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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Publications at this Location » Publication #384869

Research Project: Intervention Strategies to Predict, Prevent and Control Disease Outbreaks Caused by Emerging Strains of Virulent Newcastle Disease Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

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

item PARRIS, JOSHUA - Consultant
item KARIITHI, HENRY - Consultant
item Suarez, David

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2022
Publication Date: 7/5/2022
Citation: Parris, J.D., Kariithi, H., Suarez, D.L. 2022. Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry. Journal of Veterinary Diagnostic Investigation. 34(4):638-645.

Interpretive Summary: There have been continuing advances in being able to sequence bacteria and viruses faster and cheaper which has opened up new opportunities for diagnostic testing. These advances are often called next generation sequencing. Using random sequencing approaches, where you sequence everything in a sample, you have the potential to identify any pathogen in a sample. Unfortunately this approach often has a high percentage of the sequence produced are host sequence which reduces the sensitivity of the technique. Our approach is to selectively remove the most common host sequence which increases the percentage of sequences from everything else. We have seen an increase of 10 to 100 fold more viral sequences when using this new technique. This approach provides for the testing of clinical samples and identifying not only the pathogens in the sample, but provide detailed information on the pathogen that can aid in control of the pathogen.

Technical Abstract: PCR-based assays have become the benchmark for diagnosing 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 frequently reviewed 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. Although NGS for diagnostics offers many advantages, one of its primary limitations is low sensitivity to pathogens due to the abundance of host and other non-target sequence in sequencing libraries. In the work presented here, we explore 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. Treatment diminished host RNA from up to 40% of total reads to as low 3% and greatly reduced the total number of reads assigned to abundant bacterial classes. This resulted in up to a 700-fold increase in the number viral reads, detection of a greater number of viral agents, and higher average genome coverage for pathogens. Depletion assays added only 2 hours to the NGS library preparation workflow. Custom depletion probe design offered significant cost savings ($7-12 per sample) compared to commercially available kits ($30-50 per sample). The custom depletion strategy can be optimized for various hosts and sample types and inclusion of these enrichment steps can greatly improve sensitivity of NGS for diagnostic purposes.