A single bacterial genome sequencing method based on digital microfluidics technology

Abstract

The present invention relates to the field of single-bacterial sequencing, and more specifically to a single-bacterial genome sequencing method based on digital microfluidics technology. The present invention has developed a micro-lysis system, a digital microfluidics chip, and a single-bacterial genome sequencing technology suitable for single bacteria based on a fully automated digital microfluidics platform (DMF). Experimental results show that the lysis system rapidly lyses single bacterial cells, maintains good gene integrity, and achieves high assembly efficiency and gene coverage. Furthermore, the combination of the lysis system and the microfluidics chip based on the lysis system with DMF reduces reagent loss, lowers the risk of sample contamination, improves reaction efficiency, and reduces amplification bias, making it more suitable for single-bacterial genome sequencing.

Description

Technical Field

[0001] The present invention relates to the field of single bacterial sequencing, and in particular to a single bacterial genome sequencing method based on digital microfluidics technology. Background Art

[0002] Microorganisms are the most diverse and abundant organisms, the most widespread, and possess the most diverse survival and metabolic pathways. They play a key role in driving biogeochemical cycles and significantly impact human health. However, our understanding of bacteria largely stems from studies of a small number of culturable bacteria. However, due to the limitations of current culture techniques, most bacteria remain difficult to culture in pure culture, hindering the study of these uncultivable bacteria. This significantly limits our understanding of the vast and diverse bacterial population. The development of culture-independent research methods could overcome this limitation and provide a more comprehensive and systematic understanding of the bacterial kingdom. The genome, often called the blueprint of life, can reveal a wealth of bacterial information, enabling insights into taxonomy and function, as well as the construction of metabolic pathways. Currently, both metagenomic and single-bacterial genomic sequencing methods are culture-independent approaches. However, due to the complex data sources of metagenomic sequencing, the assembled genomes are prone to mismatches. Single-bacterial genomics, derived from a single bacterium, effectively avoids this problem and can serve as a reference genome for annotation of metagenomic datasets. Furthermore, single-bacterial genome sequencing can capture the entire DNA within a bacterium, allowing for the study of microbial interactions, such as infection, symbiosis, and predation. Furthermore, due to its single-cell resolution, single-bacterial genome sequencing can be used to investigate heterogeneity among individual bacteria and address issues such as heterogeneous bacterial resistance, which are difficult to investigate using traditional methods. However, compared to the rapid progress in single-cell genome sequencing of eukaryotic cells, the development of single-bacterial genome sequencing has lagged behind. The main reasons for this are: 1. Bacteria typically range in diameter from 0.2 to 7 μm, only one-tenth the size of eukaryotic cells. Their small size and transparency make them difficult to observe and isolate. 2. Bacteria are diverse in species, with cell walls of varying composition and thickness. Most bacterial cell walls are difficult to lyse, hindering the release of nucleic acids. 3. Bacterial DNA content is extremely low, with most bacteria containing only a few femtograms, only one-thousandth the size of a eukaryotic cell. 4. To meet the current sequencing requirements for sample loading, the nucleic acid content needs to reach at least the nanogram level, which requires amplification of the DNA by more than a million times, which can easily cause amplification deviation. At the same time, this also makes contamination protection extremely important, because even tiny amounts of contamination will be amplified.

[0003] Currently, most single-cell genome sequencing methods are developed for cells without cell walls, such as mammalian cells. These methods are not suitable for the lysis and amplification of cells with cell walls, such as bacteria. To this end, researchers have established several single-bacterial genome sequencing methods for bacteria. These methods are mainly based on flow cytometry fluorescence sorting technology (FACS) and microfluidics technology. For example, WGA-X was developed based on FACS, MIDAS was developed based on micropore microfluidics, and SiC-seq and SAG-gel platform were developed based on gel droplet microfluidics. Raman sorting technology is an emerging single-cell sorting technology in recent years. Based on this technology, single-bacterial genome sequencing methods such as RAGE-Seq have been developed.

