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 ..., 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., , # Optimizing DNA Extraction Protocols for Diet Analysis of Baleen Whales (Eubalaena australis)

[https://doi.org/10.5061/dryad.9s4mw6mrx](https://doi.org/10.5061/dryad.9s4mw6mrx)

## Description of the data and file structure

Metadata associated with this study consist of OTU tables for 18S rDNA eukaryote and 16S mtDNA crustacean datasets obtained from metabarcoding SRW faecal samples for the targeted amplification of prey DNA. These are available in the two .csv files, \"18S-asv-table.csv\" and \"16S-asv-table.csv\", which contain the OTU tables for the eukaryote and crustacean datasets respectively. A third file, \"protocol_levels.csv\", contains metadata associated with the extraction protocols, and is required to run the R script for analysis. Additionally, an excel file \"16S-18S-raw-data.xlsx\" contains the raw OTU tables with both target and non-target taxa prior to dataset cleaning. This file has been used for supplementary analyses with results available in the supplemental informat..., , **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.