CN115197840A - Single bacterium genome sequencing method based on digital microfluidic technology - Google Patents
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Abstract
The invention relates to the field of single bacterium sequencing, in particular to a single bacterium genome sequencing method based on a digital microfluidic technology. The invention develops a micro lysis system, a digital micro-fluidic chip and a single-bacterium genome sequencing technology which are based on a full-automatic digital micro-fluidic platform (DMF) and are suitable for single bacteria, and experimental results show that single-bacterium cells in the lysis system are quickly lysed, the gene integrity is good, and the assembly efficiency and the gene coverage are high; meanwhile, the cracking system and the combination of the micro-fluidic chip based on the cracking system and the DMF reduce the loss of reagents, reduce the sample pollution risk, improve the reaction efficiency, reduce the amplification deviation and make the system more suitable for single-bacterium genome sequencing.
Description
Technical Field
The invention relates to the field of single bacterium sequencing, in particular to a single bacterium genome sequencing method based on a digital microfluidic technology.
Background
The microorganisms are life bodies with highest diversity and abundance, widest distribution and most abundant survival and metabolism modes, play a key driving role in the biogeochemical cycle and have important influence on the life health of human bodies. However, our knowledge of bacteria has essentially been derived from the study of a small number of culturable bacteria. However, due to the limitations of the current culture techniques, most bacteria are still difficult to be cultured in a pure manner, which hinders the research on these non-culturable bacteria, and this greatly limits our knowledge on various and huge amounts of bacteria. Development of research methods that do not rely on culture can free us from this limitation, allowing a more comprehensive systematic understanding of the bacterial world. The genome is called a blueprint of life, and a large amount of information of bacteria can be obtained through the genome, such as analyzing classification and functions of the bacteria, constructing metabolic pathways and the like. At present, metagenome and single bacterial genome are culture-free genome sequencing methods, but because the sequencing data source of metagenome is complex, the assembled genome is easy to have mismatch, and the single bacterial genome is only from a single bacterium, so that the problem can be effectively avoided, and the assembled genome can also be used as a reference genome for annotation of a metagenome data set. In addition, single-bacterium genome sequencing can detect all DNA information in bacteria, so that the interaction among microorganisms, such as infection, symbiosis, predation and other scientific problems, can be researched. In addition, because the single-bacterium genome sequencing technology has the resolution of single cells, the method can also be used for researching the heterogeneity among bacteria individuals, analyzing the heterogeneous drug resistance of bacteria and other scientific problems which are difficult to research by using the traditional method. However, compared with the rapid development of single cell genome sequencing of eukaryotic cells, the development of single cell genome sequencing is lagged, and the main reasons can be summarized as the following points that 1, the diameter of the bacteria is generally between 0.2 and 7 μm, which is only one tenth of that of the eukaryotic cells, the cell size is too small and the whole body is transparent, so that the observation and the separation are difficult; 2. the bacteria are various in types, the composition and the thickness of cell walls are various, and most of the cell walls of the bacteria are difficult to crack, so that nucleic acid is difficult to release. 3. The DNA content of bacteria is very low, and most of the bacteria only have a few femtograms which are one thousandth of that of eukaryotic cells. 4. To meet the current sequencing requirement, the nucleic acid content needs to reach at least nanogram level, which requires over one million times amplification of DNA, and is very likely to cause amplification deviation, and at the same time, the pollution protection becomes very important because very small amount of pollution is amplified.
Most of the current single cell genome sequencing methods are developed for cell wall-free cells such as mammalian cells, and these methods are not suitable for the lysis and amplification of cells having cell walls such as bacteria. For this reason, researchers have established several methods for single bacterial genome sequencing of bacteria, based mainly on flow cytometric fluorescence sorting (FACS) and microfluidic technologies, such as development of WGA-X based on FACS, development of MIDAS based on microwell microfluidics, development of SiC-seq, SAG-gel platform, etc., based on gel droplet microfluidics. The Raman sorting technology is a single cell sorting technology emerging in recent years, and a sequencing method of single bacterial genomes such as RAGE-Seq is developed based on the technology.
