下一代IgA-Seq允许对IgA涂层细菌进行高通量，厌氧和核能评估

肠道菌群在维持全身和肠内稳态中起着重要作用，但也会影响诸如炎症性肠病（IBD）和癌症之类的疾病。肠道内的某些细菌物种可以长期激活免疫系统，从而导致低度肠炎。结果，浆细胞产生高水平的分泌抗原特异性免疫球蛋白A（IGA），该免疫球蛋白A（IgA）覆盖了免疫刺激细菌。这种针对肠道细菌的IgA免疫反应可能与维持体内平衡和健康以及疾病有关。阐明这种二分法并确定免疫刺激细菌对于理解肠道菌群与免疫系统之间的关系以及它们在健康和疾病中的作用至关重要。 IgA-Seq技术已成功鉴定出粪便材料中的免疫刺激性，IgA涂层的细菌。但是，原始技术是耗时的，并且下游应用程序有限。 In this study, we aimed to develop a next-generation, high-throughput, magnet-based sorting approach (ng-IgA-SEQ) to overcome the limitations of the original IgA-SEQ protocol.We show, in various settings of complexity ranging from simple bacterial mixtures to human fecal samples, that our magnetic 96-well plate-based ng-IgA-SEQ protocol is highly efficient at sorting and identifying IgA-coated细菌以高通量和时间效率的方式。此外，我们在不同的IgA-Seq方案之间进行了比较分析，强调了原始的基于FACS的IgA-Seq方法忽略了IGA涂层细菌的某些细微差别，这是由于分类细菌的产量较低。此外，基于磁性的NG-IGA-Seq允许新的下游应用。首先，作为概念验证，我们对10个人类粪便样品进行了元基因组shot弹枪测序，以鉴定涂有IGA的细菌菌株以及相关的途径和cazymes。其次，我们通过在厌氧条件下执行隔离方案成功地隔离和培养的IgA涂层细菌。我们的磁性96孔板的高通量下一代IgA-Seq技术有效地识别了来自粪便中的大量IGA涂抹细菌。这为分析大型队列以及新颖的下游应用（包括shot弹枪元基因组测序，培养基组学和各种功能测定法）铺平了道路。这些下游应用对于揭示免疫刺激细菌在健康和疾病中的作用至关重要。视频摘要。

The intestinal microbiota plays a significant role in maintaining systemic and intestinal homeostasis, but can also influence diseases such as inflammatory bowel disease (IBD) and cancer. Certain bacterial species within the intestinal tract can chronically activate the immune system, leading to low-grade intestinal inflammation. As a result, plasma cells produce high levels of secretory antigen-specific immunoglobulin A (IgA), which coats the immunostimulatory bacteria. This IgA immune response against intestinal bacteria may be associated with the maintenance of homeostasis and health, as well as disease. Unraveling this dichotomy and identifying the immunostimulatory bacteria is crucial for understanding the relationship between the intestinal microbiota and the immune system, and their role in health and disease. IgA-SEQ technology has successfully identified immunostimulatory, IgA-coated bacteria from fecal material. However, the original technology is time-consuming and has limited downstream applications. In this study, we aimed to develop a next-generation, high-throughput, magnet-based sorting approach (ng-IgA-SEQ) to overcome the limitations of the original IgA-SEQ protocol.We show, in various settings of complexity ranging from simple bacterial mixtures to human fecal samples, that our magnetic 96-well plate-based ng-IgA-SEQ protocol is highly efficient at sorting and identifying IgA-coated bacteria in a high-throughput and time efficient manner. Furthermore, we performed a comparative analysis between different IgA-SEQ protocols, highlighting that the original FACS-based IgA-SEQ approach overlooks certain nuances of IgA-coated bacteria, due to the low yield of sorted bacteria. Additionally, magnetic-based ng-IgA-SEQ allows for novel downstream applications. Firstly, as a proof-of-concept, we performed metagenomic shotgun sequencing on 10 human fecal samples to identify IgA-coated bacterial strains and associated pathways and CAZymes. Secondly, we successfully isolated and cultured IgA-coated bacteria by performing the isolation protocol under anaerobic conditions.Our magnetic 96-well plate-based high-throughput next-generation IgA-SEQ technology efficiently identifies a great number of IgA-coated bacteria from fecal samples. This paves the way for analyzing large cohorts as well as novel downstream applications, including shotgun metagenomic sequencing, culturomics, and various functional assays. These downstream applications are essential to unravel the role of immunostimulatory bacteria in health and disease. Video Abstract.