Optimizing DNA extraction protocols for the diet analysis of a baleen whale (Eubalaena australis)

Faecal metabarcoding is widely used for mammalian diet analysis. However,
most extraction protocols are designed to target high molecular weight
genomic DNA and not the short DNA sequences associated with digested prey
items. To examine the prey composition in southern right whale (Eubalaena
australis) faecal samples we trialled a phosphate buffer DNA extraction
method along with two commercial extraction kits (QIAamp Fast DNA Stool
Mini and QIAGEN DNEasy PowerSoil) with the following variations: 1)
incubation time in a phosphate buffer (1 and 24 hours); 2) processing both
pellet and supernatant from the phosphate buffer incubations; and 3) two
different concentrations of DNA binding buffer. We found that the choice
of extraction protocol influenced the richness, diversity and composition
of eukaryotes (18S rDNA) and crustaceans (Crust16S mtDNA) in the faecal
samples. The PowerSoil protocol performed well for both markers,
delivering the highest target richness for 18S rDNA and highest diversity
for Crust16S mtDNA, with the pellet of the phosphate buffer also
performing comparably. Taxonomic composition in the phosphate buffer
supernatant was influenced by the incubation period and concentration of
binding buffer and differed from its corresponding pellet. To maximise
taxonomic coverage, we recommend combining the extracts from both the
supernatant and pellet. In faecal studies, our findings reinforce the
importance of defining the community attributes (richness versus diversity
versus composition) of key interest prior to performing DNA extraction, as
the inference of these variables is likely to be altered by the choice of
extraction protocol.