However, these single bacterial genome sequencing methods developed to date still have deficiencies. For example, WGA-X based on FACS development, splitting single bacteria into well plates or centrifuge tubes for lysis and amplification by FACS, with the mutant Equiphi29 polymerase reducing amplification bias and increasing genome coverage. However, the FACS sorting environment is not completely sealed, which is very easy to cause contamination, and the reaction volume in the pore plate or centrifuge tube is too large, which not only consumes a large amount of reagents, but also causes larger amplification deviation. The MIDAS based on the micro-porous micro-fluidic development improves the success rate of obtaining single bacteria through a large amount of nano-upgraded micro-porous arrays, has higher flux, and in addition, the nano-upgraded reaction system can effectively reduce the amplification deviation, greatly improves the recovery rate of genome and obtains higher genome integrity. However, the capture of single bacteria by the microporous chip is random and obeys poisson distribution, so that the utilization rate of micropores is low, reagents are wasted, and the MIDAS needs to manually pick successfully amplified DNA products from the micropores, so that the operation is complicated and the difficulty is high. Based on SiC-seq developed by gel droplet microfluidics, a large number of micro-droplets wrapped with single bacteria can be generated by a droplet microfluidics chip, and more than 50000 bacteria can be sequenced at a time by combining a droplet coding technology (barcode) to realize high-throughput sequencing, however, the genome coverage rate of the method is very low and is only 0.1% -1%, and the method breaks DNA before amplification, so that de novo assembly (de novo assembly) of the genome is difficult to perform during late sequence splicing, which limits the application of the method in the uncultured bacterial genome. However, the droplet microfluidics and the microporous microfluidics are subjected to Poisson distribution, so that a large number of no-load droplets exist, the utilization rate is low, the controllability of the platforms is poor, selective cell capture cannot be realized, droplets with unhealthy, dead cells or multiple cells are difficult to remove, and reagent waste and sample cross contamination are caused to a certain extent. RAGE-Seq developed based on a Raman sorting technology is a novel single cell sorting sequencing technology, single bacteria with a specific Raman phenotype are accurately separated from a population by combining an optical tweezers and a droplet microfluidic technology, a large number of small droplets are generated by oscillation after a reagent is added, so that the reaction volume is reduced, the amplification deviation is greatly reduced, the genome coverage is improved, and the genotype and the phenotype of the bacteria can be linked together by the method, so that more bacteria information can be obtained. However, this method still has some disadvantages, such as the acquisition of raman signal takes a long time, the transfer of liquid droplet to test tube increases the possibility of contamination, etc.
Based on the problems of high sample pollution rate, serious reagent consumption, long signal acquisition time and the like, the development of a full-automatic digital microfluidic platform (DMF) for single cell genome sequencing technology is a hotspot at present. The DMF platform has the advantages of miniaturization, integration, visualization and automation, and is a dynamic controllable emerging microfluidic technology. The single cell sequencing platform based on full-automatic digital micro-fluidic is developed by Yangdong, the platform can complete selective capture on cells, the sample pollution rate in the full-automatic process is low, the assembly capability is strong, the gene coverage is high, but the same problem exists in the prior art for sequencing cell wall-free single cell genomes of most mammal cells, the full-automatic single cell sequencing technology suitable for the mammal cells is not suitable for sequencing the single cell genomes of cells with cell walls, such as bacteria, and the like, the problems of difficult cell cracking, difficult pollution amplification and protection and the like exist, and the assembly and the coverage of the genes are further influenced.
Disclosure of Invention
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 microfluidic technology.
