Supplementary Sequencing Data for Fresh Milk Timepoint Study

Raw bovine milk hosts a diverse microbiota that profoundly influences dairy product quality, safety, and shelf-life. However, current surveillance methods are time-intensive and often lack the taxonomic resolution needed for effective risk mitigation. To address this, we developed NOMAD (Nanopore-based On-site Microbiome Analysis of Diversity), a field-deployable workflow for rapid, high-resolution characterization of the raw milk microbiome using full-length 16S rRNA gene sequencing via Oxford Nanopore Technologies. Milk samples collected from a commercial dairy operation were processed using eight DNA extraction protocols, with Method 4—incorporating EDTA and TE buffer—emerging as the optimal approach for microbial richness and DNA yield. Sequencing was performed on a MinION Mk1B platform, and bioinformatic analyses revealed that a 4-hour run was sufficient to recover >90% of total community richness, with stable alpha and beta diversity metrics by this timepoint. The complete workflow, including DNA extraction, library preparation, sequencing, and analysis, was completed in 10.5 hours, enabling same-day microbiome profiling in farm-adjacent settings. Comparative analysis showed strong agreement with established milk microbiome studies, while full-length reads enhanced resolution of spoilage-associated taxa such as Pseudomonas spp. and Streptococcus spp. The NOMAD platform offers a powerful and practical tool for near real-time microbiological surveillance in the dairy industry, supporting proactive quality control and improved food safety outcomes.生鲜牛乳中栖息着多样化的微生物群落，其对乳制品的品质、安全性与货架期均具有深远影响。然而当前的微生物监测方法耗时耗力，且往往缺乏有效风险防控所需的分类学分辨率。为此，我们开发了NOMAD（Nanopore-based On-site Microbiome Analysis of Diversity，基于纳米孔的生鲜牛乳微生物组多样性现场分析）工作流，该方案可通过牛津纳米孔科技公司（Oxford Nanopore Technologies）的全长16S rRNA基因测序技术，实现生鲜牛乳微生物组的快速、高分辨率表征，且可现场部署。我们从某商业化乳制品运营场所采集牛乳样本，采用8种DNA提取方案进行处理，结果显示掺入乙二胺四乙酸（EDTA）与TE缓冲液的方案4在微生物丰度与DNA得率方面表现最优。测序在MinION Mk1B测序平台上完成，生物信息学分析显示，4小时的测序时长即可覆盖总群落丰度的90%以上，且该时间点下的α多样性与β多样性指标已趋于稳定。整套工作流涵盖DNA提取、文库制备、测序与数据分析，总耗时仅10.5小时，可在牧场周边环境中实现当日完成的微生物组分析。对比分析显示，本研究结果与已发表的牛乳微生物组研究结果高度一致；且全长读长序列提升了腐败相关分类群的分辨率，例如假单胞菌属（Pseudomonas spp.）与链球菌属（Streptococcus spp.）。NOMAD平台为乳制品行业的近实时微生物监测提供了一款高效实用的工具，可助力主动质量管控与食品安全水平的提升。