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Quake used a microfluidic method to isolate an uncultivable microorganism called TM7. The TM7 is rod-shaped, so Quake and his team specialize in capturing rod-shaped bacteria in the oral microbial community. They then track the desired cells through ribosomal RNA. Of the 35 isolated rod-shaped bacteria, 4 proved to be TM7, and they sequenced one of them.
Woyke believes that the microfluidic approach has two main advantages. First, users are able to observe the cells they are separating, which allows them to select a particular morph or phenotype and correlate this information with the genotype. The second is the amplification efficiency. A single copy of the bacterial genome is clearly insufficient for sequencing and therefore requires whole genome amplification. But not all fragments are equally amplified and some may be lost. "Some studies have shown that if the reaction volume is smaller, the recovery of the single-cell genome is better and the coverage is more uniform," Woyke said.
Despite this, microfluidic strategies are usually low-throughput and only a few laboratories have access to such equipment. A commercial option is Fluidigm's C1TM single-cell automated preparation system, which automates the isolation and preparation of 96 single-cell sequencing libraries. However, it is only suitable for mammalian cells and does not currently support bacterial isolation.
Another single cell separation method is micromanipulation, in which the researchers use a joystick-driven glass capillary to pick individual cells and transfer them to a destination container.
Woyke said that she occasionally uses this method, mainly in the processing of low-complexity samples. In an article published in 2010, she and her colleagues sequenced an insect's bacterial symbiont, according to it. The unique morphology separates it from another symbiosis 2 . “This is the main advantage,†Woyke said. "You can see the cells." However, the flux of micromanipulation is also very low. If the cells can swim or stick, it can be tricky.
Advantages and disadvantages of single cell separation strategy
According to Tanja Woyke, director of the US Department of Energy's Microbial Genomics Program, researchers use several methods to isolate single cells, each with advantages and disadvantages.