Abstract
Baker Institute for Animal Health and Department of Population Medicine & Diagnostic Sciences, Cornell University, Ithaca, NY (Goodman); Virginia Tech Animal Laboratory Services and Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA (Lahmers).
The tools used in sequencing the human genome have been classified as next-generation sequencing (NGS), a method of massively parallel, high-throughput sequencing that produces thousands to millions of individual sequences, compared to Sanger sequencing, which produces a single sequence. Use of Sanger sequencing has matured, and it has guidelines for use and monitoring. 4 NGS is currently being used in human and veterinary testing laboratories, with some assays cleared by the FDA for testing of human samples. As the technologies advance and the cost of sequencing has decreased, these types of assays will likely gain further traction in the veterinary diagnostic arena.
There are significant challenges in the application of NGS to veterinary testing. Foremost, validation of sequencing-based assays can be challenging given that variation in sequencing depth, enrichment methods, analysis pipeline, and sample types can significantly affect sensitivity and specificity. To that end, approaches to validation are being discussed and need to be a focus of the veterinary community. The economics of NGS, including equipment, service contracts, and reagent maintenance, can make these unfeasible for some laboratories and samples. There are additional challenges of analysis and storage of the massive amounts of data produced by these assays.
However, some of these challenges are offset by the potential benefits. Molecular epidemiology made possible through NGS was established ~2015 in the food safety sector, and more recently is proving to be an invaluable tool in viral disease tracking within populations and across continents. Pathogen discovery and characterization is now possible and economically feasible in a veterinary diagnostic setting. Smaller platforms are becoming available that can be run in-house for urgent cases or on the diagnostic laboratory’s own schedule as opposed to waiting for core facilities or referral laboratory send-out. Data security, version control, and lot tracking for in-house sequencing can be maintained according to accreditation standards (AAVLD, ISO, etc.). The increasing availability and application of NGS has led to open-source tools and open science applications and the ability to develop in-house pipelines or use publicly available user-friendly tools such as Galaxy, NCBI Pathogen Browser, and Nextstrain.
This issue of the Journal of Veterinary Diagnostic Investigation, the Special issue on Applied Next-Generation Sequencing, includes a series of 10 papers that represent the application of NGS to veterinary testing. These range from the most common, genome sequencing, using PCR and viral culture for enrichment, to detect and/or subtype various viral pathogens,3,5,6,9,11 to less but increasingly common applications such as shotgun metagenomics for virus detection in clinical samples. 10 Deep amplicon sequencing is presented for targeted detection of multiple pathogens with a single NGS assay. 1 There is an example of an analysis pipeline for viral pathogens of pigs. 8 We thought it important to highlight applications in cancer treatment in addition to the infectious disease approaches. Characterization of canine osteosarcoma using single-cell sequencing is an excellent illustration. 2 Scattered within these papers are discussion of the ongoing challenges of NGS and approaches to validation of assays as well as discussions of future applications. Finally, a description of the NGS capacity within the National Animal Health Laboratory Network is presented. 7 This group, along with the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN), has provided critical NGS capacity support for the veterinary diagnostic community.
NGS is an exciting new tool that has many current applications. The decrease in equipment and reagent costs as well as multiplexing has made NGS more economically feasible in a veterinary diagnostic setting. However, the analysis component of NGS does require a skillset that must be further developed in the veterinary community. In addition, there is significant work to be done in the area of assay validation to get to the point where best practices can be developed such as was published for PCR in the November 2020 issue of JVDI.
We hope that this special issue is a taste of current applications of NGS in veterinary testing and look forward to novel applications as we strive for continuous improvement in the field. It is our hope that this issue will foster discussion between those contemplating uses of NGS and those with current experience thereby increasing the availability of NGS in veterinary diagnostic laboratories.