Wiegand Sandra, Dam Hang T, Riba Julian, Vollmers John, Kaster Anne-Kristin
Institute for Biological Interfaces 5, Karlsruhe Institute of Technology, Karlsruhe, Germany.
Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.
Front Microbiol. 2021 Jun 16;12:635506. doi: 10.3389/fmicb.2021.635506. eCollection 2021.
As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as "microbial dark matter." In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique-out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages-in whole or in part-have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.
截至目前,大多数环境微生物仍未被培养。因此,它们被称为“微生物暗物质”。最近,诸如单细胞基因组学(SCG)等非培养方法使得发现了许多以前未知的微生物,其中包括Patescibacteria/候选门辐射类(CPR)。这种方法已被证明是对宏基因组学的补充,然而,除了最常用的荧光激活细胞分选(FACS)之外,开发更多精细的分选技术仍然是可取的,以便实现更多的下游应用。添加关于分选细胞数量和形态的图像信息将是有益的,尽量减少诸如染色或压力等分选条件所引起的细胞应激也会很有帮助。最近,一种新型细胞分选技术——微流控单细胞分配器已被开发出来,它通过自动光学显微镜处理来评估每个液滴中细胞的数量和形态。在此,我们首次报告了新开发的单细胞分配系统成功应用于从污水处理厂采集的复杂样本中无标记分离单个细菌,证明了该技术在单细胞基因组学和其他替代下游应用方面的潜力。使用该技术,基因组回收成功率超过80%——在880个分选细胞中,717个成功扩增。其中50.1%的细胞,16S rRNA基因分析可行,导致对50个被鉴定为Patescibacteria/CPR成员的分选细胞进行了测序。随后,可捕获并重建了15个新的、不同的Patescibacteria/CPR成员的27个单扩增基因组(SAGs),代表了尚未见过的物种、属和科。从可用的宏基因组组装基因组(MAGs)中回收的SAGs的这种系统发育独特性伴随着这样一个发现,即这些谱系——全部或部分——尚未通过同一样本的基因组解析宏基因组学得以获取,从而强调了单细胞基因组学的重要性和机遇。