Optimizing DNA extraction protocols for the diet analysis of a baleen whale (Eubalaena australis)
Data files
Oct 17, 2024 version files 330.36 KB
-
16S-asv-table.csv
80.07 KB
-
18S-asv-table.csv
223.63 KB
-
protocol_levels.csv
24.56 KB
-
README.md
2.10 KB
May 06, 2025 version files 1.25 MB
-
16S-18S-raw-data.xlsx
919.58 KB
-
16S-asv-table.csv
80.07 KB
-
18S-asv-table.csv
223.63 KB
-
protocol_levels.csv
24.56 KB
-
README.md
2.82 KB
Abstract
Faecal metabarcoding is widely used for mammalian diet analysis. However, most extraction protocols are designed to use high molecular weight genomic DNA, making short sequences of digested DNA challenging to extract. Here, we compared 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 in southern right whale (Eubalaena australis, SRW) faecal matter. 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 affected 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.
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 eukaryote and 16S 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 information.
Supplementary tables and figures (Zenodo)
Supplementary tables and figures can be found in the file “Supplemental_Information_Parikh-etal.pdf”.
Code (Zenodo)
All coding for this study was performed in R, and the associated R script can be found in the file “Parikh-et-al-2024.R”.
Files and variables
File: 16S-asv-table.csv, 18S-asv-table.csv
Description:
This file is in the format of an OTU table, with rows and OTUs and columns as samples. The first column of this file contains the unique OTU identifier, followed by columns that list the taxonomic classification of each sample (columns B-H), and a column with the total reads in each OTU (column I). The 10th column onwards (i.e. column J) consists of samples with the number of reads of each OTU detected within.
File: protocol_levels.csv
Description:
This file contains brief metadata associated with the extraction protocols which is used in the corresponding R script to perform analyses.
File: 16S-18S-raw-data.xlsx
Description:
This file contains raw OTU data for Crust16S mtDNA and 18S rDNA datasets, prior to dataset cleaning for the analysis included in the main manuscript.
Code/software
All analysis was performed in the software R (version 4.3.1). The code used can be found in the R script file “Parikh-et-al-2024.R” which lists all the R packages required to perform the analysis as well as brief comments explaining what analyses are being performed. All files and datasets required to run the analysis have been included.
Change log
- 6 May 2025, 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.
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.