Oxford MV1 sequencing of seawater eDNA: Lindblad Expeditions Iceland/Greenland Sample 001B July 2022 Cruise
PCR products were amplified using the following primer sets: 12s MarVer1 (F): ONT Rapid 4-primer PCR: 5TTTCTGTTGGTGCTGATATTGCCGTGCCAGCCACCGCG3 and 12s MarVer1 (R): ONT Rapid 4-primer PCR: 5ACTTGCCTGTCGCTCTATCTTCGGGTATCTAATCCYAGTTTG3. PCR products were generated using the following mix: 25L LongAmp taq 2X MasterMix (NEB), 16.5L nuclease-free water, 5uL 1M primer mix (0.1uM final), 1.5L barcoded Oxford Nanopore rapid compatible primer and 2L of 1:10 diluted DNA template. Samples were cycled using the following profile: initial denaturation at 94C for 1min, followed by 10 cycles of 94C for 30s, 49.5C for 30s, and 65C for 45s, followed by 30 cycles of 94C for 30s, 62C for 30s, and 65C for 45s and a final extension of 65C for 2min. After 0.8x bead cleanup, samples were then subjected to Oxford Nanopore rapid preparation (excluding fragmentation) before sequencing.