Optimizing DNA extraction protocols for the diet analysis of a baleen
whale (Eubalaena australis)
Abstract
Faecal metabarcoding is widely used for mammalian diet analysis.
However, most extraction protocols are designed to target high molecular
weight genomic DNA, making short sequences of digested DNA challenging
to extract. Here, we evaluate prey DNA metrics in baleen whale scat from
a phosphate buffer DNA extraction method along with two commercial
extraction kits (the QIAamp Fast DNA Stool Mini Kit and the PowerSoil
kit) with the following variations: 1) different durations of incubation
in a phosphate buffer (1 hour and 24 hours), 2) processing of both
pellet and supernatant from phosphate buffer incubation, and 3) two
different concentrations of DNA binding buffer to examine prey DNA. We
found that the choice of extraction protocol influenced richness,
diversity and composition of eukaryotes (18S rDNA) and crustaceans
(Crust16S mtDNA) detected in SRW faecal samples. The PowerSoil protocol
performed well for both markers, delivering the highest target richness
for 18S rDNA and highest diversity for Crust16S mtDNA, while the pellet
of the phosphate buffer yielded the highest richness for Crust16S mtDNA.
The phosphate buffer supernatant protocols produced the lowest results
for richness and diversity. Taxonomic composition in the supernatant
alone was influenced by the duration of incubation and the concentration
of binding buffer and was also distinct from the corresponding pellet.
Our results reinforce the importance of having clear targets prior to
selecting a method for faecal metabarcoding, as the specific aims (e.g.
identifying richness versus diversity) will inform the choice of
extraction protocol.