GoKawiilField collection
Fish collected for lethal sampling were euthanized with an overdose of buffered MS-222 solution (tricaine methanesulfonate, Syndel). Tissues from A. nebulosus individuals were collected (Fig. 1a) at five timepoints as normal brain or skin tissue (N) or melanistic skin tissue from fish with black lesions (T). A set of samples was collected from normal and tumour skin tissue during June of 2019 from Lake Memphremagog in Hospital Cove (2019, n = 8 (N), n = 8 (T)). A second set of Lake Memphremagog fish were collected in June of 2023 from eight locations in Lake Memphremagog for unaffected fish brain tissue only (N) and affected fish paired brain (N) and tumour tissue (T) genomic analysis (Fig. 1 and Supplementary Table 2); Fitch Bay (FB, n = 6 (N) and n = 8 (paired N and T)), Magog Area (MA, n = 6 (N) and n = 8 (paired N and T)), Hospital Cove (HC, n = 8 (N) and n = 6 (paired N and T)), Sargent’s Bay (SB, n = 6 (N) and n = 6 (paired N and T)), Scott’s Cove (SC, n = 6 (N) and n = 5 (paired N and T)), South Bay/Johns River (JR, n = 6 (N) and n = 6 (paired N and T)) and South Main Lake (SML, n = 6 (N) and n = 6 (paired N and T)). A set of reference fish (Supplementary Table 1) was collected during spring 2022 from Lake Bomoseen (BS, n = 2 (N)), Lake Champlain Larabee’s (LB, n = 2 (N)) and Sandy Point (SP, n = 2 (N)), and the Connecticut river at Hoyts Landing (HL, n = 2 Sados (N)). Additional reference fish (Supplementary Table 1) were collected in 2024 from Maine (ME, n = 1 (N)) and in 2025 from New Hampshire (VP, n = 2 (N)). A set of transcriptome samples collected from 2015 Lake Memphremagog healthy skin and tumour skin, and healthy skin from Ticklenaked Pond were analysed for mitochondrial sequence and the data are presented in Supplementary Information 1 and 5 and Supplementary Table 3.
Normal tissues were sampled, DNA extracted and whole-genome sequenced for all (normal and malignant melanoma) Lake Memphremagog fish included in this study. However, not every fish with a malignant melanoma had a tumour tissue sampled, DNA extracted or sequenced (Supplementary Table 2). Histology was performed on a subset of the fish with melanistic lesions to verify that these tumours are consistent with those in the previous study1. We note that not all of the melanistic tissues used for genomic analysis were characterized by histology39. If a tumour sample had a low variant allele frequency (>15% tumour mitochondria relative to host mitochondria, see below for details), we resampled and sequenced tumour tissue from the same fish. Collections for this study were performed under the auspices of the Vermont Fish and Wildlife Department.
Tissue processing and nucleic acid sequencing
Tissue samples were collected as soon as possible after euthanasia. All necropsy equipment was butane-flame sterilized between animals. Tissue samples used for DNA analysis at the Vermont Integrated Genomic Resource at the University of Vermont (VIGR; RRID: SCR_021775) were stored without a buffer and placed on ice. The samples were transported to VIGR on wet ice and stored in a −80 °C freezer.
All fish tissue was subsampled under sterile conditions and with equipment disinfected with 10% bleach. The 2022 reference fish were extracted using the DNeasy Blood and Tissue Kit (Qiagen). Tissues were lysed in 180 µl ATL buffer plus 20 µl proteinase K and incubated at 56 °C for 2.5 h with periodic vortexing. The lysates were extracted for DNA using the manufacturer’s protocol and eluted in 200 µl of buffer AE. The Lake Memphremagog, Maine and New Hampshire samples were extracted for DNA using the E-Z 96 Tissue DNA Kit (Omega Bio-Tek) kit but with the following modifications. The samples were lysed in 200 μl TL buffer plus 25 µl proteinase K and incubated at 60 °C overnight with periodic vortexing. The lysates were extracted according to the manufacturer’s protocol and eluted in 150 µl of the elution buffer. All extracted DNA was quantified using the Qubit spectrofluorometer (Thermo Fisher Scientific) using the dsDNA HS kit.
High-throughput sequencing
DNA short-read sequencing
Whole-genome DNA-sequencing libraries were prepared using the NEXTFLEX Rapid XP V2 DNA-Seq kit (Revvity) according to the manufacturer’s protocol with the following modifications. For the reference samples and 2019 Lake Memphremagog samples, 150 ng total DNA was used for input and the libraries were amplified for four cycles of PCR. For the 2023 Lake Memphremagog samples, 10 ng of total DNA was used for input, and the libraries were amplified for 7–9 cycles of PCR using SG UDI Primers (Singular Genomics) rather than Revvity Primer Mix V2. The final libraries were eluted in 5 mM Tris, and the library quality and concentrations were determined using the High Sensitivity DNA Kit on the Agilent Bioanalyzer 2100 and Qubit spectrofluorometer (Thermo Fisher Scientific). The samples in each group were pooled equally on the basis of total mass (ng). The reference fish and the 2019 Lake Memphremagog samples were sequenced as paired-end 150 bp or 250 bp reads on the S2 flow cell on the Illumina NovaSeq 6000 system at the UNH Hubbard Center for Genomics. The 2023 Lake Memphremagog fish were sequenced as paired-end 150 bp reads using the Singular Genomics G4 system at the VIGR and at Singular Genomics.
Long-read sequencing
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