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Microscope 2

In earlier posts, we talked about our hypothesis: falling costs and accessibility of the tools needed for science and exploration are opening up a new opportunity for amateur explorers. Well, science and exploration are much bigger ideas than just the tools.   And it’s important we be honest about the entire process as we prepare for our trip to Cortez. For us, it didn’t start with a scientific hypothesis. It started with a curiosity, with the structure, explanation, and process modeled from there.

Mac Cowell, co-founder of DIYBio.org and Genefoo, has been spearheading the scientific portion of our trip. Here he gives an explanation of how we’re thinking about it, and how it’s coming together.

Enter Mac…

As David announced, we’re heading down to the Sea of Cortez in the 59-year wake of John Steinbeck and Doc Ricketts, who sailed the Sea of Cortez in a leisurely expedition of scientific discovery the 40’s: cruising around, philosophizing, and surveying the marine life of the gulf.

Like them, we’re going to sail Baja California in the same spirit of leisure and science. Only, we’re bringing robots, modern biotechnology, and the DIY spirit. Instead of collecting and cataloging marine macrofauna, we’re going to survey some of the genomes of the microbes that live in the gulf using DNA sequencing and metagenomics. Or at least we’re going to try. We want to put our tools to the test, and find out just how much is possible.

We’re not pioneering new scientific methods or trying to run a significant scientific study (Steinbeck and Ricketts were). When we started, we didn’t even have a hypothesis to test. As hobbyists and dilettantes, we just wanted to explore the sea of genomes surrounding us using some of the fascinating – and increasingly affordable – technologies pioneered by genomics researchers, and now makers.

Side note: We’re very aware of the legal and ethical issues surrounding this trip. That’s one of the main reasons we’re going: to find the boundaries and how they will affect citizen exploration. We care a lot about doing the right thing. The next post will explore some of those regulations and challenges. That said, please do let us know in the comments if you have comments, concerns, or research ideas.

Microscope 3

Metagenomic opportunities

There’s a lot of DNA floating around in the microbes in the ocean, and the vast majority of it has never been sequenced. A single droplet of sea water is estimated to contain perhaps 1-2 trillion bases of DNA spread amongst millions of microbes. There is much, much more when considering the rest of the marine ecosystem, magnitudes more when including viruses. Scientists estimate that perhaps only 1-10% of all microbes survive for study outside of their environment and can be grown under laboratory conditions, so being able to directly sequence DNA from an environmental sample can provide a more complete, while still biased, picture of the microbial members of a particular ecosystem (Gilbert 2011).

Some scientists are starting to rethink the very boundaries that delineate an organism – instead of treating each microbe as a complete individual, perhaps it makes more sense to consider spatially distinct but functionally connected communities of microbes as a single entity – a “meta-organism” (Zarraonaindia 2013).

Thinking about this notion of meta-organisms and using DNA sequencing to directly investigate the genes and genomes of communities of microbes are core aspects of a relatively new area of study called metagenomics. According to a recent review, metagenomics can formally be described as the field of research concerned with investigating the consortia of genes and genomes from a particular niche and asking “Who’s there? What are they doing? Who is doing what? And how is evolution driving this?” (Kennedy 2010).

Falling costs of DNA sequencing have resulted in explosive growth in metagenomics studies over the last decade. Perhaps one of the most famous was the Global Ocean Sampling (GOS) Expedition, led by showman-scientist Craig Venter, which collected samples of marine microbes via sailboat from 2004 to 2006 as it was sailed around the globe. In a pilot study conducted in 2003 with samples from the Sargasso Sea, the GOS team sequenced about 1 gigabase (GB) of unique DNA with shotgun Sanger sequencing and discovered more than a million new genes. Many more were discovered and annotated during and after the main GOS expedition.

Other studies have used metagenomics techniques to investigate the complex microbial communities of the human small intestine (to better understand human health), of the riverbed of an acid-mine drainage system (investigating new approaches to environmental remediation), and of a variety of marine sponges (searching for potential new drugs). A review of the field in 2011 by Gilbert & Dupont estimated that there had been about 45 major marine metagenomic studies since the mid-nineties.

Despite the advances in sequencing, it’s still only possible to sequence a small fraction of the DNA that can be isolated from a given community, so metagenomics studies are typically designed to selectively sequence the most informative fraction of DNA from the sample. The two designs are called Environmental single-gene surveys and random shotgun studies. To quote from that recent review on marine metagenomics,

“[Environmental single-gene surveys] can be seen as a directed, focused metagenomic study. Single targets are amplified using PCR and then the products are sequenced, providing an anlaysis of the range of different orthologs… for that gene within a given community.”

Random shotgun metagenomics is a study in which total DNA has been isolated from a sample then sequenced, resulting in a profile of all genes within the community. The community coverage of both approaches is entirely dependent on the depth of sequencing, that is, how many gene fragments are obtained during sequencing.”

So what approach are we going to take? We’re not sure yet. We’re still researching the typical protocols and costs (as well as legality) of both types of studies. You can check out our collection of literature on mendeley and suggest articles we should read. (For open-access introductions to metagenomics, check out this paper, this paper, and this review).

Some ideas: Could we design a single-gene survey to look for novel fluorescent proteins? I have no idea if there are conserved sequences amongst known fluorescent protein families that could be targeted by degenerate primers, but if that’s possible, then this could be a pretty cool little study – perhaps we’d find a new fluorescent protein!

At the least, we can do “classic metagenomics” and estimate the microbial diversity and population composition of several samples with DNA barcoding approaches. This would be another single-gene survey looking at 16s ribosomal DNA sequences and perhaps one or two well-known metabolic genes.

In addition, we could look at is how microbial diversity changes along with a particular environmental parameter, such as pH, temperature, depth, dissolved iron, nitrates, oxygen, or proximity to other organisms. We’re going to measure and record each of those with each sample we collect, along with GPS coordinates and photographs.

In any case, we’re going to try to do some basic metagnomics. I’ll be back after nailing down the specific methods we’re going to use – but essentially, we’re going to collect several samples of marine microbial communities, save their DNA for metagenomic analysis, take some microscopic images of the samples, and attempt to measure some of the environmental conditions of each sampling location.

I’m off to hit the books, and the hardware store.

Lab 1

References

Gilbert, J. a., & Dupont, C. L. (2011). Microbial Metagenomics: Beyond the Genome. Annual Review of Marine Science, 3(1), 347–371. doi:10.1146/annurev-marine-120709-142811

Kennedy, J., Flemer, B., Jackson, S. a, Lejon, D. P. H., Morrissey, J. P., O’Gara, F., & Dobson, A. D. W. (2010). Marine metagenomics: new tools for the study and exploitation of marine microbial metabolism. Marine drugs, 8(3), 608–28. doi:10.3390/md8030608

Men, A., Forrest, S., & Siemering, K. (2011). Metagenomics and beyond: new toolboxes for microbial systematics. Microbiology Australia, 32, 86–89. Retrieved from http://microbiology.publish.csiro.au/view/journals/dsp_journal_file.cfm?file_id=MA11086.pdf

Zarraonaindia, I., Smith, D. P., & Gilbert, J. a. (2013). Beyond the genome: community-level analysis of the microbial world. Biology & philosophy, 28(2), 261–282. doi:10.1007/s10539-012-9357-8

David Lang

Co-Founder of OpenROV, a community of DIY ocean explorers and makers of low-cost underwater robots. Author of Zero to Maker. And on Twitter!


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