Extract and prepare DNA samples
While any fecal DNA (fDNA) extraction method should in principle be compatible with the MBD enrichment, methods that maximize the recovery of host DNA are preferable. Bead-beating methods that increase total DNA yield from feces, for example, should be avoided because the mechanical disruption increases the yield of cell-wall-bound DNA (i.e., from bacteria or plants) while fragmenting host DNA.
It is suggested to aim for a total yield of 1 µg of DNA for all samples in a maximum volume of 30 µl each, although there has been success with as little as 500 ng (the yield of host DNA is likely more important than the yield of total fDNA). If the volume is greater than 30 µl, the DNA can be concentrated via a bead cleanup (Auxiliary protocol A: Step 35 - 45).
Prior to enrichment, DNA should be quantified for the total yield (e.g., by fluorometer or spectrophotometer). Ideally, the host DNA should be quantified by qPCR (Auxiliary protocol B: Step 46 - 49).