FOCOT method: Low-cost microbial single-cell sequencing and sequencing.

In order to meet the common scientific needs of examining the "in-place function" of cells in nature, single-cell sequencing and sequencing of "on-site" and "real-time" has become an important development trend in the field of life science equipment development.
Although the third generation sequencing technology has miniaturized the instrument, the single-cell precision sorting equipment connected to sequencing is still quite bulky and expensive, and it is difficult to support on-site analysis of microbiome functions in various scientific expeditions.
Recently, a team of microflow control systems led by Ma Bo, a researcher at the Single Cell Research Center of the Qingdao Institute of Bioener energy and Process of the Chinese Academy of Sciences, achieved one cell per test tube (One-Cell-One-Tube) by designing a simple and efficient single-cell sequencing and sequencing docking device, which is expected to serve "on-site", "real-time" and even "portable" single-cell sequencing and sequencing.
Bibial cells are usually smaller (0.1-10 microns) and more difficult to manipulate precisely than human and higher animal and plant cells, so separating the "One-Cell-One-Tube" that obtains the target single cell and achieves the sequencing response requirements is a key difficulty.
Most of the current automated single-cell separation methods rely on expensive and bulky fluorescent flow cell sequencing instruments (FACS), while existing manual single-cell separation and sequencing schemes also require large and medium-sized instruments such as microcellular transfer platforms and laser photons that are equally difficult to carry with them, while relying on operator proficiency.
addition, the single-cell sequencing and nucleic acid preparation process is highly susceptible to contamination by floating microorganisms in the environment and their DNA, and this contamination is often difficult to remove completely during sequencing data processing.
, although the current MiniON and other third-generation sequencer has achieved portability, microbial single-cell sequencing and sequencing is still cumbersome operation, serious pollution interference, difficult to meet the requirements.
In response to these challenges, Qingdao Energy's single-cell center Zhang Qiang and Wang Wei have developed a method called "FOCOT" (short for Facile One-Cell-One-Tube) that isolates, acquires and separates individual microbial cells precisely, at high speed and at low cost, thus directly connecting to single-cell sequencing.
FOCOT method is as follows: First, through microflow control technology, cell dispersion is wrapped in dozens of micron diameter oil-packed water droplets; Third, single-cell encased droplets are distributed in a series of test tubes to quickly isolate individual cells, and each test tube has and has only one cell for direct docking with single-cell genome-wide amplification and sequencing.
FOCOT platform has three main features: First, in terms of simplicity and convenience, FOCOT platform in addition to self-designed chips, only need solenoid valves, tablets and ordinary optical microscopes, do not need to external any high-cost commoditized instrument platform, with easy access, easy replacement, low cost and other advantages.
At the same time, the modular, miniaturized and easy-to-use design makes the device suitable for carrying, assembling and using in the field sampling conditions of the natural environment, with little additional personnel training and technical maintenance, making it particularly suitable for scientific expeditions to a variety of extreme natural environments and conducive to the popularization and application in general laboratories.
Second, in terms of the efficiency of the selection, the FOCOT platform can effectively avoid false positives by accurately identifying and sub-selecting droplets encased in individual cells under a microscope, and its 20-second/individual splitting speed has obvious advantages over existing commercialized sub-selecting equipment such as microscopic single-cell fluid and laser photoretics.
At the same time, the yield of single cells is higher than 90%, and the success rate of culture is at least 80%, which proves that the method can effectively avoid cell loss during the transport process caused by surface adsorption of the chip, and has no or less damage to cell activity.
Third, in terms of pollution control, FOCOT platform involves fewer components, small size, relatively closed, so in the experimental process can easily achieve ultra-clean environmental space control, chip disinfection and other operations, strictly control environmental DNA pollution.
The results of genome-wide amplification and sequencing of isolated individual yeast cells showed that 99% of the effective sequences could be matched with the reference genome, indicating that the method could effectively avoid DNA contamination caused by microorganisms in the environment, with an average genome coverage of 43.3%, comparable to the sequencing results obtained by obtaining single cells using large instrument systems such as FACS in expensive ultra-clean space facilities.
Currently, through the coupling of FOCOT and the center's pre-developed single-cell Raman imaging, Raman flow cell selection and other technologies, single-cell center is building a non-labeled single-cell analysis equipment system serving the different needs of shore-based, ship-based and even hand-based, in order to serve energy, environment, health, ocean, soil and many other microbiome applications.
related research papers are published in Scientific Reports.
research work was co-chaired by Ma Bo and Xu Jian of the Single Cell Center, and was supported by projects such as the National Fund Committee's Basic Research Project on Scientific Instruments, face projects and the Chinese Academy of Sciences Bio-High-Volume Analysis Technical Service Network (STS).
paper information: Development of a facile droplet-single-cell isolation platform for developmenton and genomic analysis in microorganisms. Sci Rep, 7:41192, DOI:10.1038/srep41192.
.