[0004] However, these currently developed single-bacterial genome sequencing methods still have shortcomings. For example, WGA-X, developed based on FACS, uses FACS to sort individual bacteria into well plates or centrifuge tubes for lysis and amplification. It uses a mutant Equiphi29 polymerase to reduce amplification bias and improve genome coverage. However, the FACS sorting environment is not completely sealed, which is extremely easy to introduce contamination. Moreover, the volume of the reaction in the well plate or centrifuge tube is too large, not only consuming a large amount of reagents, but also causing greater amplification bias. MIDAS, developed based on micropore microfluidics, improves the success rate of obtaining single bacteria through a large number of nanoliter-scale micropore arrays and has a high throughput. In addition, the nanoliter-scale reaction system can effectively reduce amplification bias, greatly improve the genome recovery rate, and obtain higher genome completeness. However, the capture of single bacteria by micropore chips is random and follows a Poisson distribution, so the utilization rate of the micropores is very low, and reagents are wasted. In addition, MIDAS requires manual picking of successfully amplified DNA products from the micropores, which is a cumbersome and difficult operation. SiC-seq, developed based on gel droplet microfluidics, uses a droplet microfluidic chip to generate a large number of microdroplets containing single bacteria. Combined with droplet barcoding technology, this method can sequence more than 50,000 bacteria at a time, achieving high-throughput sequencing. However, this method has a very low genome coverage rate of only 0.1% to 1%. Moreover, this method fragments DNA before amplification, making de novo genome assembly difficult during subsequent sequence assembly, which limits its application to unculturable bacterial genomes. Due to the high-throughput advantages of droplet microfluidics, methods such as the SAG-gel platform have also been developed. However, like microwell microfluidics, droplet microfluidics follows a Poisson distribution, resulting in a large number of empty droplets and low utilization rates. Moreover, these platforms have poor controllability, making it difficult to achieve selective cell capture and remove droplets containing unhealthy, dead, or multicellular cells, resulting in reagent waste and sample cross-contamination. RAGE-Seq, based on Raman sorting technology, is an emerging single-cell sorting and sequencing technology. It precisely isolates individual bacteria with specific Raman phenotypes from a population by combining optical tweezers and droplet microfluidics. After adding reagents, the system generates a large number of small droplets through oscillation, thereby reducing the reaction volume, significantly reducing amplification bias, and improving genome coverage. Furthermore, this method can link bacterial genotype and phenotype, allowing for the acquisition of more bacterial information. However, this method still has some drawbacks, such as the long time required to acquire Raman signals and the increased potential for contamination caused by transferring droplets to test tubes.

[0005] Due to the above-mentioned problems such as high sample contamination rate, serious reagent consumption, and long signal acquisition time, the development and utilization of fully automated digital microfluidics platforms (DMF) for single-cell genome sequencing technology has become a hot topic. The DMF platform has the advantages of miniaturization, integration, visualization, and automation, and is an emerging microfluidic technology that is dynamically controllable. Yang Chaoyong and others from Xiamen University have developed a single-cell sequencing platform based on fully automated digital microfluidics. This platform can selectively capture cells, has a low sample contamination rate in the fully automated process, strong assembly capabilities, and high gene coverage. However, it has the same problems as most current single-cell genome sequencing for mammalian cells and other cells without cell walls. The fully automated single-cell sequencing technology suitable for mammalian cells is not suitable for single-cell genome sequencing of cells with cell walls, such as bacteria. It has problems such as difficulty in cell lysis and difficulty in protecting against amplification contamination, which further affects the assembly and coverage of genes. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a single bacterial genome sequencing method based on digital microfluidics technology.

[0007] The present invention provides a digital microfluidic chip, which includes a bacteria capture and lysis unit, a droplet generation channel, a liquid reservoir unit and an electrode interface; the number of the liquid reservoir units is not less than 5, and they respectively store lysis reagent A, lysis reagent B, neutralization solution, amplification reagent and bacterial solution; wherein,

[0008] Lysis reagent A includes: 800-2400 U / μL lysozyme, 400-1200 mM dithiothreitol (DTT), and 2-6 mM ethylenediaminetetraacetic acid (EDTA);

[0009] Lysis Reagent B includes: 300-500 mM KOH, 80-120 mM DTT, and 5-15 mM EDTA;

[0010] Neutralizing solution includes: 0.8-1.2M Tris-HCl solution;

[0011] Amplification reagents include random primers, DNA polymerase, dNTPs, amplification buffer, and DTT.

