Long-term viral archives are valuable sources of research data. Each archive can store hundreds of thousands of diverse sample types. In the current era of whole genome sequencing, archived samples become a rich source of evolutionary and epidemiological data that can span years, and even decades. However, the ability to obtain high quality viral whole genome sequences from samples of various types, age, and quality is inconsistent. A minimum quality threshold that helps predict the best success of obtaining high quality genomic sequences for both recent and archived samples is highly valuable. Real-time reverse transcription PCR (rrt-PCR) and droplet digital PCR (ddPCR) are useful tools to evaluate nucleic acid integrity. We hypothesized that diagnostic rrt-PCR and ddPCR data for avian influenza virus (AIV) can predict viral whole genome sequencing success. To test this hypothesis we used RNA extracted from cloacal and oropharyngeal swabs stored in the USDA-APHIS National Wildlife Disease Program Wildlife Tissue Archive. We determined that a specific rrt-PCR Cq value or ddPCR copies/μL resulted in recovery of complete sequences of all eight AIV gene segments. We used logistic regression to estimate probabilities of whole genome recovery at 0.95 (Cq = 15, copies/μL = 49,350), 0.75 (Cq = 24, copies/μL = 16,800), 0.50 (Cq = 29, copies/μL = <1), and 0.25 (Cq = 235, copies/μL = <1). We also identified values at which we predictably recovered HA and NA segments for diagnosing subtypes (Cq = 27.29; copies/μL = 757.50). This approach will allow researchers to assess the potential success of AIV whole genome recovery from diagnostic samples collected in routine AIV surveillance.

中文翻译：

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使用实时和液滴PCR预测归档的禽流感病毒（Orthomyxoviridae）样品的全基因组测序成功。

长期病毒档案是研究数据的宝贵来源。每个档案库可以存储数十万种不同的样本类型。在当前的全基因组测序时代，存档的样本成为了可能长达数年甚至数十年的进化和流行病学数据的丰富来源。但是，从各种类型，年龄和质量的样品中获得高质量的病毒全基因组序列的能力是不一致的。有助于预测获得最新样本和存档样本的高质量基因组序列的最佳成功的最低质量阈值非常有价值。实时逆转录PCR（rrt-PCR）和液滴数字PCR（ddPCR）是评估核酸完整性的有用工具。我们假设禽流感病毒（AIV）的诊断性rrt-PCR和ddPCR数据可以预测病毒全基因组测序成功。为了检验这一假设，我们使用了从USDA-APHIS国家野生动物疾病计划野生动物组织档案库中存储的泄殖腔和口咽拭子提取的RNA。我们确定特定的rrt-PCR Cq值或ddPCR拷贝/μL导致所有八个AIV基因片段的完整序列恢复。我们使用logistic回归估算了全基因组恢复的概率为0.95（Cq = 15，拷贝/μL= 49,350），0.75（Cq = 24，拷贝/μL= 16,800），0.50（Cq = 29，拷贝/μL= <1 ）和0.25（Cq = 235，拷贝数/μL= <1）。我们还确定了可预测地恢复用于诊断亚型的HA和NA片段的值（Cq = 27.29；拷贝/μL= 757.50）。