500m_Jun_4
Genomic DNA (gDNA) was extracted from sediment trap-derived environmental DNA (eDNA) samples by first washing formalin-fixed samples with PBS and centrifuging to remove formalin. Approximately 100 mg of the pellet was homogenized using TissueLyser II (Qiagen), and gDNA was extracted using the TIANamp Marine Animals DNA Kit (Tiangen Biotech) following the manufacturers protocol. The extracted gDNA was dissolved in 100 L of ultrapure water and stored at -20C.  For metabarcoding analysis of the MT-CO1 gene, a two-step PCR method based on Amplicon et al. (2013) was performed. In the first-round PCR, the MT-CO1 barcode region (313 bp) was amplified using MiSeq adapter-linked metazoan universal primers (mlCOlintF and jgHCO2198). The PCR conditions were as follows: 95C for 5 min, followed by 35 cycles of 95C for 30 sec, 46C for 30 sec, and 72C for 1 min, with a final extension at 72C for 10 min. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). In the second-round PCR, index sequences were added for sample differentiation, with PCR conditions set at 95C for 5 min, followed by 8 cycles of 95C for 30 sec, 55C for 30 sec, and 72C for 1 min, with a final extension at 72C for 10 min. The purified DNA concentration was measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific), and PCR products were pooled based on their molar concentration. The final purification was performed using AMPure XP Beads (Beckman Coulter), and sequencing was conducted on the Illumina MiSeq platform.