Optimizing DNA extraction protocols for the diet analysis of a baleen whale (Eubalaena australis)
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Springer
Abstract
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 used 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 h); (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.
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DATA AVAILABILITY STATEMENT: All data associated with this study, including metadata, code and supplementary material, are publicly available on the online repository Dryad (https://doi.org/10.5061/dryad.9s4mw6mrx).
SUPPORTING INFORMATION: TABLE S1 : List of SRW samples used in this study and associated metadata TABLE S2 : PCR conditions for 18S rDNA and Crrust16S mtDNA TABLE S3 : Classifiaction of target and non-target organisms captured by 18S rDNA and Crust16S mtDNA TABLE S4 : Summary of target zOTU richness captured by each extraction protocol for the 18S rDNA dataset TABLE S5 : Post hoc pairwise comparison of differences in target zOTU richness derived from different protocols from the 18S rDNA dataset TABLE S6 : Summary of target zOTU richness captured by each extraction protocol for the Crust16S mtDNA dataset TABLE S7 : Post hoc pairwise comparison of differences in target zOTU richness derived from different protocols from the Crust16S mtDNA dataset TABLE S8 : Post hoc pairwise comparison of significant differences in Shannon diversity of target species derived from different protocols from the 18S rDNA dataset FIGURE S1 : Comparison of Shannon diversity of target species captured by the different extraction protocols in the 18S rDNA dataset FIGURE S2 : Comparison of Shannon diversity of target species captured by the different extraction protocols in the Crust16S mtDNA dataset TABLE S10 : Post hoc pairwise comparison of significant differences in target species abundance composition derived from different protocols from the 18S rDNA dataset TABLE S11: Post hoc pairwise comparison of significant differences in target species presence-absence composition derived from different protocols from the 18S rDNA dataset TABLE S12 : Post hoc pairwise comparison of significant differences in target species abundance composition derived from different protocols from the Crust16S mtDNA dataset TABLE S13 : Post hoc pairwise comparison of significant differences in target species presence-absence compositional data derived from different protocols from the Crust16S mtDNA dataset FIGURE S3 : Proportion of reads of classes of target organisms captured by each individual sample by the 18S rDNA amplicon. Samples are arranged based on biological sample/replicate (NZ1 – SA3 on right y-axis), extraction replicate (1, 2, 3 on right y-axis), PCR replicate (a, b, c on bottom x-axis), and extraction protocol (top x-axis) FIGURE S4 : Proportion of reads for different orders of subphylum Crustacea captured by each individual sample by the Crust16S mtDNA amplicon. Samples are arranged based on biological sample/replicate (NZ1 – SA3 on right y-axis), extraction replicate (1, 2, 3 on right y-axis), PCR replicate (a, b, c on bottom x-axis), and extraction protocol (top x-axis) FIGURE S5 : Comparison of zOTU richness as captured by each protocol in the raw datasets for (a) 18S rDNA and (b) Crust16S mtDNA FIGURE S6 : Comparison of Shannon diversity as captured by each protocol in the raw datasets for (a) 18S rDNA and (b) Crust16S mtDNA TABLE S14 : Summary of zOTU richness captured by each extraction protocol for the raw 18S rDNA dataset TABLE S15 : Post hoc pairwise comparison of differences in zOTU richness derived from different protocols from the raw 18S rDNA dataset TABLE S16 : Post hoc pairwise comparison of differences in Shannon diversity derived from different protocols from the raw 18S rDNA dataset. TABLE S17 : Summary of zOTU richness captured by each extraction protocol for the raw Crust16S mtDNA dataset. TABLE S18 : Post hoc pairwise comparison of differences in zOTU richness derived from different protocols from the raw Crust16S mtDNA dataset. TABLE S19 : Post hoc pairwise comparison of differences in Shannon diversity derived from different protocols from the raw Crust16S mtDNA dataset. FIGURE S7 : Relative abundance of (a) unique classes detected in the raw 18S rDNA dataset and (b) unique species detected in the raw Crust16S mtDNA dataset. TABLE S20 : Post hoc pairwise comparison of significant differences in species abundance compositional data derived from different protocols from the raw 18S rDNA dataset TABLE S21 : Post hoc pairwise comparison of significant differences in species presence-absence compositional data derived from different protocols from the raw 18S rDNA dataset. TABLE S22 : Post hoc pairwise comparison of significant differences in species abundance compositional data derived from different protocols from the raw Crust16S mtDNA dataset. TABLE S23 : Post hoc pairwise comparison of significant differences in species presence-absence compositional data derived from different protocols from the raw Crust16S mtDNA dataset.
