Oxford Nanopore sequencing of long-range mitochondrial amplicons from paired sperm whale eDNA and tissue samples
This study aimed to determine whether long-range mitochondrial sequencing can recover reliable lineage-level genetic information from aquatic environmental DNA (eDNA) and to establish analytical thresholds for its application to low-template samples. Basecalled and demultiplexed Oxford Nanopore sequencing reads were generated from long-range PCR amplicons of sperm whale (Physeter macrocephalus) mitochondrial DNA. Environmental DNA samples were collected from surface flukeprints immediately following sperm whale dives in the Azores, Portugal, in 2023, with paired tissue biopsies obtained from the same individuals whenever possible. Up to three overlapping mitochondrial fragments of approximately 3.0-4.4 kb were amplified from eDNA samples, targeting the COX2-CYTB-control region containing much of the known mitochondrial variation in sperm whales. Five overlapping fragments spanning the mitochondrial genome were amplified from tissue samples. Amplicons from each sample were pooled, assigned a unique native barcode, and sequenced on an Oxford Nanopore Technologies MinION Mk1B device using an R10.4.1 flow cell and V14 ligation sequencing chemistry. Super-accuracy basecalling and demultiplexing produced one FASTQ dataset per barcode, corresponding to an individual eDNA or tissue sample. The reads were used for reference mapping, mitochondrial consensus polishing, variant calling, and haplogroup inference. Paired tissue samples provided biological benchmarks for evaluating eDNA-derived mitochondrial SNP and haplogroup concordance and for optimizing bioinformatic quality-control thresholds. This work supports the development of non-invasive approaches for monitoring mitochondrial diversity and population structure in cetaceans and other vertebrates that are difficult to sample using conventional tissue-based methods.