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Fascinating microbes live in the most remote areas
Transporting samples home during a 20-day expedition is difficult
What if we brought a sequencing lab to them?
Our aim
"Biology for anyone, anywhere"
Oxford Nanopore Technologies
We wanted to test this on Europe's largest ice cap, completely off-grid, pulling everything we need for 20 days on skis and sledges
We packed a sequencing lab into a sledge, dragged it over 100 km across an ice cap, sequenced microbial communities in the field and completely off-grid, then skied 100 km home with nothing but data
How we did it
Before we begin, we would like to express huge thanks to Dr Arwyn Edwards for his endless help and enthusiasm with designing an off-grid nanopore set up. Also thanks to Ingeborg Klarenberg for her local knowledge on microbial communities in moss samples in Iceland and thanks to Dr Tom Ellis for his support of this scientific work (and lending us his laptop!)
Step 1: Build a lab
Design a lab small and light enough to pull on a sledge alongside food, fuel, gear, and tent for over 200 km. Here is our 18L solution.
Additional items: Dell XPS 13 16GB RAM 1TB SSD with Pelicase 1095, the (amazing) MSC 90W solar panel and an unholy number of dry bags
Step 2: Keep flowcells warm
Nanopore sequencing utilises one-time-use flowcells to sequence extracted DNA. These must be kept above 2˚C and below 30˚C at all times.
Our first challenge was doing this for the first 11 days skiing 100 km on the Vatnajökull ice cap.
See the full temperature log.
Step 3: Extract DNA
Find your microbial community, isolate a sample, and extract the DNA using QIAGEN PowerSoil extractions. Swap the centrifuge for a DremelFuge. Add in a terralyser. Pack your handwarmers (buffers precipitate in the cold!)
Step 4: Get sequencing
Use the LRK001 Field Kit from Oxford Nanopore to prepare your sequencing library. Use a thermos mug with a meat thermometer to get some water to 80˚C (mix boiling water and snow in the right proportions).
There's a lot of volts and amps flying around here. This is simply what we used. We recommend doing your own research into each device and use at your own risk.
Step 4: Keep it sequencing
Maintaining laptop power is key. Minimise power consumption by turning off basecalling. Use a 90W solar panel to charge a Poweradd 20Ah pack while that charges the laptop. This will work for a whole day. Then back up power packs to last the night... (remember to solar charge these the day before!)
Our results
We extracted one sample of DNA from a hot spring gorge (64°40'56.3"N 16°31'40.7"W) located ~3km north of the northern edge of the ice cap. The sample consisted of soil from just below the surface of a moss patch found ~30 cm above the waterline (water at ~60˚C). 4x QIAGEN PowerSoil extractions were pooled and concentrated (AMPure XP) to obtain 6.54 µg DNA (100 µl at 65.4 ng/µl), quantified using a Qubit 4 running off a power pack outputting 12V DC.
This DNA was prepared for sequencing using the Oxford Nanopore LRK001 Field Sequencing Kit. The 80˚C incubation step was achieved using a thermos mug with boiling water and snow mixed to achieve 80˚C, measured using a meat thermometer. FLO-MIN106 flowcells were used with offline MinKNOW software. Reaching 34˚C in the MinION was aided by handwarmers and a sleeping bag. Be aware these handwarmers get VERY hot if unattended.
QC results:
Flowcell 1 (Before: 1375 pores, after: 1320 pores)
Flowcell 2 (Before: 1180 pores, after: 1183 pores)
Therfore keeping the flowcells warm using body heat on a ski traverse we maintained 96% and 100% of the active pores.
Run 1: 5.5 hrs
- Local Basecalling
- Laptop battery + 20Ah power pack
Run 2: 19.5 hrs
- No basecalling
- Laptop battery + 3x 20Ah power packs + 11-12 hrs solar power (21V output)
Combined, these runs generated 133,538 reads containing 185.9 Mbp of data.
Basecalling was done post-run with Guppy (Oxford Nanopore Technologies).
The combined reads were uploaded to Kaiju and aligned to the 'NCBI BLAST nr + euk' database using 'Greedy mode' (min match length 11, min match score 75). Shown here is a snapshot of the results from krona which graphically indicates the species of microbes identified by the DNA sequencing results.
Click here to read our open access publication
CONTACT US
If you are interested in finding out more about our expedition or conducting an expedition of your own, please get in touch:
Expedition planning/general enquiries: oliver.vince94 [at] gmail [dot] com
Nanopore sequencing: glgowers [at] gmail [dot] com
Enquiries the others can't answer: johnhenry.charles [at] gmail [dot] com
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