| Heterogeneity exists among cells.The behaviour and characteristic of individual cell,as well as the differences among different cells,play an important role in some essential biological progresses,such as cancer progresses,nerverous system and embryo developments.Single-cell analysis targeting at individual cells can reveal the differences of key properties from genomes,transcriptome,proteome and epigenenome,presenting great potential in study of disease progress,subtype classfication,lineage tracing and cell atlas.However,current single-cell analysis is limited to the poor cell capture,low cell utilization,low analysis throughput,unsatisfied sensitivity/accuracy,high cost and complex data integration.The development of microfluidics greatly improves the single-cell isolation and manipulation efficiency.It possesses nano-liter to pico-liter reaction volume which fits well to the volume of cells.Moreover,the tiny volume increases the original template concentration and reduces the reagent consumption.In the other hand,the progress of sequencing technology such as the appearance of the next generation sequencing significantly reduces the sequencing cost.Combined with the development of bioinformation technology,the single-cell omics analysis is accelerated.By integrating the highly efficient single-cell manipulation ability of microfluidics and high-throughput sequencing technology,this thesis proposes a series of new single-cell sequencing methods based on microfluidics and also extends them into different biological applications.It includes:1.Highly parallel single-cell transcriptome sequencing based on picoliter paired chamber(Paired-seq)By combining the highly efficient single-cell capture and manipulation chip with DNA barcoding beads,this system can conduct the simultaneous analysis of hundreds of single-cell transcriptome by only one sequencing,to achieve high-throughput single-cell heterogeneity analysis.Based on the least flow resistance and pump/valve controlled single-cell manipulation chip,this system can capture rare cells with high efficiency.By combining DNA barcoding technology,every cell and molecular can be labelled with unique cell label and molecular label,which helps to realize the simultaneous sequencing for a number of cells.The pump/valve controlled structure allows the active mixing,reagent exchange and cell washing in the tiny reaction system,which improves the cell lysis efficiency and removes the free RNAs.Moreover,the picoliter reaction volume largely improves the RNA template concentration so as to efficiently capture RNAs and conduct reverse transcription,which helps to enhance the sensitivity in gene detection.Compared to other single-cell sequencing platforms,Paired-seq can detect more genes in the same sequencing depth,allowing to reduce the cost significantly.Due to the advantages of high cell utilization,high sensitivity and low sequencing cost,Paired-seq presents great potential in single-cell heterogeneous analysis and the great ability of Paired-seq was further verified in study of stem cell differentiation and cancer cell resistance to drugs.2.High-throughput single-cell simultaneous transcriptome and proteome sequencing(multi-Paired-seq)This work presents a high-throughput cellular indexing of mRNAs and proteins platform,named multi-Paired-seq,by combining microfluidics,protein barcoding technology,barcoding beads technology and sequencing technology to transform transcriptome and protein analysis into efficient sequencing data readout.The protein barcoding and sequencing technology successfully transform the protein information into DNA information,allowing the great enhancement of throughput in protein analysis.Based on hydrodynamic differential flow resistance,this platform can achieve highly efficient single cell and single bead capture and pairing to improve cell utilization,in which>95%effective cell barcodes containing both mRNAs and proteins information were achieved.Combined with pump/valve structure,it can remove free DNA barcoded antibodies and cell-free mRNAs to reduce background interferences and to achieve highly accurate detection.Moreover,by developing single-cell transcriptome and protein co-measurement algorithm,the system can remove the interferences of non-specific absorption of proteins,to achieve accurate correlation information between mRNAs and proteins.By using multi-Paired-seq,different types of breast cancer cells are classified according to their mRNA and protein expressions.Also the work first explored the correlations between mRNAs and proteins among different cell types as well as those before and after drug treatment.The results witness that variations of phenotype are inconformity with those of mRNA molecules after drug treatment on a single-cell basis,which shows that multi-Paired-seq has great potential in co-measurement of mRNA and protein molecules for better study of gene expression rule,inter-omic correlations and cell heterogeneity.3.A highly automated single-cell RNA sequencing platform with digital microfluidics(DMF-RNA-seq)This work reports a digital microfluidics-based single-cell RNA sequencing(DMF-RNA-seq)for simple,efficient and low-cost single-cell mRNA measurements.DMF-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of single-cell RNA sequencing(scRNA-seq).To overcome the current problems of single-cell isolation in efficiency,integrity,selectivity and flexibility,this work proposes a new strategy,passive dispensing method,relying on well-designed hydrophilic-hydrophobic microfeatures to rapidly generate single-cell sub-droplets when a droplet of cell suspension is encountered.For sufficient cDNA generation and amplification,DMF-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 DMF-RNA-seq ensure highly accurate measurement.The whole reaction volume is 540nL,which saves 92-fold reagent consumption compared to traditional tube manipulation.This work demonstrates the functionality of DMF-RNA-seq by quantifying heterogeneity among single cells,where DMF-RNA-seq shows excellent performance in rare transcript detection,cell type differentiation and essential gene identification.With the advantages of automation,sensitivity,and accuracy,DMF-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.4.Automatic single-cell transcriptome and genome simultaneous sequencing platform(DMF-GTseq)This work proposes a digital microfluidics-based single-cell DNA and RNA simultaneous sequencing platform(DMF-GTseq).DMF-GTseq automates fluid handling as discrete droplets to sequentially perform protocols of single-cell isolation,cell lysis,DNA/RNA separation,DNA/RNA amplification,which promotes to achieve the genetics law between gene mutaions and gene expressions.By introducing well-designed hydrophilic-hydrophobic microfeatures,this system can rapidly generate single-cell sub-droplets when a droplet of cell suspension is encountered.By using low permeability lysis and magnetization,we can separate the RNA and DNA from single cells with high efficiency and reduce the nucleic interference.To improve the reaction efficiency and reduce reagent consumption,we construct nanoliter reaction system in the chip.Moreover,this system uses the hydrophobic interface to reduce the nucleic absorption and improve the library recovery,which helps to achieve highly sensitive single-cell transcrptome and genome measurements.The stable droplet handling and oil-closed reaction space featured in DMF-GTseq ensure highly accurate multi-omics measurement.By developing algorithms in bioinformatics,this system successfully associates the genomic mutations with gene expressions to investigate the genetic developments.The work further applys DMF-GTseq in study of single myeloma cell from the spinal cord and blood of patients,to investigate the correlations between gene mutation and gene expression,serving for the study of the developmental mechanism of myeloma. |
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