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).
J Vet Diagn Invest. 2022 Jul;34(4):638-645. doi: 10.1177/10406387221102430.
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 2 h 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).
基于 PCR 的检测方法已成为检测家禽和其他家畜病原体的基准方法；然而，这些技术在检测多种感染因子的能力、提供病原体的有限遗传信息以及对于 RNA 病毒，必须经常审查以确保高灵敏度和特异性方面存在局限性。相比之下，非靶向、高通量测序可以快速检测样品中的所有感染因子，同时提供基因组序列信息，以允许对病毒进行更深入的特征分析。尽管下一代测序 (NGS) 具有许多优势，但它的一个主要局限性是病原体的灵敏度较低，因为在测序文库中存在大量宿主和其他非目标序列。我们探索了从拭子样本的 RNA 提取物中提高 NGS 检测家禽呼吸道和肠道病毒的灵敏度的方法。我们采用商业和定制设计的负富集策略，有选择地从宿主和非目标细菌中去除最丰富的 rRNA 读数；从总读数中减少了高达 40%的宿主 RNA，降至低至 3%，并大大减少了分配给丰富细菌类别的总读数数量。我们的处理方法使病毒读数增加了高达 700 倍，检测到更多的病毒因子，并且病原体的平均基因组覆盖率更高。消耗检测仅在 NGS 文库制备工作流程中增加了 2 小时。与商业试剂盒（每个样本 30-50 美元）相比，定制的消耗探针设计可显著节省成本（每个样本 7-12 美元）。