SUPPORTING INFORMATION: TABLE S1 : List of SRW samples used in this study and associated metadata TABLE S2 : PCR conditions for 18S rDNA and Crrust16S mtDNA TABLE S3 : Classifiaction of target and non-target organisms captured by 18S rDNA and Crust16S mtDNA TABLE S4 : Summary of target zOTU richness captured by each extraction protocol for the 18S rDNA dataset TABLE S5 : Post hoc pairwise comparison of differences in target zOTU richness derived from different protocols from the 18S rDNA dataset TABLE S6 : Summary of target zOTU richness captured by each extraction protocol for the Crust16S mtDNA dataset TABLE S7 : Post hoc pairwise comparison of differences in target zOTU richness derived from different protocols from the Crust16S mtDNA dataset TABLE S8 : Post hoc pairwise comparison of significant differences in Shannon diversity of target species derived from different protocols from the 18S rDNA dataset FIGURE S1 : Comparison of Shannon diversity of target species captured by the different extraction protocols in the 18S rDNA dataset FIGURE S2 : Comparison of Shannon diversity of target species captured by the different extraction protocols in the Crust16S mtDNA dataset TABLE S10 : Post hoc pairwise comparison of significant differences in target species abundance composition derived from different protocols from the 18S rDNA dataset TABLE S11: Post hoc pairwise comparison of significant differences in target species presence-absence composition derived from different protocols from the 18S rDNA dataset TABLE S12 : Post hoc pairwise comparison of significant differences in target species abundance composition derived from different protocols from the Crust16S mtDNA dataset TABLE S13 : Post hoc pairwise comparison of significant differences in target species presence-absence compositional data derived from different protocols from the Crust16S mtDNA dataset FIGURE S3 : Proportion of reads of classes of target organisms captured by each individual sample by the 18S rDNA amplicon. Samples are arranged based on biological sample/replicate (NZ1 – SA3 on right y-axis), extraction replicate (1, 2, 3 on right y-axis), PCR replicate (a, b, c on bottom x-axis), and extraction protocol (top x-axis) FIGURE S4 : Proportion of reads for different orders of subphylum Crustacea captured by each individual sample by the Crust16S mtDNA amplicon. Samples are arranged based on biological sample/replicate (NZ1 – SA3 on right y-axis), extraction replicate (1, 2, 3 on right y-axis), PCR replicate (a, b, c on bottom x-axis), and extraction protocol (top x-axis) FIGURE S5 : Comparison of zOTU richness as captured by each protocol in the raw datasets for (a) 18S rDNA and (b) Crust16S mtDNA FIGURE S6 : Comparison of Shannon diversity as captured by each protocol in the raw datasets for (a) 18S rDNA and (b) Crust16S mtDNA TABLE S14 : Summary of zOTU richness captured by each extraction protocol for the raw 18S rDNA dataset TABLE S15 : Post hoc pairwise comparison of differences in zOTU richness derived from different protocols from the raw 18S rDNA dataset TABLE S16 : Post hoc pairwise comparison of differences in Shannon diversity derived from different protocols from the raw 18S rDNA dataset. TABLE S17 : Summary of zOTU richness captured by each extraction protocol for the raw Crust16S mtDNA dataset. TABLE S18 : Post hoc pairwise comparison of differences in zOTU richness derived from different protocols from the raw Crust16S mtDNA dataset. TABLE S19 : Post hoc pairwise comparison of differences in Shannon diversity derived from different protocols from the raw Crust16S mtDNA dataset. FIGURE S7 : Relative abundance of (a) unique classes detected in the raw 18S rDNA dataset and (b) unique species detected in the raw Crust16S mtDNA dataset. TABLE S20 : Post hoc pairwise comparison of significant differences in species abundance compositional data derived from different protocols from the raw 18S rDNA dataset TABLE S21 : Post hoc pairwise comparison of significant differences in species presence-absence compositional data derived from different protocols from the raw 18S rDNA dataset. TABLE S22 : Post hoc pairwise comparison of significant differences in species abundance compositional data derived from different protocols from the raw Crust16S mtDNA dataset. TABLE S23 : Post hoc pairwise comparison of significant differences in species presence-absence compositional data derived from different protocols from the raw Crust16S mtDNA dataset.
Keywords
Faecal metabarcoding, Southern right whale (SRW), Phosphate buffer extraction, PowerSoil, QIAamp fast DNA stool mini kit, Baleen whale (Eubalaena australis), Eubalaena australis, Southern right whale ( (Eubalaena australis)
Sustainable Development Goals
SDG-14: Life below water
Citation
Parikh, A., O’Rorke, R., Carroll, E.L. et al. 2026, ‘Optimizing DNA extraction protocols for the diet analysis of a baleen whale (Eubalaena australis)’, Conservation Genetics Resources, vol. 18, art. 25, pp. 1-14, doi: 10.1007/s12686-026-01429-6