The invention provides a digital microfluidic chip, which comprises a bacteria capturing and cracking unit, a liquid drop generating channel, a liquid storage pool unit and an electrode interface, wherein the bacteria capturing and cracking unit is connected with the liquid storage pool unit; the number of the liquid storage tank units is not less than 5, and a cracking reagent A, a cracking reagent B, a neutralizing solution, an amplification reagent and a bacterial solution are respectively stored in the liquid storage tank units; wherein,
the cracking reagent A comprises: 800-2400U/microliter lysozyme, 400-1200 mM Dithiothreitol (DTT) and 2-6 mM Ethylene Diamine Tetraacetic Acid (EDTA);
the cracking reagent B comprises: 300-500 mM KOH, 80-120 mM DTT and 5-15 mM EDTA;
the neutralizing liquid comprises: 0.8-1.2M Tris-HCl solution;
the amplification reagents include: random primers, DNA polymerase, dNTP, amplification buffer and DTT.
Further, in some embodiments, the lysis reagent a, the lysis reagent B, the neutralization solution, and the amplification reagent are independently selected from the following i) to iv) to achieve a good lysis effect and high gene integrity:
i) The cracking reagent A comprises: 1600U/. Mu.L lysozyme, 800mM DTT and 4mM EDTA;
ii) lysis reagent B comprises: 400mM KOH,100mM DTT and 10mM EDTA;
iii) The neutralizing solution comprises a 1M Tris-HCl solution;
iv) amplification reagents including random primers, DNA polymerase, dNTPs, amplification buffer and DTT.
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, bst DNA polymerase and Equiphi29 DNA polymerase, but the present invention is not limited thereto.
Further, the bacterium single genome is amplified by using the Equiphi29 DNA polymerase as a subject. The high fidelity of the EquiPhi29 DNA polymerase is combined with the advantages of a full-automatic digital microfluidic platform, so that the amplification deviation can be further reduced, and the genome coverage can be improved.
In the present invention, the amplification buffer includes a commercially available or self-prepared amplification buffer suitable for the above DNA polymerase amplification, which is not limited in the present invention.
In the invention, the digital microfluidic chip also comprises fluorescent dye, filling oil and a purification reagent;
the fluorescent dye is selected from the group consisting of PI, DAPI, SYTO-9, FM 1-43FX, FM 4-64, FM 4-64FX, bacLigh Bacterial strains, alexa Fluor 647NHS Ester, cellTracker TM Red CMTPX or FM 1-43;
the filling oil is selected from mineral oil, fluorine oil, silane oil and dimethyl silicone oil;
the purification reagent is selected from a column purification reagent or a magnetic bead purification reagent.
Furthermore, the invention uses FM 1-43 fluorescent dye to carry out fluorescent staining on bacteria, thereby facilitating the observation of single cell capture condition and the acquisition of cell morphological information; the invention uses the dimethyl silicone oil as a tested object for sealing the chip; the invention uses magnetic bead purification reagent to purify.
In the invention, the volume ratio of the cracking reagent A to the cracking reagent B to the neutralization solution is 1 (1-1.5) to 1-1.5; the volume ratio of the amplification reagent to the mixed solution of the lysis reagent A, the lysis reagent B and the neutralization solution is 1.
Furthermore, when the volume ratio of the lysis reagent A to the lysis reagent B to the neutralization solution is 1.5.
The micro-unicellular lysis system based on mammals and the like is not suitable for lysis of a single bacterium having a cell wall such as a bacterium. The invention develops a unicellular miniature cracking system suitable for bacteria, the cracking system provided by the invention comprises a cracking solution A, a cracking solution B and a neutralizing solution, the cracking solution A is an enzyme cracking reagent, the cracking solution B is an alkali cracking reagent, and the cracking solution A is firstly subjected to enzyme cracking and then alkali cracking and finally neutralized, so that a good cracking effect is obtained. Compared with other cracking schemes or selection of cracking reagents, the obtained cracking effect is good, the gene integrity is high, and the subsequent library construction and sequencing reaction are facilitated.
