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.

Southern right whale faecal samples were collected opportunistically over decades of research. Faecal samples underwent DNA extraction using three different methods (phosphate buffer extraction, PowerSoil kit and QIAamp FAST DNA Stool Mini Kit) with modifications in each leading to 12 unique protocols. The modifications included: incubating samples for 1 hr and 24 hrs in a phosphate buffer, processing both the pellet and supernatant from phosphate buffer incubation, and the addition of 1x and 2x DNA binding buffer to the silica column. Extracted samples were amplified via a universal 18S and crustacean-specific 16S marker and sequenced. Analyses for taxonomic richness, diversity, and composition were performed in R to compare results from the different protocols.

Changes after Oct 17, 2024: An additional Excel file with raw reads data for the full 18S rDNA and Crust16S mtDNA datasets was added, and the Supplementary material was updated to reflect reviewer feedback with additional analyses on the raw data included.

Changes after May 6, 2025: Supplemental_Information_Parikh-etal.pdf was added and some copyediting of the Abstract.