Single-cell Mitochondrial Genome Polymorphism Research Based On Agarose Gel Microdroplet

Through extensive application and exploration,people gradually realize that mitochondrial DNA(mtDNA)heterogeneity not only found in populations and individuals,but also in tissues,intercellular and intracellular.As the non-coding region of mtDNA,D-loop is the regulatory hub of mtDNA replication and transcription.Its hypermutability has been proved to be seriously related to many diseases.Compared with bulk sequencing,single-cell sequencing data can restore the low-frequency mutation information not observed in bulk sequencing and accurately count through cell barcode.Droplet microfluidic can generate isolated droplets in the form of single-cell,then high-throughput single-cell libraries are constructed.However,the existing droplet-based cell barcoding and library construction are cumbersome,requiring multi-step droplet manipulation.This may superimpose droplet demulsification,decrease the effective droplet ratio and increase intercellular cross-coding.And the sequence sampling of mtDNA based on the transposase-accessible also has the inhomogeneous phenomenon.According to the above problem,the advantage of agarose gel in droplet manipulation was utilized in this theise.The D-loop region amplification primer with barcode sequence was generated by PCR pre-amplification,which was used to amplify the D-loop region of single-cell mtDNA and simultaneously realize large-scale single-cell barcoding.A novel single-cell D-loop heterogeneity detection technique with few manipulation steps,high cell barcoding capacity and controllable cost was developed.1.Construction of droplet microfluidic platform and preparation of monoclonal multi-copy barcode dropletsThe amplification primers for the D-loop region containing the 4+12N elongated barcode sequence were designed.The single-molecule amplification primer was used as template to perform the first round of ddPCR in droplets.Thereby,single-cell barcoding droplets containing monoclonal multi-copy amplification primers were obtained.In the preparation of barcode droplets,the stability of the droplets during PCR was greatly improved by of 1%Tween-20 and 5%008-FluoroSurfactant FC-40.The average diameter of droplet before PCR was 22.92 μm,the coefficient of variation was 3.69%;the average diameter of droplet after PCR was 23.42 m,the coefficient of variation was 4.58%.The ratio of positive droplets(containing monoclonal multi-copy amplification primers)to negative droplets(not containing monoclonal multicopy amplification primers)was as expected.The electrophoretic bands were correct after demulsification.2.Generation of single-cell agarose gel microdroplet,"one-to-one" pairing and fusionSingle cell was encapsulated in agarose gel microdroplet and fused with the single-cell barcoding droplet containing monoclonal multi-copy amplification primers by "one-to-one" pairing.Low melting point agarose gel was used as the solid medium for cells in droplets.K562 cell line as the research object.A "slender" laminar flow chip was specially designed to assist the generation of single-cell agarose gel microdroplet.The single microsphere encapsulation rate of laminar flow chip was increased by 121.3%and the multi-microsphere encapsulation rate was reduced by 64.6%compared with that of ordinary flow focusing chip under the same input,achieving super-Poisson distribution.On this basis,single-cell agarose gel microdroplets were generated.The average diameter of the droplets was 29.65 μm and the coefficient of variation was 5.51%.The solidified gel beads can be easily lysed,washed and preserved.Then the beads were regenerated into droplets and barcode droplets for "one-to-one" pairing and fusion.According to diameter statistics,the success rate of droplet fusion was～64%.3.Large-scale single-cell barcoding and cross-coding analysis of mix sampleAfter fusion,the barcode sequence was introduced into the product through the second round of ddPCR to achieve large-scale single-cell barcoding and D-loop region amplification.The product was clone sequencing and alignment.All clones matched the reference sequence,and the library structure was complete for next-generation sequencing.Cross-coding analysis was performed using the mixed sample of human(K562)and mouse(BALB/3T3 clone A31).The results showed that,due to the demulsification of droplets and small amount of mtDNA escaped from the gel,there was a certain background amplification in the experiment,the background amplification data accounted for 7.48%.4.Single-cell D-loop heterogeneityIn the K562 single-cell D-loop heterogeneity data,we set the filter condition that the proportion of ReadPairs in each single-cell was greater than 1%and the number was not less than 30 in order to remove background amplification data.We found heterogeneity in all cells by GATK.Twenty-two mtDNA_types were obtained from statistics.MtDNA_type 1 accounted for 75.56%,and mtDNA_type 1 dominated in 95.52%of cells.99.07%of cells contained 4 or more mtDNA_types.T293G,C306A and C377A were found to be new mutations by MITOMAP search.And all Deletions were concentrated in the D310 region.This may indicate that D310 polymorphism is associated with myelogenous leukemia and may be a future research direction.Therefore,the single-cell D-loop heterogeneity detection technique in this thesis has shown great the potential in the field of singlecell sequencing.It can provide new means for the diagnosis of D-loop heterogeneity and the treatment of mitochondrial diseases and related cancers.