Improved Resolution of Highly Pathogenic Avian Influenza Virus Haemagglutinin Cleavage Site Using Oxford Nanopore R10 Sequencing Chemistry. Influenza A virus

Highly pathogenic avian influenza viruses continue to pose risks to "One Health" including agriculture and public and animal health globally. Rapid and accurate genomic surveillance is critical for monitoring viral mutations, tracing transmission, and guiding interventions in near real time. Oxford Nanopore sequencing holds promise for real-time influenza genotyping, but data quality from R9 chemistry has limited its adoption due to challenges resolving low-complexity regions such as the biologically critical hemagglutinin cleavage site, a homopolymer of basic amino acids that distinguish highly pathogenic strains. In this study, human and avian influenza isolates (n=45) from Cambodia were sequenced using both R9.4.1 and R10.4.1 flow cells and chemistries to evaluate performance between approaches. Overall, R10.4.1 yielded increased data output with higher average quality compared to R9.4.1, producing improved consensus sequences using a reference-based bioinformatics approach. R10.4.1 had significantly lower minor population insertion and deletion frequencies, driven by improved performance in low sequence complexity regions prone to insertion and deletion errors, such as homopolymers. Within the hemagglutinin cleavage site, R10.4.1 resolved the correct motif in 90% of genomes compared to only 60% with R9.4.1. Further examination showed reduced frameshift mutations in consensus sequences generated with R10.4.1 that could result in incorrectly classified virulence. Improved consensus genome quality from nanopore sequencing approaches, especially across biologically important low-complexity regions, is critical to reduce subjective hand-curation and will improve local and global genomic surveillance responses.

高致病性禽流感病毒仍对全球范围内涉及农业、公共卫生与动物健康的同一健康（"One Health"）体系构成持续威胁。快速精准的基因组监测对于近实时监测病毒变异、追踪传播路径以及指导干预措施至关重要。牛津纳米孔（Oxford Nanopore）测序技术在流感病毒实时基因分型方面展现出应用前景，但受限于R9测序化学试剂的数据质量短板，其推广应用曾遭遇瓶颈——这一短板源于该技术难以解析低复杂度区域，例如具备关键生物学功能的血凝素（hemagglutinin）切割位点：该位点是一段由碱性氨基酸构成的均聚物（homopolymer），可作为区分高致病性毒株的标志。本研究针对来自柬埔寨的45株人类与禽流感分离株（n=45），分别采用R9.4.1与R10.4.1测序流通池（flow cell）及配套化学试剂开展测序，以对比两种测序方案的性能差异。总体而言，相较于R9.4.1，R10.4.1的测序数据产出量更高、平均质量更优，通过基于参考序列的生物信息学分析流程可获得质量更优异的一致序列。R10.4.1的次要变异群体插入缺失频率显著更低，这得益于其在易发生插入缺失错误的低序列复杂度区域（如均聚物区域）的性能优化。在血凝素切割位点区域，R10.4.1可在90%的基因组样本中解析出正确的基序（motif），而R9.4.1的解析正确率仅为60%。进一步分析显示，采用R10.4.1生成的一致序列中移码突变比例更低，此类突变曾可能导致病毒毒力分类出现错误。纳米孔测序技术所实现的一致基因组质量提升，尤其是在具备重要生物学意义的低复杂度区域的质量优化，对于减少人工手动校正的主观性至关重要，同时也将提升本地与全球的基因组监测响应效能。