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Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry

Journal Article · · Journal of Veterinary Diagnostic Investigation
 [1];  [1];  [1]
  1. Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, Agricultural Research Service, USDA, Athens, GA, USA (Parris, Kariithi, Suarez), Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, Loresho, Nairobi, Kenya (Kariithi)
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PCR-based assays have become the benchmark for detecting pathogens of poultry and other livestock; however, these techniques are limited in their ability to detect multiple infecting agents, provide limited genetic information on the pathogen, and, for RNA viruses, must be reviewed frequently to assure high sensitivity and specificity. In contrast, untargeted, high-throughput sequencing can rapidly detect all infecting agents in a sample while providing genomic sequence information to allow more in-depth characterization of viruses. Although next-generation sequencing (NGS) offers many advantages, one of its primary limitations is low sensitivity to pathogens given the abundance of host and other non-target sequences in sequencing libraries. We explored methods for improving the sensitivity of NGS to detect respiratory and enteric viruses in poultry from RNA extracts of swab samples. We employed commercial and custom-designed negative enrichment strategies to selectively deplete the most abundant rRNA reads from the host and non-target bacteria; host RNA was diminished from up to 40% of total reads to as low as 3%, and the total number of reads assigned to abundant bacterial classes were reduced greatly. Our treatment resulted in up to a 700-fold increase in the number of viral reads, detection of a greater number of viral agents, and higher average genome coverage for pathogens. Depletion assays added only 2h to the NGS library preparation workflow. Custom depletion probe design offered significant cost savings (US$7–12 per sample) compared to commercial kits (US$30–50 per sample).

Research Organization:
Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
DOE Contract Number:
SC0014664
OSTI ID:
1983030
Journal Information:
Journal of Veterinary Diagnostic Investigation, Vol. 34, Issue 4; ISSN 1040-6387
Publisher:
SAGE
Country of Publication:
United States
Language:
English

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