单细胞测序技术在微生物生态领域中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2020-0335

摘要/Abstract

摘要：

单细胞测序技术是指在单细胞水平上对核酸分子进行测序的技术。近年来,单细胞测序技术异军突起,成为分子生物学研究的热点,在医学、生物化学、生命科学等领域取得了卓越的成果,也成为了单细胞生态学的重要组成部分。单细胞测序与扩增子或宏基因组技术结合可以更准确地识别微生物物种,探究种群异质性,进一步研究特定物种的功能,获得稀有物种的完整基因组。简述了单细胞测序的产生及发展历程,重点介绍了细胞分离和基因组扩增的前沿技术,并详细论述了单细胞测序技术在微生物生态领域中的应用。

关键词: 单细胞测序, 单细胞基因组, 环境微生物, 种群异质性, 单细胞生态学

Abstract:

Single-cell sequencing technology,allowing nucleic acid molecules sequenced at the level of single cell,has become a hot spot in molecular biology,and from it there are remarkable achievements in medicine,biochemistry,life science,thus it become an important part of single-cell ecology. The combination of single-cell sequencing and amplicon or metagenomic techniques can more accurately identify microbial species,explore population heterogeneity,intensively study the function of specific species,and obtain the complete genome of rare species. Here,we briefly review the generation and development process of single-cell sequencing,focus on introducing the new technologies of cell isolation and genome amplification,and illustrate the application of single-cell sequencing in microbial ecology.

Key words: single-cell sequencing, single-cell genomics, environmental microbiology, population heterogeneity, single-cell ecology

王丹蕊, 沈文丽, 魏子艳, 王尚, 邓晔. 单细胞测序技术在微生物生态领域中的应用[J]. 生物技术通报, 2020, 36(10): 237-246.

WANG Dan-rui, SHEN Wen-li, WEI Zi-yan, WANG Shang, DENG Ye. Applications of Single-cell Sequencing Technology in Microbial Ecology[J]. Biotechnology Bulletin, 2020, 36(10): 237-246.

图/表 2

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细胞分离技术	技术简介	优点	局限性	应用案例
有限稀释法	培养液稀释至约每0.1mL含有1个菌或细胞,根据泊松分布计算	简单廉价	准确性差,不具有针对性	对HIV-1病毒的全长序列进行测序^[23]
显微操作法	机械显微操作,用毛细管从各类样品中捕获单个细胞	对细胞进行可视性评估	通量极低,易对细胞造成机械损伤	分离并检测食物中的致病菌^[24]
激光捕获显微分离技术	将细胞进行固定和显色处理后直接分离	显色标记与分离结合,可分离复杂基质中的单细胞	通量低,易引入杂质或导致遗传信息丢失	研究动植物宿主-微生物的相互作用,鉴定未培养细菌,对单核原核细胞进行分析^[25]
拉曼镊子	通过拉曼显微镜区分生化特性不同的细胞,再用激光将其捕获	不需标记处理	只能分离生化特性显著不同的细胞	结合拉曼光镊和单细胞芯片分离癌细胞、红细胞、淋巴细胞和大肠杆菌^[26]
涡旋与相分隔	通过高速涡旋形成油包水体系	通量高,效率高,成本低	难以保证过程的精确性	用于探究硫酸盐还原功能类群^[20]
荧光细胞分选技术	基于所需细胞属性,结合多参数对特定细胞进行检测分离,同时评估生理学和分类学特性	灵敏度高,自动化程度高,可依据大小、粒度、荧光等属性分选	通量中等,成本高	分离丝状真菌并进行分类^[27]
微流控技术	通过微流控芯片原件等设备包裹和分离单细胞样品	通量高,精确稳定	需要特定仪器装备,开发周期较长	单细胞全基因组测序及抗性基因分析^[21]

细胞分离技术	技术简介	优点	局限性	应用案例
有限稀释法	培养液稀释至约每0.1mL含有1个菌或细胞,根据泊松分布计算	简单廉价	准确性差,不具有针对性	对HIV-1病毒的全长序列进行测序^[23]
显微操作法	机械显微操作,用毛细管从各类样品中捕获单个细胞	对细胞进行可视性评估	通量极低,易对细胞造成机械损伤	分离并检测食物中的致病菌^[24]
激光捕获显微分离技术	将细胞进行固定和显色处理后直接分离	显色标记与分离结合,可分离复杂基质中的单细胞	通量低,易引入杂质或导致遗传信息丢失	研究动植物宿主-微生物的相互作用,鉴定未培养细菌,对单核原核细胞进行分析^[25]
拉曼镊子	通过拉曼显微镜区分生化特性不同的细胞,再用激光将其捕获	不需标记处理	只能分离生化特性显著不同的细胞	结合拉曼光镊和单细胞芯片分离癌细胞、红细胞、淋巴细胞和大肠杆菌^[26]
涡旋与相分隔	通过高速涡旋形成油包水体系	通量高,效率高,成本低	难以保证过程的精确性	用于探究硫酸盐还原功能类群^[20]
荧光细胞分选技术	基于所需细胞属性,结合多参数对特定细胞进行检测分离,同时评估生理学和分类学特性	灵敏度高,自动化程度高,可依据大小、粒度、荧光等属性分选	通量中等,成本高	分离丝状真菌并进行分类^[27]
微流控技术	通过微流控芯片原件等设备包裹和分离单细胞样品	通量高,精确稳定	需要特定仪器装备,开发周期较长	单细胞全基因组测序及抗性基因分析^[21]