After months of planning to get here and weeks of work, both in the field and the lab, team G-062 has finally generated the first next-generation DNA sequence data in situ on the seventh continent from samples collected right here in Antarctica.
This story begins on the morning of December 10, 2016 when the five of us boarded a Bell 212 helicopter and set off from McMurdo base across the Ross Ice Shelf, destination: the Dry Valleys. After a 45 minute flight our pilot dipped the helicopter into Wright Valley and dropped us off on the shore of Lake Vanda, a 5km long, 69 meter deep hypersaline lake with a salinity 10 times that of seawater. There, high above the shore, we dug into sediment that at one time was a lakebed covered in water, but dried up two or three thousand years ago as the lake receded. At a depth of roughly 10 cm under the surface we discovered ancient microbial mats that had once lived happily in their watery environment, but had since desiccated and been buried as the water receded. Were any of these cells still alive, hunkered down and biding their time? If so, how were they doing it – how could they survive? There was only one way to find out.
We collected the samples, flew them back to our lab at the Crary Science Center and isolated DNA from cells that had not seen the light of day for a very long time. We assessed the DNA quality on our trusty Agilent Bioanalyzer, and successfully constructed paired-end 150-bp libraries from the ancient DNA using Illumina Nextera XT chemistry. We chose a single sample to perform massively parallel sequencing of millions of DNA fragments on the Illumina MiniSeq sequencing system housed in our lab space at Crary. After nearly 24 hours the sequencer delivered our first run results: 8.1 billion base-pairs of data at a cluster density of 166K/mm2, with 92.7% of clusters passing filter (>Q30). Not bad. We’re slowly (very slowly) offloading some of this data from the continent to do more sophisticated bioinformatics than our laptops allow down here. We’ll analyze most of this data back home, but we’re eager for a quick look as soon as we can get one!
Figure 1. First MiniSeq metagenomics run.
When I first told friends, family and colleagues that I was going to Antarctica to dig up ancient microorganisms and bring them back to the U.S. to sequence their DNA, folks obviously had many questions. But what surprised me the most is that every last one of them, all different people from all walks of life all asked me the same question – “Aren’t you worried you’ll bring back some new alien life form that will take over the world and unleash upon us the demise of humanity?”
However, this resembles more the plot of a good horror movie, and as we know these plots are rarely based in reality. In fact, it is these ancient little organisms that need to be protected from us humans. In the field and in the lab, our team goes to great lengths to protect them from being contaminated with our DNA. After our helicopter pilots drop us off in our field sampling sites in the Dry Valleys, we suit up in white full-body Tyvek suits with hoods that make us look less like scientists on an Antarctic expedition and more like Jet-Puffed marshmallows lost in the largest wilderness in the world. We don nitrile gloves and facemasks and dig holes in ancient lake beds using digging tools that have been cleaned with solvents and bleach as well as “ashed”– heated to 550°C for 8 hours (ouch!). Our samples are collected in cryotubes and plastic “Whirl Paks” that are completely sterile. We place the tubes into a liquid nitrogen primed cryoshipper and the Whirl Paks into a very cold cooler, then fly back to the Crary Science and Engineering Lab at McMurdo Station. We then bring them into a laboratory space that has had every surface (even the floors) cleansed daily in a bleach solution, which is really good at – you guessed it – obliterating any traces of DNA. We work in gloves, lab coats, and Tyvek booties to cover our shoes. We do all of this because we do not want to contaminate these precious samples with our human DNA. Why? Because the answers to the fundamental scientific questions we are asking are found in the sequences of the nucleic acids we isolate from these ancient beings. And the last thing we want to see in these sequence data are traces of Angela, Dave, Elena, Scott or Sarah.
Figure 1: Helping Scott suit up. Just a little more tucking, and he’ll be ready to go.
After a long journey from Singapore, through Auckland and Christchurch New Zealand, the Agilent Bioanalyzer 2100 has also arrived at McMurdo Station, Antarctica and has been successfully installed into the Crary lab space assigned to team G062M. The instrument was generously donated to us from Agilent (thanks Agilent!) and will be a critical instrument for our mission of sequencing DNA and RNA isolated from the field samples we will collect in the coming days from the McMurdo Dry Valleys.
The Agilent Bioanalyzer is the gold standard for sizing and analysis of DNA and RNA isolated from biological samples, and is a critical component for quality assessment of DNA libraries for next-generation sequencing. The instrument is a unique analysis tool which uses a DNA “chip” comprised of wells to load microliter volumes of DNA or RNA samples, along with a sieving polymer matrix and an external “ladder” control. Micro-channels are fabricated in glass to create interconnected networks among these wells.
To prepare the chip, the micro-channels are first filled with the sieving polymer and fluorescence dye. Then, the experimental samples and ladder with marker are loaded in each well. Once the wells and channels are filled, the chip becomes an integrated electrical circuit. The chip then contacts a 16-pin electrode array arranged to fit into the wells of the chip, and a power supply passes a current through the electrodes to create a voltage gradient. As DNA and RNA are electrically charged, the molecules migrate through the gel matrix, electrophoretically driven by the voltage gradient, similar to slab gel electrophoresis. Because of a constant mass-to-charge ratio and the presence of the sieving polymer, the molecules are separated by size, with smaller fragments migrating faster than larger ones. During migration the dye molecules intercalate into the DNA or RNA strands and these complexes are detected by laser-induced fluorescence. The software automatically compares the unknown samples to the ladder fragments and the results are translated into gel-like images (bands) and electropherograms (peaks) that contain data such as fragment length and the concentration of the DNA or RNA samples.
We really couldn’t do our work down here without this instrument, and for that we thank the generosity of Agilent, Inc.
Figure 1: An Agilent High Sensitivity DNA Chip.