Category Archives: Antarctica 2016

Nanopore Sequencing in the Dry Valleys

We’ve spent the last couple of days sequencing in the Dry Valleys, and we’re getting beautiful results!  Our biggest concerns were library prep in the field and whether the tiny MinION would maintain temperature, but good weather and good hand warmers have averted any problems.  We’re sequencing ancient microbial mats, some over 10,000 years old, and we’re getting astonishingly long reads, up to 68kb.  Reads this long are certainly from viable cells, as ancient DNA floating around in the environment would be highly fragmented, even under these excellent preservation conditions.  So who are these hardy microbes, and how are they surviving?  We’ll be analyzing the data over the next few days and weeks… can’t wait to see what it tells us!

DSC_3117Figure 1: MinION primed and ready to go.

DSC01750Figure 2: Hand warmers help!

DSC01677Figure 3: 68kb reads! 

Quick Break to Climb Observation Hill

After a really long day in the lab, Sarah, Dave, Angela, Elena and I grabbed dinner and decided to go hiking up Observation Hill. Well, it’s not really a hill like we’re used to. This hill is a 1500 ft, super steep climb that is all loose volcanic rocks and boulders. It’s a great post-dinner activity down here at McMurdo, or a great way to accidentally break a leg after eating too much. JK.  During our hike up there were three stopping points for picture taking and to catch your breath. First is a plateau where there are several communication repeaters and a plaque dedicated to the PM-3A medium power nuclear power plant that once supplied energy to McMurdo in the early 1970s.

The rest of the hike was challenging. It was steep with loose lava stones, and there was little room for error. Once we got to the top, we could see the never-ending horizon over the Ross ice sheet and could see Willy, Pegasus, and Phoenix Air Fields in the distance. The weather was crystal clear, and we had the most phenomenal view of the open blue sky and the white continent. We took a bunch of team photos and admired the breathtaking views. But the real challenge was the way down. Loose lava stones had us skidding all the way and holding on to anything for dear life. Except for crazy Scott, who decided it was a ski hill and decided to run down it! We all made it down safely and were so happy we squeezed in this breathtaking 90-minute hike…

20161218_201912Figure 1: Elena and Angela at the summit.

Well, it looks like we finally did it

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!

20161216_223500Figure 1. First MiniSeq metagenomics run.

 

Extractions

We’ve been processing samples as soon as we collect them in our little Southern outpost lab, making for some very busy days! I could not contain my excitement this morning, so I woke up early, went to the gym, met the team for some breakfast and then we headed off to the lab to extract us some nucleic acids! I started with some RNA extractions using a Trizol-based protocol on paleomats we found buried upslope of Lake Vida in the haltingly beautiful Victoria Valley. We placed the tubes directly into a liquid nitrogen primed cryoshipper in the field, which immediately froze them to -190°C. As we began to process them, the tubes containing the samples turned many shades of pink! The RNA is currently precipitating in absolute ethanol in the -80°C. And of course, we’re also extracting DNA from the same site. Because of variation in lysis and extraction efficiencies of different methods on different types of cells, we’ve been evaluating the relative efficiencies of four different methods protocols (good ol’ Phenol-chloroform, MoBio’s PowerSoil Kit, MP Biomedical’s Ancient DNA Kit, and an Omega Biotek Metagenomics Kit (custom built with our input for extremely long fragment lengths). We’re also testing for differences between lysozyme and a blend of multiple lytic enzymes (aka “polyzyme”) for methods making use of enzymatic cell lysis. So why are we testing so many methods? Because these microbes are known to be difficult to DNA from, if you get any at all. These puppies can be tough to crack open! We quantified the DNA on the Qubit and Agilent to tell us more about the quality of the DNA we’ve extracted. We’ll be using this information to select samples to sequence.

Eventually, we walked out into a beautiful sunlit night to get some sleep. We’ve got sampling to do tomorrow!

DSC_1794Figure 1: Grabbing samples out of the -80.

DSC_1839Figure 2: Lots of tubes!

DSC_2877Figure 3: Angela checking the protocol.

Happy Birthday Angela!

Our youngest team member turned 22 today! We were all very excited to embarrass her, like writing her name on the activities board in the Galley.  As per birthday girl’s request, we all – well, almost all (Scott!) – went to Yin Yoga together.  It was taught by one of the researchers here, and it was great fun.  Then we snuck off to pick up Angela’s cake.  Although we couldn’t make it ourselves, the kitchen here at McMurdo makes custom cakes upon request. Yay cake! The pastry chef whipped up a lovely chocolate cake with coffee icing and beautiful lettering.  Then it was time for Angela’s party.  We hosted it at Gallagher’s here at the base (right in the middle of Two-Step Tuesday). The whole Crary Lab showed up to wish her a Happy Birthday!

We’re so happy to share this special day with Angela and so grateful to have her with us.

DSC01109 (1)Figure 1: They forgot to write down the most important event of the day – Angela’s Birthday!

