- Compare to other ecosystems where microbial alpha-diversity is rather low and more is known about the functions of these microbiota (such as termite intestines, mammals gut, rumen etc)
- When alpha-diversity is high but spatially more homogeneous such as ocean water and other aquatic environments
In addition to high biological diversity, researchers interested in the microbial composition of soils are confronted with technical challenges throughout the sample processing workflow. The general workflow of amplicon sequencing includes 1) careful planning of the experimental design, 2) nucleic acid extraction, 3) quality control and sequence library preparation, 4) primer choice, PCR amplification, sequencing and 5) analysis of sequence data (Figure 2b). At each of these steps, information can be lost as a result of the techniques applied (ie nucleic acid extraction method, primer selection, statistical approaches), with consequences for data interpretation in the context of ecological questions.
As with any scientific experiment, the experimental design is essential for determining the specific hypotheses that can be addressed. In amplicon sequencing experiments, one must
Soil scientists face additional challenges in selecting the appropriate spatial scale (ie aggregate/microscale, centimeter scale, meter scale) and temporal scale for sampling in order to address specific questions regarding community dynamics (discussed in sections XYZ).
The physiochemical properties of soils make nucleic acid extraction from this matrix particularly challenging. Numerous extraction protocols and kits have been developed to circumvent challenges with DNA extraction from soil, however each method introduces distinct bias on the subset of the microbial community retrieved \cite{Terrat_2011}.
- extraction efficiency - acidic pH, humic substrates/physiochemical properties, cell lysis efficiency
- primer choice - diverse organisms (cross-domain), how do we cover this diversity, while also dealing with highly degenerate primers?
- PCR efficiency, i.e. inhibitors limit PCR reaction efficiency
- sparsity of datasets, filtering rare taxa
- These factors make ecological interpretation more complicated