A novel eDNA approach for rare species monitoring: Application of long-read shotgun sequencing to Lynx rufus soil pawprints

Abstract

Environmental DNA (eDNA) sampling is a relatively new technique that has been employed in biodiversity surveys around the world. Environmental DNA can be an effective, non-invasive method of identifying the presence of target host species. Furthermore, advances in DNA deep sequencing technologies are enabling in-depth information to be recovered from eDNA. Here we report the development of a bobcat (Lynx rufus) species-specific probe-based qPCR assay for eDNA studies, which was validated on wild samples and those from a bobcat housed at the Jacksonville Zoo in Florida. Furthermore, we show that long-read shotgun sequencing of eDNA extracted from pawprint soil samples, using an Oxford Nanopore Technologies MinION, could successfully detect bobcat DNA, including enabling correct species level identification and phylogenetic placement within regionally distanced bobcats. This was all achieved without utilizing any DNA enrichment approaches. The long read shotgun sequencing simultaneously recovered genetic information from microbes known to be part of the bobcat microbiome, indicating that potential health-status-related microbial information can be obtained alongside wildlife eDNA. This study revealed that non-targeted (no metabarcoding or enrichment) long-read shotgun sequencing of eDNA samples can be sufficient for species identification, phylogenetic analyses, and population genetics applications. This paves the way for the rapid deployment of eDNA approaches for any species of interest, without requiring laborious development of targeted approaches, thereby increasing the ease and utility of eDNA research for endangered species conservation and management.