[0012] Furthermore, in some specific embodiments, the lysis reagent A, lysis reagent B, neutralization solution and amplification reagent are independently used as shown in the following i) to iv) to achieve good lysis effect and high gene integrity:

[0013] i) Lysis reagent A includes: 1600 U / μL lysozyme, 800 mM DTT and 4 mM EDTA;

[0014] ii) Lysis Reagent B includes: 400 mM KOH, 100 mM DTT, and 10 mM EDTA;

[0015] iii), neutralizing solution including 1M Tris-HCl solution;

[0016] iv) Amplification reagents include random primers, DNA polymerase, dNTPs, amplification buffer and DTT.

[0017] In the present invention, the DNA polymerase in the amplification reagent includes but is not limited to: any one or a combination of two or more of vent DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, DNA polymerase I, Sulfolobus DNA polymerase IV, phi29 DNA polymerase, BstDNA polymerase, and Equiphi29 DNA polymerase, and the present invention is not limited to this.

[0018] Furthermore, the present invention uses Equiphi29 DNA polymerase as a test subject to amplify a single bacterial genome. The high fidelity of EquiPhi29 DNA polymerase combined with the advantages of a fully automated digital microfluidics platform can further reduce amplification bias and improve genome coverage.

[0019] In the present invention, the amplification buffer includes commercially available or self-prepared amplification buffer suitable for the above DNA polymerase amplification, and the present invention is not limited to this.

[0020] In the present invention, the digital microfluidic chip further comprises a fluorescent dye, a filling oil and a purification reagent;

[0021] The fluorescent dye is selected from PI, DAPI, SYTO-9, FM 1-43FX, FM 4-64, FM 4-64FX, BacLigh Bacterial Stains, Alexa Fluor 647NHS Ester, CellTracker TM Red CMTPX or FM 1-43;

[0022] The filler oil is selected from mineral oil, fluorine oil, silane oil, and dimethyl silicone oil;

[0023] The purification reagent is selected from a column purification reagent or a magnetic bead purification reagent.

[0024] Furthermore, the present invention uses FM 1-43 fluorescent dye to fluorescently stain bacteria, which facilitates the observation of single-cell capture status and the acquisition of cell morphology information; the present invention uses dimethyl silicone oil as a test object for sealing the chip; and the present invention uses magnetic bead purification reagent for purification.

[0025] In the present invention, the volume ratio of the lysis reagent A, lysis reagent B, and neutralization solution is 1:(1-1.5):(1-1.5); the volume ratio of the amplification reagent to the mixed solution of lysis reagent A, lysis reagent B, and neutralization solution is 1:6.5.

[0026] Furthermore, when the volume ratio of the lysis reagent A, lysis reagent B, and neutralization solution is 1:1.5:1.5, the single bacterial lysis effect is good and the gene integrity is high.

[0027] Micro single-cell lysis systems based on mammals and the like are not suitable for the lysis of single bacteria with cell walls, such as bacteria. The present invention has developed a single-cell micro lysis system suitable for bacteria. The lysis system provided by the present invention includes lysis solution A, lysis solution B and neutralization solution. Lysis solution A is an enzyme lysis reagent, and lysis solution B is an alkaline lysis reagent. Enzyme lysis is performed first, then alkaline lysis, and finally neutralization is performed, thereby obtaining a good lysis effect. Compared with other lysis schemes or the selection of lysis reagents, the lysis effect obtained is good, the gene integrity is high, and it is more conducive to the construction of subsequent libraries and the conduct of sequencing reactions.