In the invention, the digital microfluidic chip comprises a bacteria capturing and cracking unit, a liquid drop generating channel, a liquid storage pool unit and an electrode interface. The bacteria capture and lysis unit comprises: the device comprises an upper polar plate substrate, an ITO coating arranged on the lower surface of the upper polar plate substrate, and an upper hydrophobic layer arranged on the lower surface of the ITO coating, wherein the upper polar plate and the upper hydrophobic layer are light-transmitting, so that observation of single bacterium capture is facilitated; the device comprises a lower polar plate substrate, a medium layer arranged on the upper surface of the lower polar plate substrate, an electrode embedded in the medium layer, a lower hydrophobic layer arranged on the upper surface of the medium layer, and a support body supported between the lower hydrophobic layer and the upper hydrophobic layer, wherein a liquid drop generating channel is formed among the upper hydrophobic layer, the lower hydrophobic layer and the support body, a hydrophilic groove is embedded in the lower hydrophobic layer, and an upper notch of the hydrophilic groove is communicated with the liquid drop generating channel; the port of the liquid drop generating channel is communicated with the liquid storage tank unit; the number of the liquid storage tank units is not less than 5, and a cracking reagent A, a cracking reagent B, a neutralization solution, an amplification reagent and a bacterial solution are respectively stored in the liquid storage tank units; the electrode interface is connected with the electrode.
Further, the electrode layer requires the use of conductive materials, including but not limited to metals, alloys, graphene, indium tin oxide. The hydrophobic layer requires the use of a material that is hydrophobic, including but not limited to polytetrafluoroethylene or polyvinyl alcohol. The upper plate substrate and the upper hydrophobic layer are made of transparent materials. The upper polar plate substrate and the lower polar plate substrate are independently selected from glass, quartz and plastic materials.
The hydrophilic groove is of a hollow cylindrical structure, has the diameter of 150-300 mu m, and has a distance of more than 2 electrodes with the liquid storage tank; the size of the liquid storage tank is 1mm multiplied by 1mm, and the size ratio of the liquid storage tank to the channel is (5-8): 1.
the invention provides a digital microfluidic device which comprises the digital microfluidic chip, an integrated circuit and an imaging system, wherein the integrated circuit is connected with an electrode interface of the digital microfluidic chip.
In the digital microfluidic device, the integrated circuit is a circuit control system of the digital microfluidic chip, and the liquid drops on the electrodes move by switching on and off the adjacent electrodes on the integrated circuit chip. The imaging system is positioned right above the digital microfluidic chip or can be moved to the position right above the digital microfluidic chip to observe the separation condition of bacteria in the hydrophilic groove on the chip.
The invention provides a method for sequencing a single bacterial genome, which comprises the steps of carrying out separation and capture, nucleic acid amplification, library construction and whole genome sequencing on single bacteria by using a digital microfluidic chip and/or a digital microfluidic device.
Furthermore, the steps of sequencing the genome of a single bacterium according to the present invention are as follows:
a. staining the bacterial sample with a fluorescent dye;
b. b, placing the bacterial suspension subjected to dyeing treatment in the step a into a digital microfluidic chip, adding filling oil to seal the chip, moving the bacterial liquid to a hydrophilic groove through a droplet generation channel, and observing, separating and capturing single bacteria through a fluorescence microscope;
c. controlling the electrode to be powered on and powered off, and enabling the cracking reagent A, the cracking reagent B and the neutralizing liquid to enter the hydrophilic groove through the droplet generating channel in sequence to crack the single bacterium captured in the step B on the digital microfluidic chip to obtain the bacterial genome DNA;
d. controlling the electrode to be powered on or powered off, and allowing the amplification reagent to enter the hydrophilic groove through the droplet generation channel to amplify the genome DNA;
e. and d, transferring the amplification product obtained in the step d into a centrifugal tube for nucleic acid purification and sequencing library construction, and finally, performing machine sequencing.
In the present invention, the cleavage method in step c includes, but is not limited to, one or a combination of two or more of an enzymatic cleavage method, an extreme pH cleavage method, a chemical reagent cleavage method, and a physical cleavage method, which is not limited in the present invention. Further, in the invention, cell lysis is performed by combining enzyme lysis and alkali lysis; the first step is enzyme cracking, the added reagent cracking reagent A comprises lysozyme, DTT and EDTA mixed liquor, the second step is alkali cracking B, the added reagent is KOH and EDTA mixed liquor, and finally, the neutralization solution Tris-HCl solution is added for neutralization.