 

Staying Clean

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?”

Fair question.

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.

DSC_2016 (1)Figure 1: Helping Scott suit up.  Just a little more tucking, and he’ll be ready to go.

 

Helo Ops, Helo Ops, this is Golf Zero Six Two…

We’ve had a thrilling few days… ten helicopter rides, three valleys, and four beautiful paleolakes!  There’s nothing more exhausting than this field work though, especially when the wind kicks up.   Our boots each weigh five pounds, and there’s a ton of ground to cover.  But we’ll upload more pictures soon!

DSC01044

Around Town

Couldn’t resist posting these!DSC00141
Figure 1: A seal on the sea ice.

DSC00327 Figure 2: A fat tire bike parked at the Galley.

DSC00317 Figure 3: Derelict Junction, where you catch the shuttle to Scott Base.

20161208_080101Figure 4: The taxi of science!

Hut Point Calibration

This morning we woke up to snow! It made for some beautiful views, and the mountains looked especially pretty with the dusting of snow. Sadly, this also meant unsafe conditions for flight, and so our first day in the field was delayed – i.e. cancelled. I’m glad that Helo Ops (helicopter operations) made that call as the wind and snow picked up later in the day.

Not to worry – we find plenty to keep ourselves busy with, testing our lab equipment with DNA standards to make sure everything’s working properly and ready for samples as soon as we are able to collect and extract them. For our work into testing mechanisms of long-term cell survival, we are planning to do some sequencing in the field. That’s right, sequencing out in the Dry Valleys in real time. The sequencer we’d use for this is Oxford Nanopore’s MinION that we are running offline with our laptop. To make sure that the laptop and sequencer wouldn’t complain too much about the cold when we’re at our sample sites, we needed to verify the setup in the cold. What better day for this than today? Angela and I packed up the laptop and MinION (that we’ve yet to name) and hiked out to Hut Point. It doesn’t get better than combining science and recreation!

DSCN1487 editedFigure 1. A misty view of our dorms and the energy powerhouses of McMurdo in the snow. 

DSCN1516 editedFigure 2. Elena with Dasse the penguin, our lab manager.

Hut Point is a narrow peninsula on Ross Island where McMurdo Station is located. Less than half a kilometer from the Station is the Discovery Hut built by Robert Falcon Scott in 1902 during the Discovery Expedition. The hut has been designated as a Historic Site, so we couldn’t go in, but it was great to see this amazing witness to exploration.

Once there, we marveled at the beauty around us, thanked our lucky stars, saw a Weddell seal, and then got down to business testing our sequencer setup. We connected the MinION to the laptop, launched the MinKNOW software, and ran a test using the configuration test cell. We were impressed at how quickly and smoothly it ran. We got a warning that it could not “ping”, but the software continued running as we were running it locally (not using the online server). The test completed successfully within a few minutes, so now we know it is doable in the extreme cold. Can’t wait to run some ancient microbial DNA on a Nanopore flow cell!

DSCN1518 editedFigure 3. Configuring the minION sequencer

DSCN1522 editedFigure 4. It works! 

 

Quirky McMurdo

Our field season falls near the height of the austral summer. As we are far below the Antarctic Circle, we get 24 hours of sunlight per day. Not only do the ~900 summer residents of McMurdo have to adjust to the remoteness of the location, they must also reconcile with the strangeness of passing time under perpetual sunlight. Those who are crazy enough to plant themselves in such a place take pride in their weirdness and go above and beyond to make this ecosystem thrive.

It’s immediately evident that McMurdoans have a quirky sense of humor, and they’re not afraid to show it. Hidden among announcements of upcoming helo schedules and base-wide maintenance events are little gems of humor that never fail to bring a smile to my face.

IMG_0524smFigure 1. Make sure you stay safe. 

IMG_3591smFigure 2. We don’t want to spook them! 

IMG_3709Figure 3. Your daily dose of poetic desperation. 

IMG_3731Figure 4. Specializing in Cool Ranch flavor

A serendipitous conversation with Dave, a military Chaplain deployed here for the season, revealed how truly wonderful this community is. Because it is so difficult to obtain employment here, people who do get to work here radiate extraordinary passion and vibrancy. Not only do they express their colors without fear, they strive to create a warm place that encourages everyone here to do the same. Just a couple days here, and I’m already in love with this place!

Agilent magic

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.

DSC_1607 (2)

Figure 1: An Agilent High Sensitivity DNA Chip.

Setting Up Our Genomics Lab

After landing in Antarctica and spending the first day in training, we finally got escorted to our new lab in the Crary Lab facility; we got room 146. It’s an awesome lab, and you just wouldn’t believe the view. It is to die for! We have breathtaking vistas of Mt. Discovery and the Dry Valleys, and every time we look up, there’s a helicopter flying right by our window.

