Single-cell Sequencing Based On Digital Microfluidics

Single-cell sequencing is a technique for single-cell analysis by high-throughput sequencing,which can resolve cellular networks and intracellular molecular regulatory patterns,providing a discriminative analytical perspective in the single-cell level for our understanding of the cellular networks of life.Microfluidics affords the integrated workflow of complex single-cell processing on a chip,which is the main tool for singlecell sequencing currently.However,most microfluidic systems for single-cell sequencing are limited by complex structures and equipments,inefficient single-cell isolation,highly skilled requirements and limited integration and scalability,making them difficult to be widely applied.Digital microfluidics is a novel discrete droplethandling technology based on electrowetting-on-dielectric,which does not rely on complex microstructures,such as pumps,valves,and microfluidic channels.Digital microfluidics features automation,easy compatibility,scalability and dynamic configuration,which is particularly suitable for the highly integrated,high-performance and complex system of single-cell sequencing.Based on this,we developed some new methods for single-cell sequencing with digital microfluidics:(1)Construction of the digital microfluidic platform for droplet manipulationCurrent digital microfluidic platforms are difficult to keep the balance of high stability,low voltage,operability and scalability,which cannot adapt to most complex biochemical systems.We developed an integrated digital microfluidic system coupled with the corresponding fabricating workflow of the digital microfluidic chip.Based on the homemade digital microfluidic system,droplet dispensing and splitting are well homogeneous;droplet mixing is quickly and efficiently;droplet washing is sufficiently against the wetting structure.This provides an excellent platform for high-performance single-cell sample preparation.(2)Construction of the single-cell digital microfluidic platformHigh throughput,selectivity,low damage and addressable single cell isolation and manipulation are key challenges in the field of single cell sequencing.Currently,methods for single-cell isolation suffer from complex operation,low efficiency and high loss.This chapter develops an efficient single-cell processing platform based on digital microfluidics.The platform consists of two single-cell isolation systems,a hydrophilic structure based on local-wetting properties and a micro-capture structure combined hydrodynamic principles with local-wetting properties.The hydrophilic structure can achieve efficient single cell capture by the dynamical control,while the micro-capture structure can achieve fully automated 100%single-cell capture.By characterising capture efficiency,structural versatility,applicability,scalability and robustness of single cells based on the two isolation structures,the platform is proved to be a solid foundation for subsequent single-cell sequencing.(3)Single cell genome sequencing based on digital microfluidicsSingle-cell whole-genome sequencing(WGS)is critical for characterizing dynamic intercellular changes in DNA.Current sample preparation technologies for single cells are operationally complex,expensive,and suffer from high coverage bias despite recent advances,lagging behind in the impressive progress of novel sequencing technologies.Here we describe Digital-WGS,an automated and efficient single-cell sample preparation platform for whole-genome amplification(WGA)based on digital microfluidics.Digital-WGS enables streamlined parallel nanoliter-volume single-cell WGA with automatic processing from high-efficiency single-cell isolation to excellent amplification performance.We show that Digital-WGS provides high single cell capture efficiency(100%)for any amount and types of cells.Digital-WGS achieves up to 88.7%coverage of a single-cell genome at 10× sequencing depth,and enables simultaneous detection of copy number variants(CNVs)with smallest bin of 150 kb and single-nucleotide variants(SNVs)with alleles drop out(ADO)rate of 5.2%in an individual human cell.As such,the method provides an efficient and robust method for performing single-cell sequencing,which overcomes current technical barriers,holding great promise for broader applications for single cell genomics.(4)Single cell transcriptome sequencing based on digital microfluidicsSingle-cell RNA sequencing(scRNA-seq)is a powerful method in investigating single-cell heterogeneity to reveal rare cells,identify cell subpopulations and construct a cell atlas.Conventional bench-top methods for scRNA-seq including multistep operations,are labor intensive,reaction inefficient,and reagent consuming.Here we report a digital microfluidics-based single-cell RNA sequencing(Digital-RNA-seq)for simple,efficient and low-cost single-cell mRNA measurements.Digital-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of scRNA-seq.To overcome the current problems of single-cell isolation in scRNA-seq,we used droplet-based wettability trapping to rapidly generate single-cell subdroplets.For sufficient cDNA generation and amplification,Digital-RNA-seq uses nanoliter reaction volumes and hydrophobic reaction interfaces,achieving high sensitivity in gene detection.Additionally,the stable droplet handling and oil-closed reaction space featured in Digital-RNA-seq ensure highly accurate measurement.We demonstrate the functionality of Digital-RNA-seq by quantifying heterogeneity among single cells,where Digital-RNA-seq shows excellent performance in rare transcript detection,cell type differentiation and essential gene identification.With the advantages of automation,sensitivity,and accuracy,Digital-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.(5)A digital microfluidic mass spectrometry platform for single cell genotype profilingGenotype profiling of heterogeneous circulating tumor cells(CTCs)offers comprehensive and real-time molecular information of tumors for targeted therapy guidance,but the lack of efficient and multiplex genotyping techniques for single CTC analysis greatly hinders its development and clinical application.This paper reports a single-CTC mass spectrometry analysis platform for efficient and multiplex genotype profiling based on digital microfluidics(DMF-scMS).Digital microfluidics affords integrated single-CTC manipulation,from selective cell isolation to high-performance whole genome amplification,via nanoliter droplet-based wettability trapping and hydrodynamic adjustment of cell distribution.Coupled with matrix-assisted laser desorption/ionization tim e-of-flight mass spectrometry,the multiplex mutation information of individual CTCs can be efficiently and accurately identified by the inherent mass differences of different DNA sequences.This platform achieved KRAS mutation profiling of heterogeneous CTCs at single-cell level from cancer patient samples,offering new avenues for genotype profiling of single CTCs and cancer therapy guidance.