Metatranscriptomes from chicken host (oral and cloacal swabs) Transcriptome. Metatranscriptomes from chicken host (oral and cloacal swabs)

AbstractPCR-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 of 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).