[0028] In the present invention, the digital microfluidic chip includes a bacteria capture and lysis unit, a droplet generation channel, a liquid reservoir unit and an electrode interface. The bacteria capture and lysis unit includes: an upper electrode substrate, an ITO coating arranged on the lower surface of the upper electrode substrate, an upper hydrophobic layer arranged on the lower surface of the ITO coating, the upper electrode and the upper hydrophobic layer are light-transmissive, which is conducive to the observation of captured single bacteria; a lower electrode substrate, a dielectric layer arranged on the upper surface of the lower electrode substrate, an electrode embedded in the dielectric layer, a lower hydrophobic layer arranged on the upper surface of the dielectric layer, a support body supported between the lower hydrophobic layer and the upper hydrophobic layer, a droplet generation channel is formed between the upper hydrophobic layer, the lower hydrophobic layer and the support body, the lower hydrophobic layer is embedded with a hydrophilic groove, the upper notch of the hydrophilic groove is connected to the droplet generation channel; the opening of the droplet generation channel is connected to the liquid reservoir unit; the number of the liquid reservoir units is not less than 5, which respectively store lysis reagent A, lysis reagent B, neutralization solution, amplification reagent and bacterial solution; the electrode interface is connected to the electrode.

[0029] Furthermore, the electrode layer is required to be made of a conductive material, including but not limited to metals, alloys, graphene, and indium tin oxide. The hydrophobic layer is required to be made of a hydrophobic material, including but not limited to polytetrafluoroethylene or polyvinyl alcohol. The upper electrode substrate and the upper hydrophobic layer are made of transparent materials. The upper electrode substrate and the lower electrode substrate are independently selected from glass, quartz, and plastic materials.

[0030] The hydrophilic groove is a hollow cylindrical structure with a diameter of 150 to 300 μm and a distance of more than two electrodes from the liquid reservoir; the size of the liquid reservoir is 1 mm × 1 mm, and the size ratio of the liquid reservoir to the channel is (5 to 8):1.

[0031] The present invention provides a digital microfluidic device, which comprises the digital microfluidic chip, an integrated circuit and an imaging system according to the present invention, wherein the integrated circuit is connected to the electrode interface of the digital microfluidic chip.

[0032] In the digital microfluidic device, the integrated circuit is a circuit control system for a digital microfluidic chip. By switching power on and off between adjacent electrodes on the integrated circuit chip, droplets on the electrodes are moved. The imaging system is located directly above the digital microfluidic chip, or can be moved directly above the digital microfluidic chip to observe bacterial separation within the hydrophilic grooves on the chip.

[0033] The present invention provides a method for single bacterial genome sequencing, comprising using the digital microfluidic chip and / or the digital microfluidic device of the present invention to perform single bacterial isolation and capture, nucleic acid amplification, library construction and whole genome sequencing.

[0034] Furthermore, the steps of single bacterial genome sequencing according to the present invention are as follows:

[0035] a. staining the bacterial sample with a fluorescent dye;

[0036] b. Place the bacterial suspension stained in step a above into a digital microfluidic chip, add filler oil to seal the chip, allow the bacterial suspension to move through the droplet generation channel to the hydrophilic groove, and observe the separation and capture of single bacteria using a fluorescence microscope;

[0037] c. Controlling the on / off power of the electrodes, lysing reagent A, lysing reagent B, and neutralizing solution are sequentially passed through the droplet generation channel into the hydrophilic groove to lyse the single bacteria captured in step b above on the digital microfluidic chip to obtain bacterial genomic DNA;

[0038] d. Control the electrodes to turn on and off, and the amplification reagents enter the hydrophilic grooves through the droplet generation channel to amplify the genomic DNA;

[0039] e. The amplified product obtained in step d above was transferred to a centrifuge tube for nucleic acid purification and sequencing library construction, and finally sequenced on a sequencing machine.

[0040] In the present invention, the lysis described in step c includes, but is not limited to, one or a combination of two or more of enzymatic lysis, extreme pH lysis, chemical reagent lysis, and physical lysis, and the present invention is not limited thereto. Furthermore, in the present invention, cell lysis is performed by combining enzymatic lysis with alkaline lysis; the first step is enzymatic lysis, in which the added reagent is lysis reagent A, comprising a mixture of lysozyme, DTT, and EDTA; the second step is alkaline lysis B, in which the added reagent is a mixture of KOH and EDTA; and finally, a neutralizing solution, Tris-HCl solution, is added for neutralization.

[0041] In the present invention, the nucleic acid purification described in step e, the purification method includes but is not limited to magnetic bead purification method, centrifugal column purification method, the present invention does not limit this. Further, the present invention uses a magnetic bead purification kit Nucleic acid purification was performed using DNA CleanBeads (Vazyme biotech co., ltd.).

