From Field to Genome: Non-Lethal, On-Site Nanopore-Only Assembly of the Endangered Grenada Frog (Pristimantis euphronides)

The Grenada frog (Pristimantis euphronides), a critically endangered amphibian endemic to Grenada, West Indies, has an estimated population of fewer than 300 individuals. It is threatened by habitat loss due to farming and tourism development, as well as potential risks from climate change, disease, and an invasive species, Eleutherodactylus johnstonei, a member of the same superfamily (Hyloidea). Despite the importance of genomic resources for conservation, only seven annotated whole-genome assemblies at chromosome-scale resolution exist for Hyloidea frogs. No genome data exist for the family Strabomantidae, to which P. euphronides was recently reassigned based solely on morphological characteristics, and only one distantly related reference genome is currently available. Whole-genome sequencing of P. euphronides is critical for phylogenomic validation, conservation planning, and understanding genetic adaptations relevant to climate resilience and biodiversity preservation. A 1.75 Gb genome assembly for P. euphronides was generated using standalone nanopore sequencing, conducted entirely in Grenada, a low- and middle-income country (LMIC) in the Eastern Caribbean. Genome sequencing and assembly were performed using a total of 56 Gb of sequence data from twelve MinION flow cells, achieving 32× sequencing coverage. The Flye assembly and RagTag scaffolding workflow achieved a contig N50 value of 302 kb, a scaffold N50 values of 118 Mb, and 89.7% BUSCO completeness for Tetrapoda-specific genes. The sequencing effort was conducted using a mobile, field-ready setup without access to high-profile next-generation sequencing (NGS) and computational facilities, demonstrating the feasibility of in-country genomic research for conservation. Given the species' conservation status and respective ethical restrictions, minimally invasive sampling was conducted on a single female Grenada frog using toe clips, blood, and oral swabs. The challenges of obtaining high-quality, high-yield DNA from a live amphibian specimen, rather than from a sacrificed individual, required optimized extraction and assembly strategies to maximize genome quality. Out of the seven existing annotated complete Hyloidea genomes the proteome of P. euphronides showed the highest similarity to Eleutherodactylus coqui. High genome collinearity with E. coqui enabled alignment-based scaffolding, with P. euphronides contigs spanning all 13 chromosomes of this related reference genome. The annotated scaffold-level Grenada frog assembly provides the first whole genome for the anuran family Strabomantidae and will facilitate further studies on molecular systematics, biogeography, genetic differentiation, and conservation genetics.