Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species.
Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA justinlee337@gmail.com.
Justin S
JS
Lee
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA.
Ryan S
RS
Mackie
Department of Computer Science, Colorado State University, Fort Collins, Colorado, USA.
Thomas
T
Harrison
Department of Computer Science, Colorado State University, Fort Collins, Colorado, USA.
Basir
B
Shariat
Department of Computer Science, Colorado State University, Fort Collins, Colorado, USA.
Trey
T
Kind
German Cancer Research Center, Research Program in Infection, Inflammation and Cancer, Heidelberg, Germany.
Timo
T
Kehl
German Cancer Research Center, Research Program in Infection, Inflammation and Cancer, Heidelberg, Germany.
Martin
M
Löchelt
Department of Computer Science, Colorado State University, Fort Collins, Colorado, USA.
Christina
C
Boucher
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA.
Sue
0000-0001-9227-1622
S
VandeWoude
D000818:Animals; D001424:Bacterial Infections / Q000175:diagnosis / Q000662:veterinary*; D002371:Cat Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000821:virology; D002415:Cats; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary*
multiplex diagnostic assay; next-generation sequencing; pathogen genomics; targeted enrichment