Going Mobile: Using Portable Genomic Technologies for PCR-Free In Situ Species Identification and Real-Time Molecular Systematics.

Across the globe, anthropogenic environmental changes are threatening animal biodiversity and contributing to the emergence of vector-borne and zoonotic pathogens through host range shifts. To combat these challenges, accurate and timely biodiversity assessments and molecular species monitoring efforts are critical. Here, we document how the implementation of a portable laboratory in combination with targeted long-read nanopore sequencing can facilitate in situ genomic and systematic analyses across several animal taxa. Working at two ecologically divergent field sites in Guyana, South America, we collected small mammals and blood-feeding insects, including bats, rodents, a marsupial, mosquitoes, and a phlebotomine sand fly. For each specimen sampled, genomic DNA was extracted in the field and used for the preparation of nanopore sequencing libraries. For field sequencing, we utilized a novel software-based targeted sequencing approach-nanopore adaptive sampling (NAS)-that enabled the selective sequencing of mitochondrial reads using mitogenome assemblies of related taxa as enrichment targets. Basecalled reads from our field sequencing experiments were used to assemble complete mitogenomes and to generate mitochondrial biomarker consensus gene sequences for all nine small mammals and four blood-feeding insects sequenced. Confirmatory molecular identifications were made with a combination of local nucleotide BLAST queries and maximum likelihood analyses using biomarker consensus sequences. Importantly, the mitogenome-based targeted sequencing strategies outlined here are amplification-free and allowed us to bypass time-consuming and potentially troublesome PCR-based methods in the field, streamlining library preparation, sequencing experiments, and on-site analyses. Our findings describe targeted sequencing with NAS as an effective tool for implementation into portable laboratories to widely enhance field-based biodiversity monitoring and rapid molecular species assessments across vertebrate and invertebrate hosts of consequential emerging pathogens.