Although microbes are small, they play an important part in both biogeochemical cycles in the ocean as well as on land. However, as they are not so easy to observe by eye, and in many cases can’t be cultured in the laboratory, sequencing-based methods are essential in the field of microbial ecology.
There are plenty of different sequencing approaches that might be implemented ranging from sequencing a single gene from an environmental DNA extract (or set of isolates, if you’re lucky enough to get them growing), to quantitative PCR, to metagenomics. As a recent article by Carini and colleagues points out, these tools have revolutionized how we understand microbiology, but the resulting data should be examined with caution.
A few of the questions microbial ecologists currently are asking include: who is doing what (function)? and how are the microbes interacting with each other (connectivity)? In order to find answers, sequences from the microbes actually living and growing in the environment should be used. There are plenty of dead cells in soil and often extracellular DNA that can hang around for days, weeks or multiple years, depending on the environmental conditions. We’ve pointed out before (in a different study by many of the same authors), there are pitfalls to not considering relic DNA (as defined by Carini et al., as either extracellular or in non-intact cells).
Using a viability PCR the authors compared samples treated with or without propidium monoazide (PMA). Essentially, PMA attaches to extracellular DNA or DNA in dead cells (because the membrane is compromised, so it can get in there), then when the treated DNA is exposed to light, PMA actually alters the DNA, which then cannot be amplified by PCR. The authors used high-throughput sequencing to measure the abundance and diversity of treated vs non-treated samples, and found that in the majority of the 31 soil samples tested, relic DNA accounted for a huge portion (40.7 ± 3.75%) of the overall DNA.
“Our finding that relic DNA can lead to significant overestimation of soil microbial diversity and reduce the ability to accurately quantify prokaryotic and fungal community structure has several important implications.”
The authors found that in most soils, removal of relic DNA changed overall richness estimates, but there were also several cases where the diversity assessment after relic removal didn’t change significantly. They highlight that the influence of relic DNA depends on a variety of factors, and even in the same environment, it’s abundance might change over time depending on different conditions (including the concentration of exchangeable base cations, and pH values).
“..relic DNA may obscure subtle spatiotemporal patterns of treatment effects in experimental manipulations of soil conditions.”
This study emphasizes that environmental conditions are important to determine not only to understand what role the resident microbes might have but also the potential risk of including relic DNA in the analysis. It is not absolutely, totally, or in all other ways inconceivable, that we will continue to develop current tools as well as refine new ones to sort through the challenges of what sequences to analyze, and ultimately understand what the intricate processes occurring in an environment on the microbial scale. That challenge is, in fact, what makes the field of microbial ecology so darn exciting.
Carini, P., Marsden, P.J., Leff, J.W., Morgan, E.E., Strickland, M.S. and Fierer, N., 2016. Relic DNA is abundant in soil and obscures estimates of soil microbial diversity. Nature Microbiology. 19 December 2016. Vol. 2, 16242 | DOI: 10.1038/nmicrobiol.2016.242