Non‐invasive reconstruction of complete mitochondrial genomes from aquatic environmental DNA using PCR‐free long‐read sequencing

Environmental DNA (eDNA) enables non‐invasive assessment of aquatic biodiversity. Because eDNA is typically diluted and fragmented, most applications rely on short mitochondrial amplicons, which often fail to resolve closely related species and do not support individual‐level inference. However, intact or near‐full‐length mitochondrial genomes may persist in some waters. Advances in long‐read sequencing (LRS) may enable recovery of whole mitogenomes from eDNA; however, current protocols are not optimised to detect or utilise longer fragments. To address this, we developed an LRS workflow to reconstruct complete mitogenomes from eDNA. Using aquarium water from Carassius auratus and Oryzias latipes , we optimised DNA extraction and filter pore sizes to maximise recovery of high‐molecular‐weight target DNA. We then evaluated two strategies: PCR‐free sequencing of native eDNA and long‐range PCR followed by nanopore sequencing. Finally, using single‐fish aquaria, we compared the resulting full‐length sequences across samples to assess the feasibility of non‐invasive, mitogenome‐wide polymorphism analysis. Using the selected extraction method and filters (1.2‐μm pore size), PCR‐free datasets from single‐fish C. auratus aquaria yielded hundreds of mitochondrial reads, including >16 kb molecules, enabling de novo assembly of full‐length mitogenomes in several cases. PCR‐based datasets sometimes produced >100,000 mitochondrial reads, and, after careful preprocessing, these data could also be assembled successfully. Assemblies closely matched tissue‐derived references, and cross‐sample comparisons resolved two C. auratus haplotypes. These results reveal that full‐length mitogenomes can be reconstructed from eDNA using both PCR‐free and PCR‐based strategies under appropriate conditions. Our results show that LRS can recover near‐complete mitogenomes directly from eDNA, including through PCR‐free workflows. This capability advances eDNA‐based genetic polymorphism analysis, supporting aquaculture management and non‐invasive genetic monitoring of captive conservation populations. With further refinement and field validation, it could also enable detailed assessment of genetic diversity in natural populations.