In the present invention, the nucleic acid purification in step e includes, but is not limited to, magnetic bead purification and spin column purification, which are not limited in the present invention. Further, the present invention uses a magnetic bead purification kit
DNA Clean Beads (Vazyme biolech co., ltd.) were subjected to nucleic acid purification.
In the present invention, the Library construction method in step e includes, but is not limited to, NGS Fast DNA Library Prep Set for Illumina, collibri TM ES DNA Library Prep Kits for Illumina Systems、TruePrep TM DNA Library Prep Kit V2 for
And the invention is not limited in this regard. Further, the present invention uses truePrep TM DNA Library Prep Kit V2 for
And constructing a library of the amplification products.
The invention provides the digital microfluidic chip and/or the application of the digital microfluidic device in bacterial library construction.
Further, the bacterial library includes, but is not limited to, a cloning library, an expression library, a plasmid library or a phage library, which is not limited by the present invention.
The invention provides the digital microfluidic chip and/or the application of the digital microfluidic device in a bacterial genome sequencing platform.
Further sequencing platforms of the present invention include, but are not limited to, illumina Hiseq-2000, illumina Hiseq-2500, illumina Novaseq 6000, pacBio sequence II, promehION. In the present invention, 2 × 150bp paired-end sequencing is preferably performed using the Illumina NovaSeq 6000 sequencing platform.
Further, the bacteria of the present invention include gram-positive bacteria and gram-negative bacteria, and the present invention uses gram-negative bacteria escherichia coli as a subject to perform single-bacterium genome sequencing based on a digital microfluidic platform. The experimental result shows that compared with the sequencing results of test tubes (Tube) and group bacteria (Bulk) genomes, the single bacteria genome sequencing based on the digital microfluidic platform has the advantages of highest single bacteria genome sequencing comparison rate, small pollution rate, high genome integrity and coverage, and remarkable advantage of the method in single cell genome sequencing.
The invention develops a micro lysis system, a digital micro-fluidic chip and a single-bacterium genome sequencing technology which are based on a full-automatic digital micro-fluidic platform (DMF) and are suitable for single bacteria, and experimental results show that single-bacterium cells in the lysis system are quickly lysed, the gene integrity is good, and the assembly efficiency and the gene coverage are high; meanwhile, the cracking system and the combination of the micro-fluidic chip based on the cracking system and the DMF reduce the loss of reagents, reduce the sample pollution risk, improve the reaction efficiency, reduce the amplification deviation and make the system more suitable for single-bacterium genome sequencing.
Drawings
Fig. 1 shows a side view of a digital microfluidic chip structure, wherein 1: an upper plate substrate; 2: an ITO coating layer; 3a: an upper hydrophobic layer; 3b: a lower hydrophobic layer; 4: a support body; 5: a hydrophilic groove; 6: a dielectric layer; 7: an electrode layer; 8: a lower plate substrate;
fig. 2 shows a top view of the microfluidic chip, wherein 1: a lower plate circuit connection point; 2 is an electrode layer array;
figure 3 shows a schematic diagram of single bacterial genome sequencing based on a digital microfluidic device.
Detailed Description
The invention provides a single bacterium genome sequencing method based on a digital microfluidic technology, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the description of the present invention, the terms "upper", "lower", and the like indicate orientations or positional relationships based on orientations or positional relationships shown in the drawings, which are merely for convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.
