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.