[0042] In the present invention, the library construction in step e includes but is not limited to NGS Fast DNA LibraryPrep Set for Illumina, Collibri TM ES DNA Library Prep Kits for IlluminaSystems、TruePrep TM DNA Library Prep Kit V2 for etc., the present invention does not limit this. Further, the present invention uses TruePrep TM DNA Library Prep Kit V2 for The amplified products were used to construct a library.

[0043] The present invention provides the application of the digital microfluidic chip and / or the digital microfluidic device in the construction of a bacterial library.

[0044] Furthermore, the bacterial library includes but is not limited to a clone library, an expression library, a plasmid library or a phage library, and the present invention is not limited thereto.

[0045] The present invention provides the application of the digital microfluidic chip and / or the digital microfluidic device in a bacterial genome sequencing platform.

[0046] Further sequencing platforms of the present invention include but are not limited to Illumina Hiseq-2000, Illumina HiSeq-2500, Illumina NovaSeq 6000, PacBio Sequel II, and Promeh ION. In the present invention, preferably, the Illumina NovaSeq 6000 sequencing platform is used for 2×150 bp paired-end sequencing.

[0047] Furthermore, the bacteria described in this invention include both Gram-positive and Gram-negative bacteria. The present invention used Gram-negative Escherichia coli as the test subject for single-bacterial genome sequencing using a digital microfluidics platform. Experimental results demonstrated that, compared with tube and bulk genome sequencing results, single-bacterial genome sequencing based on the digital microfluidics platform achieved the highest alignment rate, the lowest contamination rate, and high genome integrity and coverage, demonstrating the significant advantages of this method for single-cell genome sequencing.

[0048] The present invention has developed a micro-lysis system, digital microfluidic chip and single-bacterial genome sequencing technology suitable for single bacteria based on a fully automatic digital microfluidic platform (DMF). Experimental results show that the lysis system can rapidly lyse single bacterial cells, maintain good gene integrity, and achieve high assembly efficiency and gene coverage. At the same time, the combination of the lysis system and the microfluidic chip based on the lysis system with DMF reduces reagent loss, lowers the risk of sample contamination, improves reaction efficiency and reduces amplification deviation, making it more suitable for single-bacterial genome sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Figure 1 shows a side view of the digital microfluidic chip structure, where 1: upper electrode substrate; 2: ITO coating; 3a: upper hydrophobic layer; 3b: lower hydrophobic layer; 4: support; 5: hydrophilic groove; 6: dielectric layer; 7: electrode layer; 8: lower electrode substrate;

[0050] Figure 2 Figure 2 shows a top view of the microfluidic chip, where 1 is the lower plate circuit connection point; 2 is the electrode layer array;

[0051] Figure 3 Schematic diagram of single bacterial genome sequencing based on digital microfluidics device. DETAILED DESCRIPTION

[0052] The present invention provides a single bacterial genome sequencing method based on digital microfluidics technology. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0053] In the description of the present invention, the terms "upper" and "lower" etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention.

[0054] In the present invention, the steps of single bacterial genome sequencing are as follows:

[0055] a. Use 5 μg / mL of fluorescent dye FM 1-43 and incubate on ice for 30 minutes to fluorescently stain the bacterial sample;

[0056] b. The bacterial suspension stained in step a was placed in a digital microfluidic chip. Filler oil was added to seal the chip. The electrodes were powered on and off to generate 30 nL bacterial droplets that moved through the droplet channel to the hydrophilic groove. Single bacteria were isolated and captured using a fluorescence microscope.

[0057] c. The single bacteria captured in step b above were lysed on a digital microfluidic chip. The electrodes were powered on and off, and 30 nL of lysis reagent A droplets, 45 nL of lysis solution B droplets, and 45 nL of neutralization solution were introduced into the hydrophilic groove through the droplet generation channel to lyse and obtain bacterial genomic DNA.

[0058] d. The control electrode is turned on and off, and the amplification reagent enters the hydrophilic groove through the droplet generation channel. The amplification reagent droplet size is 3 × 250 nL. The genomic DNA is amplified by incubating at 45°C for 6 hours.

