Sequence-based microbial surveys

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In a typical molecular phylogenetic analysis, you start wih genomic DNA isolated from a pure culture an organism (or enriched sample, as in the case of the T. ruber). The ssu-rRNA sequence obtained by PCR amplification is used to determined the place of that organism in the 'big tree' - it's "phylotype".

It is also possible, however, to start a molecular phylogenetic analysis with DNA extracted directly from an environmental sample instead of a pure culture. The PCR amplification products in this case are a collection of ssu-rRNA sequences representing the population of rRNA genes in the DNA, in turn representing the population of organisms in the original sample. The ssu-rRNA sequences are separated by cloning, and then ssu-rRNA sequences from each are determined, in order to survey the microbial inhabitants of an environment. This can be a relatively simple survey, as in the case of the pink filaments, or more thorough surveys, as we shall see in the example of the hydrothermal vent scaly snail microbial symbionts.

More modern methods push this approach over the limit, obtaining not dozens, not hundreds, not thousands, not even tens of thousands, but hundreds of thousands of sequences from environmental samples. This is possible because sequencing technology is improving very, very quickly; dropping in cost by about a factor of 10 every year. We'll have a look at a great example of this kind of analysis of the human "microbiome".

Whether using the old-fashioned clone-and-sequence approach, or the more modern high-throughput sequencing approach, molecular phylogenetic surveys are far superior to the older cultivation-based approaches, and are used a lot.

However, it has become clear that several aspects of PCR/cloning/sequencing of microbial populations limits their quantitative interpretation. In fact, bias exists in every step of the process, and so estimation of the relative abundance of organisms from molecular phylogenetic "surveys" just is not quantitative. Some cells will not be opened using standard methods (or will not be opened as efficiently), some will be selectively lost to some extent in the DNA purification (perhaps tightly bound by protein), some are easier than others to amplify rDNAs from (depending on primer sequences, PCR conditions, etc), different organisms have different rDNA copy numbers, some will clone more readily than others, &c. Nevertheless, even a qualitative molecular phylogenetic survey of the microbial population is very useful, and far more informative than examination of whatever happens to grow on plates.