Author Archives: Elena Zaikova

From Sample to Species Identity

The roads to El Tatio remained closed through yesterday and today, which unfortunately meant that we could not see the geysers or silica sinters. The heavy rains and snow were part of the worst storm in the region in over 60 years. I suppose we should be glad we witnessed such a rare event. Luckily for us, the Atacama Desert is a beautiful place that’s used as an analog for Mars and to test landers, rovers and other Mars-bound instruments. Between the extreme aridity (just an average of 15 mm of rain per year), red rocks, and spectacular sand dunes, it is not difficult to imagine that you’re standing on a martian surface! So, we decided to try out our DNA extraction, sequencing and analysis in this remote setting.

We picked two sites, one yesterday and one today, to test remote field sequencing with offline bioinformatics. Our first site was in cactus-lined hills between Machuca and Guatín. The presence of vegetation and a small stream below suggested that there might be enough biomass in this area for our very first test. Our second site had no vegetation or water, beautiful large sand dunes, and veins of gypsum throughout. The veins looked just like those on images taken by the Curiosity rover!

For the in situ extractions, we collected ~200 mg sandy material into a 2ml tube containing DNA/RNA shield and bashing beads, bead-beat the sand using the TerraLyzer, and then recovered the DNA in the lysate using a spin column. Next, we made a Nanopore rapid sequencing library (RAD002), loaded it onto the MinION and sequenced without live basecalling or “pinging” ONT. After 60 (yesterday) or 90 (today) minutes of sequencing, we paused sequencing, basecalled the reads, and classified the resulting sequences using Centrifuge software with archaeal and bacterial reference genomes. We then restarted sequencing for later analyses. We’ll bring some the DNA back with us to look at the fragment length distribution and DNA purity (A260/280 ratio). To check for contamination, we extracted negative controls, and will sequence them once we return. In addition to being completely offline, we used portable power – a powerbank to run the miniPCR for incubations at 30°C, 75°C and 22°C (“RT”) and the laptop’s battery for laptop and MinION.

A brief look at the taxonomic assignments indicated that we sequenced different communities at the two sites. There were >100 distinct genera at the first site and ~200 at the second. While some assignments overlapped, the distribution and identities were different from each other (and Thursday’s community standard). Looking forward to comparing these field-based assignments with longer lab runs and more in-depth analyses!

Elena  Screen Shot 2017-07-13 at 4.35.37 PMDSC_3623

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!

 

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! 

 

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!