[0059] e. The amplified product obtained in step d above was transferred to a centrifuge tube for nucleic acid purification and sequencing library construction, and finally sequenced on a sequencing machine.

[0060] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0061] The present invention will be further described below in conjunction with the embodiments:

[0062] Example 1 Single bacterial genome sequencing method based on digital microfluidics technology

[0063] 1. Digital Microfluidic Device Structure

[0064] The digital microfluidic device consists of a digital microfluidic chip, an integrated circuit, and an imaging system. The imaging system, located directly above the digital microfluidic chip, features multi-channel imaging capabilities and is used to capture images of the chip, droplets, and bacteria. In brightfield imaging, it captures the position and volume of droplets; in fluorescence imaging, it observes the number of bacteria captured at the hydrophilic sites. The integrated circuit serves as the circuit control system for the digital microfluidic chip. It activates and deactivates adjacent electrodes on the integrated circuit chip to move droplets on the electrodes.

[0065] The digital microfluidic chip includes a bacteria capture and lysis unit, a droplet generation channel, a liquid reservoir unit and an electrode interface. Figure 1 As shown, it includes: an upper plate substrate ( Figure 1 1), an ITO coating ( Figure 1 2), an upper hydrophobic layer ( Figure 1 3a), the upper plate substrate, ITO coating and upper hydrophobic layer are light-transmitting; the lower plate substrate ( Figure 1 8), a dielectric layer ( Figure 1 6), the dielectric layer has hydrophilic properties and can adhere to tiny droplets; the electrode layer ( Figure 1 7), the electrode layer requires the use of conductive materials, the electrode layer includes the electrode interface ( Figure 2 1), liquid reservoir electrode unit, and electrode layer array ( Figure 2 2); a lower hydrophobic layer ( Figure 1 3b); a support body supported between the lower hydrophobic layer and the upper hydrophobic layer ( Figure 1 4); a droplet generation channel is formed between the upper hydrophobic layer, the lower hydrophobic layer and the support; the lower hydrophobic layer is embedded with a hydrophilic groove ( Figure 1 5), the hydrophilic groove is a hollow cylindrical structure with a diameter of 150 to 300 μm. The hydrophilic groove is more than 2 electrodes away from the liquid reservoir so that small droplets can be generated when the hydrophilic site is reached. The upper notch of the hydrophilic groove is connected to the droplet generation channel, and the integrated circuit is controlled to generate 10nL to 250nL droplets in the droplet generation channel as needed. The opening of the droplet generation channel is connected to the liquid reservoir unit, and the number of liquid reservoir units is not less than 5, which respectively store lysis reagent A, lysis reagent B, neutralization solution, amplification reagent and bacterial solution (such as Figure 3 The reservoir is typically 1 mm x 1 mm and should be larger than the channel. The reservoir to channel size ratio is typically 5:1 to 8:1. The electrode interface is connected to the electrode.

[0066] 2. Single Bacterial Genome Sequencing

[0067] like Figure 3 As shown, the steps of the single bacterial genome sequencing method based on digital microfluidics technology include bacterial staining, single bacterial isolation, cell lysis, whole genome amplification, library construction and sequencing. The present invention uses Escherichia coli as the test subject to perform bacterial single genome sequencing. The specific steps are as follows:

[0068] 1. Prepare the E. coli sample into a bacterial suspension. Add the fluorescent dye FM 1-43 to the bacterial suspension at a final concentration of 5 μg / mL and incubate on ice for 30 minutes.

[0069] 2. Lysis Reagent A consists of 1600 U / μL of Ready-Lyse lysozyme, 800 mM dithiothreitol (DTT), and 4 mM ethylenediaminetetraacetic acid (EDTA); Lysis Reagent B consists of 400 mM KOH, 100 mM DTT, and 10 mM EDTA; and Neutralizer is 1 M Tris-HCl. Add 300 nL of stained bacterial culture, 300 nL of Lysis Reagent A (a mixture of lysozyme, DTT, and EDTA), 450 nL of Lysis Reagent B (a mixture of KOH and DTT), 450 nL of Neutralizer (HCl solution), and 3000 nL of MDA Amplification Reagent (a mixture of random primers, Equiphi29 polymerase, dNTPs, Reaction Buffer, and DTT, prepared according to Table 1) to different reservoirs on the DMF chip.

