Milk Sequencing Supplementary Data Methods Raw Reads (Part 2)

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.生牛乳（Raw bovine milk）定植有多样的微生物群落，其对乳制品的品质、安全性与货架期具有深远影响。然而当前的监测方法耗时冗长，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（基于纳米孔的现场微生物组多样性分析，Nanopore-based On-site Microbiome Analysis of Diversity）——一种可现场部署的分析流程，可借助牛津纳米孔科技公司（Oxford Nanopore Technologies）的全长16S rRNA基因测序技术，对生牛乳微生物组进行快速、高分辨率的表征。我们从某商业化乳品加工厂采集牛乳样本，采用8种DNA提取方案进行处理，其中整合乙二胺四乙酸（EDTA）与TE缓冲液（TE buffer）的方法4，在微生物丰富度与DNA得率方面表现最优。测序在MinION Mk1B平台上完成，生物信息学分析显示，4小时的测序时长即可回收超过90%的总群落丰富度，且该时间点下α多样性（alpha diversity）与β多样性（beta diversity）指标已趋于稳定。整套流程涵盖DNA提取、文库制备、测序与生物信息学分析，总耗时仅10.5小时，可在牧场周边环境实现当日完成的生牛乳微生物组谱分析。对比分析表明，该方法与已发表的牛乳微生物组研究结果具有高度一致性，且全长读长可提升对腐败相关类群的分类分辨率，如假单胞菌属（Pseudomonas spp.）与链球菌属（Streptococcus spp.）。NOMAD平台为乳品行业的近实时微生物监测提供了一款高效且实用的工具，有助于实现主动质量管控并改善食品安全水平。