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Microbial Dark Matter single cell genomics protocol published

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The technique employed to extract and amplify the DNA from single microbial cells for the microbial dark matter study is now detailed in a Nature Protocols. We describe all necessary steps from sample preservation and preparation, to single cell separation, fluorescence-activated cell sorting (FACS), cell lysis and finally the whole genome amplification.

The procedure takes four days, can be perfomerd in a standard molecular biology lab, and yields up to a microgram of DNA per single microbial cell, an amount considered “suitable” for PCR amplification and shotgun sequencing. And please note,  that although the protocol was applied to microbial cells, it can also be adapted for microbial eukaryotes and mammalian cells.
Single-cell whole-genome amplification workflow.

Rinke C., J. Lee, N. Nath, D. Goudeau, B. Thompson, N. Poulton, E. Dmitrieff, R. Malmstrom, R. Stepanauskas, and T. Woyke, “Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics,” Nat. Protocols, vol. 9, no. 5, pp. 1038–1048, May 2014.

What is Microbial Dark Matter and why should we explore it?

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Life as we know it on our planet would not be possible without microbes. Much of the oxygen we breathe is produced by microbes, they are necessary to create our food such as yogurt, and cheese. Also beer and wine would not exist without their ability to ferment sugar to alcohol, and in industry microbes are used as tiny living factories, for example to produce human insulin for people with diabetes.

Microbes comprise ~60% of the earth’s biomass and they are everywhere. They are abundant in the oceans and in soil – there are more microbes in a teaspoon of soil than there are humans on earth -  they live in deserts, on mountains in Antarctica, near boiling hydrothermal vents at the seafloor, and in the acid stomachs of mammals including us humans.

Scientists are very curious to learn more about this microbial world, but there is one big problem. We can only study about 1% of all the microbes in our laboratory, since most of the little critters just don’t grow on artificial substrates in the lab. Traditionally one would grow the microbes - millions of them - to get enough DNA for sequencing, because one cell has so little DNA. Since we can't do this for the majority of microbes, they remained a mystery to us and are known as the Microbial Dark Matter (MDA).

Exploring Microbial Dark Matter
Exploring Microbial Dark Matter

The inability to culture this microbial dark matter has led to a very skewed view of the microbial world. The two largest groups of microbes (Bacteria and Archaea) have many members which are only known because we found a small piece of DNA, the 16S rRNA gene, in environmental samples. We have almost no genomic information about those microbes, so we don’t know what they are doing or what they are capable of.

We apply a method, called single cell genomics, which omits the culturing step and allows to amplify the DNA of a single microbial cell a billion fold, more than enough to sequence its genome. The first step is to take an environmental sample and to sort individual cells into tiny droplets. This is done on a cell sorter which detects the cells by a laser and separates them into droplets by electrostatic forces, similar to how an ink-jet printer directs individual droplets of ink to print a letter on a page. Next we break open the cell envelope, very carefully so we don't damage the DNA. Then we add a cocktail with the enzyme phi29 which has the ability to amplify very long stretches of DNA, and after several hours we have billions of copies from the microbial genome and can start sequencing.