Environmental flora single-cell RACS-Seq technology comes out

Environmental flora single cell RACS-Seq technology comes out

Environmental flora single-cell RACS-Seq technology comes out Environmental flora single-cell RACS-Seq technology comes out

Raman analysis with accuracy to one cell—sorting—sequencing ( Photo courtesy of Qingdao Institute of Energy, Chinese Academy of Sciences )

Chinese Academy of Sciences

Single cell Raman sorting coupled sequencing (RACS-Seq) is an important means to analyze the functional mechanism of environmental flora, but the coverage of the single bacterial cell genome after Raman sorting is usually less than 10%, which greatly limits its application
.

Recently, the Single Cell Center of Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences, based on the self-developed RAGE (Gravity-Driven Single Cell Droplet Wrapped Raman Sorting) technology, instruments and corresponding kits, for the first time achieved accuracy to one bacterial cell, complete The environmental microbiota scRACS-Seq, with a genome coverage of more than 92%, provides a powerful new tool for the study of in situ metabolic functions of the environmental microbiome
.

Soil is one of the most important ecosystems on the earth.
The metabolic activities of the soil microbiome support agriculture and animal husbandry, and also play a key role in the earth's element cycle and global climate change
.

At the same time, soil flora is also one of the most diverse and complex microbiomes on the planet.

However, scRACS-Seq for environmental flora has always had two major bottlenecks.
One is that it is difficult to obtain single cells with a specific Raman phenotype without damage and quickly; the other is that it is difficult to obtain single-cell genome data with high coverage
.

This has become a key bottleneck for the widespread application of the scRACS-Seq technology system in complex flora

In response to this common difficulty, a joint research team composed of Jing Xiaoyan, Gong Yanhai, and Xu Teng from the Single Cell Center of the Institute, based on the RAGE-Seq technology invented in the earlier stage, analyzed the soil fed with stable isotope substrates from liquid phase Raman.
Starting from the flora, the bacterial single cells of a specific Raman phenotype are accurately separated and packaged in the skin upgrade droplets, which are then coupled with downstream genome sequencing
.

The results of the study show that low-abundance species with active cell metabolism in the soil flora can be accurately identified and sorted by RAGE-Seq coupled with heavy water feeding and labeling, and its single-cell genome coverage is as high as 93%; similarly, based on RAGE -Seq, soil microbial cells containing carotenoids can realize the metabolic reconstruction of single cell resolution and high genome coverage, so as to fully and deeply explore their carotenoid synthesis pathways; these "in-situ" carotenoid synthesis soils Among the microbial cells, both metabolically active and a considerable part are inert, indicating that the strategy based on pure culture is bound to miss these metabolically inert functional microorganisms.
Therefore, "in-situ", single-cell precision functional cell identification and separation is essential for comprehensive , Objective microbial function analysis and resource mining are of great significance
.

In addition, this work also established a systematic and strict scRACS-Seq quality evaluation and control system through artificial flora whose composition and state are precisely controllable
.

Based on this system, the team found that the technology can quickly and accurately separate bacterial single cells of different Raman phenotypes from the flora, while ensuring the quality of single-cell Raman spectra, the sorting accuracy can reach 100%
.

In addition, using empty droplets from the aqueous phase around the target cells as a negative control, it was found that the probability of the target cell sequence being contaminated with DNA from other cells in the flora is extremely low

The reporter learned that, as a new member of the RACS technology family, scRACS-Seq can establish a link between metabolism and genome in a complex flora at the resolution of a single microbial cell, thereby accurately answering "who is doing what and why"
.

On this basis, the Single Cell Center has developed and industrialized a single cell Raman sorting-sequencing coupling system (RACS-Seq instrument), as well as corresponding RAGE chips and single cell analysis kits
.

The kit includes applications for the extraction and preparation of microbial single cells in environmental samples, stable isotope feeding of cells, and single-cell nucleic acid lysis and amplification

The work won the China Ke Xueyuan - Support "function and regulation of soil microbial system" pilot projects, the National Natural Science Foundation of China, Qingdao Star Game Biotechnology Co.
, Ltd.
, etc.

.

He won the China Kexue Yuan - Support "function and regulation of soil microbial system" pilot projects, the National Natural Science Foundation of China, Qingdao Star Game Biotechnology Co.
, Ltd.
, etc.

.

Related paper information: https://journals.

https://journals.
asm.
org/doi/10.
1128/mSystems.
00181-21