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 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}.
Maybe talk about the Figure 2 here (Joanas) and the workflow including the technical challenges associated with soil
Loss of information throughout
- 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
- filtering rare taxa
- These factors make ecological interpretation more complicated
The advent of next-generation sequencing technologies has facilitated rapid growth in the number of studies investigating communities of bacteria by sequencing the partial ribosomal RNA gene, and of fungi and microbial eukaryotes by sequencing amplicons of internal transcribed spacers (ITS) located between the nuclear ribosomal RNA genes (Nilsson et al. 2019, other REFs).
These challenges also bring to light the biases associated with applying diversity metrics in sequencing studies. Address challenges with calculating and interpreting alpha-diversity metrics in amplicon datasets...
We might address this by stating that using alpha diversity metrics with 16S data is reasonable due to the availability of reference data for this particular marker gene. However, when we consider ITS or other marker genes with relatively more incomplete reference genome sets, these metrics may become inaccurate and misleading. Discussion of the use of alpha and other diversity metrics in sequencing studies have been addressed in X, Y, and Z...