Once we got to the lab, we wasted no time moving in. Our mission was to unpack all the instruments and lab gear as soon as possible and create our Antarctica Genomics Facility. This is the first time anything like this has ever been attempted on the Continent! We stayed up until all hours–not surprising if you know our personalities–and got all the equipment installed and up and running within three days. It was no small task. We brought down an Illumina MiniSeq, three Oxford Nanopore Minions, an Agilent Bioanalyzer 2100, and all the supporting laptops, accessories, and reagents so we can do every type of DNA and RNA extraction under the sun… and the sun never goes down here in the summer!

 

View

Take nothing, leave nothing

Our second day of training and safety and protocol lectures. But only three today. Whew! The first was an Environmental Field brief. Antarctica is a pristine continent with many unique sites. Treating our sampling sites with respect and practicing good environmental stewardship is a must in Antarctica. That means packing everything that brought to a sampling site, and bringing it all back. Everything. There’s nowhere in Antarctica that you can pee on the ground, they reminded us, and while it’s gross to wash a pee bottle, it’s even grosser to wash someone else’s.

IMG_3603Figure 1. The golden rule: clean your own pee bottles. 

Since we will be working in the Dry Valleys, we were given some additional information specifically for working within the McMurdo Dry Valleys ASMA, which contains five ASPAs. ASPAs are Antarctic Specially Protected Areas, and an ASMA is an Antarctic Specially Managed Area. Science (and other) activities in these areas are managed and coordinated to minimize human impacts to protect the ecological, environmental, and aesthetic qualities.

Our third and final safety talk was the Outdoor Safety Lecture, which is necessary for any recreation that involves being outside. We immediately put this training to good use. We went to see marine life thriving under the ice in McMurdo Sound (McMurdo Station is on the Southern tip of Ross Island). How can you do that without donning dive gear? The “Ob Tube” (observation tube)! The tube is narrow, and about 20 feet long, with windows all around at the bottom. There, you can sit and enjoy the marvels of the sea – the schools of silvery fish, jellies, a sponge and some crustaceans. We could hear seals and were hoping to see them, but sound can travel far distances under water so they may have been nowhere near us. I guess we’ll just have to hike around this beautiful landscape to catch a glimpse of the seals.

20161203_130610_069Figure 2. Whoa Dave, where’d ya come from?!

 

It’s all about the buddy system

The theme of the day was training, training and more training! In comparison to our last few mornings, we had the luxury of sleeping in today, with our first class at the late hour of 7:30 am. We started off with briefing and then went on to Core Training, which consisted of Light Vehicle (F350 “light” not Civic “light”), Fire Safety, Waste Management, and Health training. Most of what was covered involved cold weather considerations, and proper water and waste management associated with remote locations. Some topics were less obvious or expected, although they make perfect sense. For example, always park facing the wind since an open door is essentially a large sail when the wind is behind it. Also, writing or creating art with a finger on a muddy car, is literally etching musings into the side of the vehicle for years to come (the mud is made of sharp volcanic rock). Wonder if there’s a book of the “best” efforts somewhere…

After a break for lunch, we were in for a few hours of Field Safety and Training, which was a lot of fun! We got to start a fire, put up a tent and practice our knots. These would all be helpful should we ever have to dip into our emergency kits filled with survival equipment.

The day can be summarized in two statements that inevitably go hand in hand:

  • Safety is the priority
  • Communication is key

The importance of communication and safety planning, both within the team and with support staff, before hitting the field, while out in the field and a debrief after are the best ways to ensure that science is done successfully and safely (injured scientist = no science). Most injuries are found by other team members rather than the affected person, so it is important not only to pay attention to your own body (am I dehydrated? unfocused? cranky?) but the moods and well-being of others as well. Much like any other activity where even momentary inattention can have severe consequences, the buddy system is key.

McMurdo Station functions like a small town – a special and wonderful small town dedicated to understanding the world around us. It’s everyone’s job to make sure everyone is safe and happy. Looking forward to what the coming days will bring!

 

20161202_135725 edited
Figure 1. Angela can save us from dehydration and cold!

A “warm” welcome from Provost Groves

We arrived at McMurdo (finally) after the longest day, which started at 4:00 AM and included 8-hour flight followed by a 1-hour drive. We got the most amazing welcome from Provost Groves.  He’s so personable, funny, and genuinely curious!  He was inspecting McMurdo and Amundsen-Scott as a member of the National Science Board. In the interests of this responsibility, he visited scientists of all stripes at both stations, and he even took a Georgetown banner to the South Pole! I was moved by the probing and insightful questions he asked us and all the scientists here. It’s clear that he cares deeply about scientific endeavors both at Georgetown and in the world at large, and that he does not dismiss these visits as merely duties but as the opportunity to have meaningful conversations about scientific progress and potential. I’m thankful for Provost Groves as well as the other NSB members for their unceasing support of the work that we do!

DSC_1356Figure 1: With Provost Groves (and Rear Admiral Byrd)!