Extending eDNA capabilities via long-range PCR and third-generation sequencing

Katrina M West1, Alastair Harry2, Gabby Mitsopoulos2, Michael Travers2, Sharon A. Appleyard1, Bruce Deagle1

1 CSIRO Australian National Fish Collection, National Research Collections Australia, CSIRO, Hobart, TAS, Australia

2 Western Australian Fisheries and Marine Research Laboratories, Department of Primary Industries and Regional Development, Government of Western Australia, North Beach, WA, Australia

Abstract:

The emergence of environmental DNA (eDNA) metabarcoding, a genetic detection technique that can non-invasively profile ecosystem biota from environmental samples, is revolutionising our approach to biodiversity surveying. However, there are very few studies which have retrieved long eDNA sequences from the environment; if this was routinely possible it would facilitate non-invasive, population haplotype analyses and, potentially progress, to population genetics from eDNA. In this study, we aim to expand current eDNA capabilities by attempting to retrieve longer mitochondrial sequences (1-16 kb) and subsequent population haplotype information for critically endangered and elusive elasmobranchs found in the inshore and offshore waters of the Kimberley Marine Park and Roebuck Marine Park, Western Australia. To facilitate this approach, we firstly modelled the length distribution of mitochondrial eDNA from tiger shark (Galeocerdo cuvier), collected in the field, to reveal eDNA size lengths that could potentially be targeted via long-range PCR. We then conducted long-range metabarcoding for elasmobranchs, sequenced our longest retrievable fragments via third-generation, Oxford Nanopore Minion sequencing, and validated these results against known elasmobranch haplotypes in the region. We additionally assess whether targeting longer eDNA fragments produces a similar presence-absence detection profile compared to targeting shorter fragments (i.e. 100-600 bp). This case study provides a proof of concept for long-range PCR in conjunction with third-generation sequencing that can be adapted for any targeted eDNA application.


Biography:

Katrina is an early career researcher; she has recently completed her PhD at Curtin University, Western Australia where she focused on the development and application of eDNA metabarcoding for aquatic biomonitoring in Australia’s Indian Ocean region. She is now a postdoctoral fellow at the CSIRO, Hobart where she aims to extend eDNA metabarcoding capabilities and applications for marine fish and elasmobranch monitoring.

Date

Mar 21 - 23 2022
Expired!