Study examining the diet of Galapagos sea lion and Galapagos fur seal by applying metabarcoding (mt16S) on scats samples
Scats were collected immediately after defecation was observed. The remaining samples were gathered opportunistically, targeting adult scats and always prioritizing those that were fresh to ensure high-quality DNA. It is important to emphasize that GFS scats differ from those of GSL by their softer consistency, usually with a more yellowish color, and being smaller in size. All collections took place exclusively at sunrise and sunset to minimize exposure to sunlight, which could degrade the quality of the fresh samples. Each scat sample (full scat) was collected onto clean aluminum foil, with the outer layer (ca. 3–5 mm) removed. The inner content was then homogenized by mixing the whole scat while still fresh with a sterile popsicle stick. Samples were sealed in plastic bags and immediately frozen at -20°C using a portable freezer in the field and later stored in a laboratory freezer until DNA extraction.  Approximately 220 mg of wet fecal material was subsampled from each selected scat by cutting multiple random sections of the frozen sample using a disposable scalpel. These segments were then combined for DNA extraction, using the “human DNA analysis protocol” from the QIAGEN QIAamp® Fast DNA Stool Mini Kit. Adjustments made compared to the manufacturer's protocol (based on initial extraction trials) included lowering the incubation temperature from 70°C to 50°C and using 100 µL of TAE buffer for the final DNA elution. DNA concentration was measured with a Nanodrop 2000® spectrophotometer, and DNA integrity was evaluated via 1% agarose gel electrophoresis. Each DNA extraction batch included a negative control (blank extraction). A fragment of approximately 250 bp of the mitochondrial 16S rRNA gene was amplified with primers Vert-16S-eDNA-F1 (5' - AGA CGA GAA GAC CCY DTG GAG CTT - 3') and Vert-16S-eDNA-R1 (5'-GAT CCA ACA TCG AGG TCG TAA - 3'). These primers can amplify fish and invertebrate DNA such as cephalopods as demonstrated in an earlier work. Here, we also tested these primers for amplifying arthropod DNA, specifically a Drosophila virilis, which we employed as a positive control. For metabarcoding, the PCR primers were modified with a dual-indexing system to enable sample multiplexing for Illumina sequencing [34]. To minimize amplification of GSL/GFS host DNA, we employed the sea lion blocking primer (5' - TGG AGC TTC AAT TAA CTT ACC CAA TCA GAA TTT ATT C - 3'). Every PCR was performed in duplicate, including the positive control, extraction negative controls, and a PCR negative controls. Each PCR reaction was prepared as follows: 1) 4µl of “Hot Start FIREPol® Blend Master Mix” (Ready to Load with 2.0 mM MgCl2; Solis BioDyne, Estonia), 2) 0.7 µl of forward primer 10 µM, 3) 0.7µl of reverse primer 10 µM, 4) 7 µl of blocking primer 20 µM, 5) 2 µl of template DNA, and 6) 5.6 µl of PCR-grade H2O, to complete a total of 20 µl of reaction mix. The thermocycler program included the following steps: 1) Initial denaturation at 95°C for 15 min (hot start), 2) denaturation at 94°C for 45s, 3) annealing at 53°C for 45s, 4) elongation at 72°C for 90s, and 5) final elongation at 72°C for 5min. Steps 2–4 were iterated for 35 times. We assessed PCR success by loading the PCR products in a 1.5% agarose gel via electrophoresis. Samples that didn't generate a visible band were subject to a new PCR round with 3 µl of DNA template (and 4.6 µl of H2O, rest of the PCR mix remained the same). Based on the PCR product intensity on an agarose gel, (as a proxy for amplicon concentration) we pooled samples into a single library (1 µL for very clear, shiny bands to 9 µL for the negative controls). The pooled library was purified using Favor-Prep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp., Austria), following the manufacturer's instructions.
Scats were collected immediately after defecation was observed. The remaining samples were gathered opportunistically, targeting adult scats and always prioritizing those that were fresh to ensure high-quality DNA. It is important to emphasize that GFS scats differ from those of GSL by their softer consistency, usually with a more yellowish color, and being smaller in size. All collections took place exclusively at sunrise and sunset to minimize exposure to sunlight, which could degrade the quality of the fresh samples. Each scat sample (full scat) was collected onto clean aluminum foil, with the outer layer (ca. 3–5 mm) removed. The inner content was then homogenized by mixing the whole scat while still fresh with a sterile popsicle stick. Samples were sealed in plastic bags and immediately frozen at -20°C using a portable freezer in the field and later stored in a laboratory freezer until DNA extraction.  Approximately 220 mg of wet fecal material was subsampled from each selected scat by cutting multiple random sections of the frozen sample using a disposable scalpel. These segments were then combined for DNA extraction, using the “human DNA analysis protocol” from the QIAGEN QIAamp® Fast DNA Stool Mini Kit. Adjustments made compared to the manufacturer's protocol (based on initial extraction trials) included lowering the incubation temperature from 70°C to 50°C and using 100 µL of TAE buffer for the final DNA elution. DNA concentration was measured with a Nanodrop 2000® spectrophotometer, and DNA integrity was evaluated via 1% agarose gel electrophoresis. Each DNA extraction batch included a negative control (blank extraction). A fragment of approximately 250 bp of the mitochondrial 16S rRNA gene was amplified with primers Vert-16S-eDNA-F1 (5' - AGA CGA GAA GAC CCY DTG GAG CTT - 3') and Vert-16S-eDNA-R1 (5'-GAT CCA ACA TCG AGG TCG TAA - 3'). These primers can amplify fish and invertebrate DNA such as cephalopods as demonstrated in an earlier work. Here, we also tested these primers for amplifying arthropod DNA, specifically a Drosophila virilis, which we employed as a positive control. For metabarcoding, the PCR primers were modified with a dual-indexing system to enable sample multiplexing for Illumina sequencing [34]. To minimize amplification of GSL/GFS host DNA, we employed the sea lion blocking primer (5' - TGG AGC TTC AAT TAA CTT ACC CAA TCA GAA TTT ATT C - 3'). Every PCR was performed in duplicate, including the positive control, extraction negative controls, and a PCR negative controls. Each PCR reaction was prepared as follows: 1) 4µl of “Hot Start FIREPol® Blend Master Mix” (Ready to Load with 2.0 mM MgCl2; Solis BioDyne, Estonia), 2) 0.7 µl of forward primer 10 µM, 3) 0.7µl of reverse primer 10 µM, 4) 7 µl of blocking primer 20 µM, 5) 2 µl of template DNA, and 6) 5.6 µl of PCR-grade H2O, to complete a total of 20 µl of reaction mix. The thermocycler program included the following steps: 1) Initial denaturation at 95°C for 15 min (hot start), 2) denaturation at 94°C for 45s, 3) annealing at 53°C for 45s, 4) elongation at 72°C for 90s, and 5) final elongation at 72°C for 5min. Steps 2–4 were iterated for 35 times. We assessed PCR success by loading the PCR products in a 1.5% agarose gel via electrophoresis. Samples that didn't generate a visible band were subject to a new PCR round with 3 µl of DNA template (and 4.6 µl of H2O, rest of the PCR mix remained the same). Based on the PCR product intensity on an agarose gel, (as a proxy for amplicon concentration) we pooled samples into a single library (1 µL for very clear, shiny bands to 9 µL for the negative controls). The pooled library was purified using Favor-Prep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp., Austria), following the manufacturer's instructions.