基于稳定同位素拉曼定向筛选和多层次基因组学分析的微生物群落功能解析新方法研究

Metagenomics and single-cell genomics are among the most important approaches for functional dissection of microbial consortia. However, critical limitations still persist, such as the difficulty in measuring and identifying consortium phenotypes, the lack of links between organismal markers and functional markers, and the typically low resolution of functional characterization. To tackle these key challenges, here we propose to develop a novel method for functional dissection of microbiota based on Raman spectroscopic Stable-Isotope-Probing (Raman-SIP) and multi-layer genomics analysis. Firstly, stable isotope probing coupled with single-cell Raman spectroscopy would be employed to identify the cells of specific metabolic function within a microbial consortium, so as to formulate a practically operable foundation where the targeted metabolic function can be discriminated among the plethora of component cells at the single cell resolution. Secondly, a novel device that enables Raman-activated laser ejection and collection of targeted cells in an “all-in-one” integrated device will be developed, to sort either individual cells with targeted metabolic phenotype as described by their Raman signal or a small number of cells with identical or similar metabolic phenotype. The sorting of cells will be directly coupled to genome amplification and sequencing, so as to generate both the “single-cell” genome data and the “precision metagenome” data. Finally, the two layers of genomic data will be integrated with the “whole-consortium metagenome” data from the initial microbiota sample, so as to reconstruct the microbiota structure and functional genome that target a particular metabolic function and that are more complete and precise than conventional approaches. This project is expected to provide new, widely applicable methods and tools for resource mining and mechanistic dissection of the vast array of microbiota in nature.

宏基因组及单细胞基因组技术是当前进行微生物群落功能解析的重要研究手段，但其仍存在着表型表征困难、功能联系缺失、解析度低等诸多缺陷。针对这些瓶颈问题，本项目提出了一种基于稳定同位素拉曼单细胞定向筛选和多层次基因组学分析的新方法用于微生物功能群落的深度解析。首先，利用稳定同位素拉曼标记技术从微生物群落中快速筛选出具有特定代谢功能的细胞，建立单细胞精度、个体功能明确的分析体系；其次，发展细胞即时弹射技术和一体化集成装置，定向分选功能细胞个体以及具有同类功能的小型细胞群体并进行基因扩增及测序，从而分别获得功能定向的”单细胞基因组”数据及“精准宏基因组”数据；最后，通过整合包括“原始样本宏基因组”在内的三个层次组学数据进行分析重建，实现针对特定功能的、更为完整和准确的微生物功能基因组及群落结构解析。本项目将为海洋微生物及其他环境微生物资源的功能研究和机制解析提供共性的新方法和新工具。

宏基因组及单细胞基因组技术是当前进行微生物群落功能解析的重要研究手段，但其仍存在着表型表征困难、功能联系缺失、解析度低等诸多缺陷。针对这些问题，本项目提出并成功完成采用稳定同位素拉曼单细胞定向筛选和多层次基因组学分析的新方法对微生物特定功能群落进行深度解析的工作。首先，项目开发和优化了稳定同位素拉曼标记技术，用于微生物群落特定代谢功能单细胞及细胞群体的精确识别；其次，项目开发了多种基于单细胞拉曼表型的功能定向分选技术，并实现了对海洋光合固碳微生物、土壤菌群中细胞代谢活跃物种等环境微生物群落中特定功能表型细胞的分选；同时，项目开发和优化了基于多重置换扩增技术的单细胞及微群体细胞基因组扩增技术，并通过结合微液滴包裹及乳化扩增方式，大幅提升了微生物单细胞基因组的覆盖率（>90%）；最后，利用拉曼同位素标记及单细胞拉曼分选，项目完成了土壤菌群单细胞基因组学解析和海洋固碳微生物多层次基因组学解析，并对固碳分子机制进行了研究，首次发现SAR11类型海洋微生物原位利用二氧化碳的表型证据，为解答SAR11原位自养争议提供了新的实验证据。本项目的顺利完成，为复杂环境微生物群落的功能解析提供了新的实验工具和组学分析思路。