following mostly the SOPs I had developed on Svalbard. Opportunity #2 presented itself in the form of the fearsome Professor Andy Hodson. Andy had come out to play and had brought his fave toy, an infra-red gas analyser with which to estimate cryoconite net ecosystem productivity with him. Later, two Brit male scientists left unsupervised with beer and a device to measure gas composition led to its natural conclusions: My farts are twice as potent as The Hod’s, it takes 10 square metres of an Arctic glacier to make them carbon netural, and he has yet to forgive me for the trauma inflicted on his IRGA. I digress: NEP and radiometric analyses of productivity suggested the cryoconites were typically heterotrophic. This provided essential context for our DNA work.
Back in the UK, I extracted the DNAs and did some other analyses and archived the DNA for posterity in -80*C. The story goes cold then for a little over a year. In the meantime, Aberystwyth University was investing heavily in next generation sequencing kit and expertise with the support of the BBSRC and the Welsh Government. Opportunity #3 presented itself when Dr. Justin Pachebat phoned up: The maiden run of an Illumina HiScanSQ was happening in 36h. Did I have anything interesting to sequence?
An hour later, the DNAs from Rotmoosferner were thawed enough to be quantified fluorometrically. If pooled, we had enough to make a paired-end library for sequencing. Justin immediately set about making the library: shearing, end-repairing, ligating, amplifying, quantifying and qualifying the library like a man possessed. By 4AM and after takeout pizza, tons of tips and scalpel blades, we had a library. My role in this was marginal: I can just about work spin columns and courier stuff in ice boxes.
In the morning, I took the library off to the Translational Genomics Facility on our other campus, and left it in some capable hands. Good news and bad news followed: Of eight libraries loaded onto the sequencer, ours was the one that worked, no doubt due to Justin’s extensive experience in making Illumina libraries and some luck.
The bad news was, the sequencer had blown up halfway through the run.
I exaggerate, but I gather somewhere inside an Illumina sequencer, there’s an USB dongle which must stay in contact at all times with the rest of the machine. Like the iPhone port on an Ion Torrent, I don’t know why that feature seemed like a good idea at the time for the designers. It lost the plot, and some short circuits followed.
We could salvage what amounted to 7 Gbp of single-end reads. Fortunately, once the sequencer was fixed, Opportunity #4 knocked. Our library, by virtue of having worked, could be used to titrate loadings of the chip. It seemed more interesting that just re-sequencing a phage genome standard, so the sequencing people just did it.
We now had more data than I could make sense of. 27 billion base pairs of it. Nearly 10x of a human genome.
Fortunately, Opportunity #5 presented itself: a 152-core, 1.5TB RAM high performance computing cluster fresh out the box. Assembling the metagenome took some clever work on the part of an expert next-gen bioinformatician which let me do the easy stuff on a seven year old laptop from PC World.
And so the plan came together.
Certain caveats should be applied. Nevertheless, opportunities multiply as they are seized. We may have sequenced and assembled a lot of cryoconite DNA into one metagenome, but it dates from only one time and place. But we now have dozens of glacier metagenomes in the works at Aber.
The analysis pipelines are less than perfect, particularly when dealing with microbes from poorly-explored habitats such as this. Subglacial flyfishers beware! Thus, problematic novelty is a particular challenge. Dedicated efforts to sequence, annotate and experiment with genomes of key organisms are required.
I remember looking at a KEGG map (biochemist’s wallpaper showing the metabolic circuitry of a cell) annotated with metabolic pathways reconstructed from a oceanic metagenome and pondering if/when we would ever achieve something similar for cryoconite. Two years later we published one. But does the presence of contigs affiliated to various functional categories mean anything in practice? Downstream -omics can help provide answers, which is why we are working with colleagues expert in metabolomics and metatranscriptomics. It was particularly gratifying to read that the paper had captured the imagination of another young researcher interested in cryoconite, leading him to ponder about transcriptomics experiments with cryoconite.
It’s clear that next-gen sequencing offers many opportunities – and challenges. My next post will consider Opportunity #6: How did I Stop Worrying and Learn to Love Next-Gen?