Progress of Analysis of e-DNA Samples and Sampling Activities in Less Potential Site of Bali Sardine

We have conducted laboratory analysis for Environmental DNA (e-DNA) samples to detect Bali Sardine (Sardinella lemuru) obtained from Sembulungan Point (Potential Fishing Ground of Bali Sardine) and Ujung Pasir Point (Potential Fishing Ground Record of Bali Sardine 12 years ago). The e-DNA extraction, PCR, Nanopore sequencing, and initial bioinformatic analysis were performed at the specialized eDNA laboratory (Biodiversitas Indonesia (BIONESIA) in Bali) in September 2024. The use of laboratory analysis protocols was based on optimizing the experimental trial from BIONESIA and previous eDNA protocol studies, especially for the Nanopore Sequencing processes.

The e-DNA extraction steps refer to the protocol of Minamoto et al. (2020), Wu and Minamoto (2023), and Qiagen Kit Manufacture Instruction. Technical optimization by modification of published protocol based on the BIONESIA-Bali laboratory protocol (Figure 1): cutting the membrane filter paper into the smallest parts before transferring Buffer ATL Qiagen from the sample tube and adding Proteinase K (Fig. 1a), incubation process using a rotating incubator with a temperature of 56°C overnight (Fig. 1b), and pre-heated (70°C) treatment of Buffer AE Qiagen onto the center of the spin column to release or elute e-DNA extract into the final collection tube. The extraction results were then checked for quality by Gel Electrophoresis and their quantity and purity by Qubit Fluorometer (Figures 2a and 2b).

 

Figure 1. (a-b) Technical optimization by modification of published protocol based on the BIONESIA-Bali laboratory protocol, (c) the centrifugation setting, and (d) adding extraction reagents kit following the manufacture instruction of Qiagen.

 

Figure 2. The extracted eDNA genome. 2 µl of extraction results of each eDNA sample were checked for quality and quantity by (a) 1% Gel Electrophoresis and (b) Qubit Fluorometer.

The number of PCR reactions is the number of samples per site with three replicates at each depth, one positive control, and one negative control (no DNA added to the reaction). The eDNA primers used were 12S MiFish-U-F (10 µM; 1.25 µl) and MiFish-U-R (10 µM; 1.25 µl) (Miya et al. 2015). The PCR reaction volume per sample used Bioline HS Ready Mix (12.5 µl) and BSA (0.2 µl), ddH2O molecular grade (9.8 µl) and 2 µl e-DNA template. To obtain the best-yielded amplification of the targeted e-DNA band, all e-DNA templates (10X) from extracted e-DNA are diluted with a ratio of 1:9 (Figure 3b), except for two e-DNA templates with sample ID S2-3M-3L and boat blanko (negative control). Moreover, PCR conditions refer to the thermocycling program in Figure 3a. Furthermore, the PCR results were then visualized by running a 1% agarose gel (140 volts; 200 A; 35 minutes) with a 100 bp hyper-ladder. Look for the targeted fragment size of ~ 260 bp in Fig. 3b.

 

Figure 3. The PCR reactions amplify the specific PCR product (12rRNA Mitochondrial DNA). (a) Thermocycling program with 40 cycles run in a 2720 thermocycler (Applied Biosystems, USA). (b) Single bands of PCR product with a targeted DNA fragment size of fish around 260 bp.

The eDNA sample preparation stage before priming and loading into the SpotON flow cell was based on the ONT Library Preparation Protocol. Before the End-Prep stage in the ONT Library Preparation protocol, each PCR product concentration was normalized to 200 fmol. The Nanopore sequencing process used Native Barcode Ligation and was run for 24 hours (Figure 4). The sequencing results were then directly base called with high-accuracy model v4.3.0, 400 bps by the MinKNOW App ver. 24.06.8.

 

Figure 4. Nanopore Sequencing Process. The passed reads are close to the total number of reads, indicating good sequencing performance. A Q score of more than 9 also indicates good read quality.

At the end of October, e-DNA sampling was conducted at two different depths for the less potential fishing ground of Bali Sardine (Banjubiru Bay-Bali Strait) (Figure 5a and Figure 5b). The boat trip to the point takes 2 hours, depending on weather conditions. Banjubiru Bay Point has many human activities, such as tourist activities, fishing with lift nets, and pearl cultivation. Interestingly, Banjubiru Bay Point has a high level of water clarity, so seawater filtration per sample can be reached up to 7 liters.

 

Figure 5a. eDNA sampling activities at two different depths in BanjuBiru Bay-Bali Strait.

 

Figure 5b. Banjubiru Bay-Bali Strait represents the less potential fishing ground of Bali Sardine.