Simultaneous identification of host, ectoparasite and pathogen DNA via in‐solution capture

Abstract

Ectoparasites frequently vector pathogens from often unknown pathogen reservoirs to both human and animal populations. Simultaneous identification of the ectoparasite species, the wildlife host that provided their most recent blood meal(s), and their pathogen load would greatly facilitate the understanding of the complex transmission dynamics of vector-borne diseases. Currently, these identifications are principally performed using multiple polymerase chain reaction (PCR) assays. We developed an assay (EctoBaits) based on in-solution capture paired with high-throughput sequencing to simultaneously identify ectoparasites, host blood meals and pathogens. We validated our in-solution capture results using double-blind PCR assays, morphology and collection data. The EctoBaits assay effectively and efficiently identifies ectoparasites, blood meals, and pathogens in a single capture experiment, allowing for high-resolution taxonomic identification while preserving the DNA sample for future analyses.

Interacting effects of land use and climate on rodent-borne pathogens in central Kenya

“…We screened for all pathogens using polymerase chain reactions (PCR) following Campana et al . [61]. All positive apicomplexan and Coxiella PCR products, and a representative sampling of positive Anaplasma , Bartonella Borrelia and Rickettsia products were Sanger sequenced on an ABI 3130 (Life Technologies, Carlsbad, CA).…”

The Expectations and Challenges of Wildlife Disease Research in the Era of Genomics: Forecasting with a Horizon Scan-like Exercise

“…Additional innovations include the development of sequencing libraries with specific targets for genomic investigations. Targeted capture techniques, which utilize hybridization oligonucleotide probes to enrich for specific nucleic acids, have become popular for their array of uses in addition to their application in noninvasive or ancient sample specimens (Perry et al 2010;Campana et al 2016;Lee et al 2017). The primary limitation of these techniques is that target sequences, from pathogens for example, must be known a priori, making the discovery of novel pathogens more difficult.…”

BaitsTools: Software for hybridization capture bait design

“…To benchmark typical baitstools performance, baits were generated and filtered using sequence data from previously sequenced African wild dog ( Lycaon pictus ) genomes (Campana, Hawkins et al., , Campana, Parker et al., ), reference sequences from GenBank (accessions: , , , ) (Bjornerfeldt, Webster, & Vilà, ; Kim, Lee, Jeong, & Ha, ; Koepfli et al., ; Lindblad‐Toh et al., ), and simulated Stacks data and ipyrad loci (available: ipyrad.readthedocs.io/output_formats.html). Benchmarked data sets are included in the example data within the baitstools repository, except for the Canis familiaris X chromosome sequence (GenBank accession: ) due to file size limitations.…”

“…Furthermore, to compare performance between baitstools tilebaits and BaitDesigner ( picard version 2.9.4), baits were generated from a 16,725 bp Lycaon pictus mitogenome (GenBank accession: ; Campana, Hawkins et al., , Campana, Parker et al., ) under analogous settings. Each program generated 120 bp baits with a 60 bp offset between baits.…”

“…Targeted high‐throughput sequencing using hybridization capture (e.g., Gnirke et al., ) is a critical tool in molecular ecology and genomics. Applications include genomic investigations of nonmodel organisms using ultra‐conserved elements (Faircloth et al., ; Lim & Braun, ), exome capture (e.g., Ng et al., ), single nucleotide polymorphism (SNP) analysis (e.g., Burbano et al., ), targeted metagenomics (e.g., Campana, Hawkins et al., , Campana, Parker et al., ), and ancient DNA enrichment and museomics (e.g., Burbano et al., ; Hawkins et al., ; Lim & Braun, ), among others. Hybridization capture utilizes oligonucleotide baits to enrich target molecules from nucleic acid libraries through hybridization of the baits to complementary nucleotide sequences in the libraries, isolation of the hybridized molecules and removal of the nontarget library molecules.…”