In order to study microbial processes in the selected environments, we need to know who is there, what can they do, and what are they doing. The way of addressing these questions is by using DNA sequencing techniques. There are two main approaches to retrieve sequences.
a) Microbial cells are first isolated either in pure culture or by cell sorting. Then, their nucleic acids can be sequenced.
The genome of a pure culture can be sequenced directly. The different DNA pieces (reads) have to be assembled into larger fragments (contigs and scaffolds). Eventually, the whole genome can be put together in a single closed circle. Genome recovery is usually very good, but many of the abundant bacteria in nature are difficult to isolate in pure culture.
Single sorted cells
Each cell is sorted into a well in a plate. The cells are lysed and their genome amplified before it can be sequenced. Amplification is not uniform and usually only a part of the genome can be obtained. The advantage is that it belongs to a naturally abundant bacterium. These genomes are called SAGs (single amplified genomes).
b) Nucleic acid (DNA or RNA) molecules are retrieved directly from the ecosystem.
Next, a particular gene can be selected for study, for example the 16S rDNA gene. In this case an amplification step is needed with primers specific for the gene and the result is a collection of amplicons, all of the same length and containing just the desired gene. This approach is very useful for studies of diversity, to know ‘who is there.’
Another approach is to sequence the whole mixture of nucleic acids. This provides the metagenome (when DNA is sequenced) or the metatranscriptome (when the mRNA is used). One advantage is that the usually biased amplification step is avoided. But the main advantage is that we can study the function of the genes in the community. The genes present (metagenome) and the genes being expressed (metatranscriptome) become available to study what capabilities do the microorganisms have, and which genes are being expressed at each time.