[0070] Table 1 Amplification mixture preparation

[0071] Reagents Final concentration volume 1 Equiphi29 DNA polymerase (10 U / μL) 1U / μL 1.2 μL 2 10× Equiphi29 amplification buffer 1× 1.2 μL 3 dNTP (10mM) 0.4mM 0.48μL 4 Random primers (500 μM) 40 μM 1 μL 5 Dithiothreitol (DTT, 100mM) 10mM 1.2 μL 6 <![CDATA[H2O]]> 4.92μL

[0072] 3. The controller and circuit control module control the electrode drive circuit, turning the electrodes on and off in a preset sequence. This generates a bacterial suspension droplet (30 nL) from the electrode unit in the bacterial reservoir area. This droplet is then moved to the hydrophilic site, where it passes through. Surface tension leaves behind small droplets, which may contain varying numbers of bacteria. These droplets repeatedly pass through the hydrophilic site until only a single bacterium remains, capturing the single bacterium. The original cell suspension droplet is then removed from the wastewater area by controlling the electrode drive circuit.

[0073] 4. According to the method in step 3, generate a droplet of lysis reagent A (30nL) from the electrode unit of the lysis reagent 1 reservoir area, move this droplet to the electrode where the hydrophilic site is located and mix it with a single bacterium, move the droplet back and forth on the electrode to fully mix the reagents, and incubate at room temperature for 20 minutes.

[0074] 5. According to the method in step 3, generate a droplet of lysis reagent B (45nL) from the electrode unit of the lysis reagent 2 reservoir area, move this droplet to the electrode where the hydrophilic site is located to mix with the above droplet, move the droplet back and forth on the electrode to fully mix the reagents, and incubate at room temperature for 20 minutes.

[0075] 6. According to the method in step 3, a neutralizing liquid droplet (45nL) is generated from the electrode unit of the neutralizing liquid storage area, and this droplet is moved to the electrode where the hydrophilic site is located to mix with the above droplet. The droplet is moved back and forth on the electrode to fully mix the reagents and terminate the lysis.

[0076] 7. According to the method in step 3, three MDA amplification droplets (3×250nL) were generated from the electrode unit in the MDA amplification liquid storage area. The droplets were moved to the electrode where the hydrophilic site was located to mix with the above droplets. The droplets were moved back and forth on the electrode to fully mix the reagents. The DNA was amplified by incubating at 45°C for 6 hours.

[0077] 8. Terminate the amplification reaction at 75°C for 15 minutes. The entire process is completely sealed and not prone to contamination.

[0078] 9. Recover the amplified droplets into a test tube and use a purification kit The amplified products were purified using DNA Clean Beads (Vazyme biotech co., ltd.).

[0079] 10. The purified product was quantified using the Equalbit dsDNA HS Assay Kit.

[0080] 11. Use the TruePrep library preparation kit TM DNA Library Prep Kit V2 for Construct a library of the amplified products.

[0081] 12. Finally, 2×150bp paired-end sequencing was performed using the Illumina NovaSeq 6000 sequencing platform.

[0082] We compared the results of single bacterial genome sequencing on DMF with those of single bacterial genome sequencing in test tubes (Tube) and genome sequencing of bacterial groups (Bulk). The results are shown in Table 1. The read matching rate and contamination rate of the three are basically the same, while the matching rate of DMF is the highest and the contamination rate is less than 5%, indicating that DMF performs well in preventing contamination. In terms of the most critical indicator, genome integrity, DMF is much higher than Tube and close to Bulk. The average genome integrity is around 90%, and some single bacterial genomes are close to 100%. This shows that the small-volume reaction system of DMF can indeed reduce amplification bias and thus improve genome integrity. In terms of the key indicators of genome assembly, maximum contig and N50, DMF is much higher than Tube, and some are comparable to Bulk. Overall, the results of single bacterial genome sequencing based on digital microfluidics technology DMF established by the present invention are significantly better than those of single bacterial genome sequencing in test tubes, showing the significant advantages of this method in single-cell genome sequencing.

[0083] Table 2 Single bacterial genome sequencing results and evaluation

[0084]

[0085] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A digital microfluidic chip, characterized in that: It includes a bacteria capture and lysis unit, a droplet generation channel, a liquid reservoir unit and an electrode interface; the number of the liquid reservoir units is not less than 5, which respectively store lysis reagent A, lysis reagent B, neutralization solution, amplification reagent and bacterial solution; wherein, Lysis reagent A is: 800-2400 U / μL lysozyme, 400-1200 mM dithiothreitol, and 2-6 mM ethylenediaminetetraacetic acid; Lysis reagent B is: 300-500 mM KOH, 80-120 mM dithiothreitol, and 5-15 mM EDTA; Neutralizing solution: 0.8-1.2M Tris-HCl solution; Amplification reagents were: random primers, DNA polymerase, dNTPs, amplification buffer, and dithiothreitol; The invention also includes: a fluorescent dye, a filling oil and a purification reagent; the fluorescent dye is FM 1-43; the filling oil is selected from mineral oil, fluorinated oil, silane oil, and dimethyl silicone oil; the purification reagent is selected from a column purification reagent or a magnetic bead purification reagent; The bacteria capture and lysis unit includes: an upper electrode substrate, an ITO coating arranged on the lower surface of the upper electrode substrate, and an upper hydrophobic layer arranged on the lower surface of the ITO coating; a lower electrode substrate, a dielectric layer arranged on the upper surface of the lower electrode substrate, an electrode embedded in the dielectric layer, a lower hydrophobic layer arranged on the upper surface of the dielectric layer, and a support body supported between the lower hydrophobic layer and the upper hydrophobic layer. A droplet generation channel is formed between the upper hydrophobic layer, the lower hydrophobic layer and the support body. The lower hydrophobic layer is embedded with a hydrophilic groove, and the upper notch of the hydrophilic groove is connected to the droplet generation channel; the opening of the droplet generation channel is connected to the liquid storage tank unit; the electrode interface is connected to the electrode, and the hydrophilic groove is a hollow cylindrical structure with a diameter of 150 to 300 μm, and is more than 2 electrodes away from each liquid storage tank.

2. The digital microfluidic chip according to claim 1, characterized in that: The DNA polymerase in the amplification reagent includes any one or a combination of two or more of vent DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, DNA polymerase I, Sulfolobus DNA polymerase IV, phi29 DNA polymerase, Bst DNA polymerase, and Equiphi29 DNA polymerase.

3. The digital microfluidic chip according to claim 1 or 2, characterized in that: The volume ratio of the lysis reagent A, lysis reagent B, and neutralization solution is 1:(1-1.5):(1-1.5); the volume ratio of the amplification reagent to the mixed solution of lysis reagent A, lysis reagent B, and neutralization solution is 1:6.

5.

4. The digital microfluidic chip according to claim 1, characterized in that: The size of the liquid reservoir is 1mm × 1mm, and the ratio of the reservoir diameter to the channel diameter is (5-8):

1.

5. A digital microfluidic device, characterized in that The invention comprises the digital microfluidic chip, integrated circuit and imaging system according to any one of claims 1 to 4; the integrated circuit is connected to the electrode interface of the digital microfluidic chip.

6. A method for sequencing a single bacterial genome, characterized in that: The digital microfluidic chip according to any one of claims 1 to 4 and / or the digital microfluidic device according to claim 5 are used for separation and capture of single bacteria, nucleic acid amplification, library construction and genome sequencing.

7. The method according to claim 6, characterized in that Specifically include: After being stained, the bacterial solution is placed in the liquid reservoir of the digital microfluidic chip. After the chip is sealed with oil, the integrated circuit controls the power on and off of the electrodes, allowing the bacterial solution to move through the droplet generation channel to the hydrophilic groove, isolating and capturing individual bacteria. Controlling the on and off of the electrodes allows the lysis reagent A, lysis reagent B, neutralization solution, and amplification reagent to enter the hydrophilic groove through the droplet generation channel in sequence, and then amplification is performed; The amplified products were purified and the library was constructed before sequencing.

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