In the invention, the steps of sequencing the genome of the single bacterium are as follows:
a. incubating the bacteria sample on ice for 30min by using a fluorescent dye FM 1-43 with the concentration of 5 mu g/mL to perform fluorescent staining on the bacteria sample;
b. b, placing the bacterial suspension subjected to dyeing treatment in the step a into a digital microfluidic chip, adding filling oil to seal the chip, controlling the on-off of an electrode, moving the generated 30nL bacterial liquid drop to a hydrophilic groove through a drop channel, and observing, separating and capturing single bacteria through a fluorescence microscope;
c. c, cracking the single bacteria captured in the step B on a digital microfluidic chip, switching on and off a control electrode, allowing a cracking reagent A liquid drop of 30nL, a cracking liquid B liquid drop of 45nL and a neutralizing liquid of 45nL to enter a hydrophilic groove through a liquid drop generating channel, and cracking to obtain bacterial genome DNA;
d. switching on and switching off the control electrode, allowing the amplification reagent to enter the hydrophilic groove through the droplet generation channel, allowing the droplets of the amplification reagent to be 3 × 250nL, and incubating at 45 ℃ for 6h to amplify the genomic DNA;
e. and d, transferring the amplification product obtained in the step d into a centrifugal tube for nucleic acid purification and sequencing library construction, and finally, performing machine sequencing.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 Single bacterium genome sequencing method based on digital microfluidic technology
1. Digital microfluidic device structure
The digital microfluidic device comprises a digital microfluidic chip, an integrated circuit and an imaging system. The imaging system is positioned right above the digital microfluidic chip, has a multi-channel imaging function, is used for acquiring image information of the chip, liquid drops and bacteria, and is used for acquiring position and volume information of the liquid drops in a bright field; under a fluorescent field, the number of bacteria captured at the hydrophilic site is observed. The integrated circuit is a circuit control system of the digital microfluidic chip, and the liquid drops on the electrodes move by switching on and off adjacent electrodes on the integrated circuit chip.
The digital microfluidic chip comprises a bacteria capturing and cracking unit, a liquid drop generating channel, a liquid storage pool unit and an electrode interface. The bacterial capture and lysis unit is shown in figure 1 and comprises: the structure comprises an upper plate substrate (1 in figure 1), an ITO coating (2 in figure 1) arranged on the lower surface of the upper plate substrate, and an upper hydrophobic layer (3 a in figure 1) arranged on the lower surface of the ITO coating, wherein the upper plate substrate, the ITO coating and the upper hydrophobic layer are light-transmitting; the liquid crystal display panel comprises a lower plate substrate (8 in figure 1) and a dielectric layer (6 in figure 1) arranged on the upper surface of the lower plate substrate, wherein the dielectric layer has hydrophilic characteristics and can be adhered with tiny liquid drops; an electrode layer (7 in figure 1) embedded in the dielectric layer, wherein the electrode layer requires a conductive material, and comprises an electrode interface (1 in figure 2), a liquid storage battery electrode unit and an electrode layer array (2 in figure 2); a lower hydrophobic layer (3 b in fig. 1) disposed on the upper surface of the dielectric layer; a support (4 in fig. 1) supported between the lower hydrophobic layer and the upper hydrophobic layer; a liquid drop generating channel is formed among the upper hydrophobic layer, the lower hydrophobic layer and the support body; the lower hydrophobic layer is embedded with a hydrophilic groove (5 in figure 1), the hydrophilic groove is a hollow cylindrical structure with the diameter of 150-300 μm, and the distance between the hydrophilic groove and the liquid storage tank is more than 2 electrodes, so that small liquid drops are generated when the hydrophilic groove reaches the hydrophilic sites. The upper notch of the hydrophilic groove is communicated with the liquid drop generating channel, and 10 nL-250 nL of liquid drops are generated in the liquid drop generating channel as required through the control integrated circuit. The through holes of the droplet generation channels are communicated with the liquid storage tank units, the number of the liquid storage tank units is not less than 5, the liquid storage tank units are respectively used for storing lysis reagent A, lysis reagent B, neutralization solution, amplification reagent and bacteria solution (as shown in FIG. 3), the size of the liquid storage tank is usually 1mm multiplied by 1mm and is larger than the size of the channel, and the size ratio of the liquid storage tank to the channel is usually 5 to 1. The electrode interface is connected with the electrode.
2. Sequencing of single bacterial genomes
As shown in FIG. 3, the steps of the single-bacterium genome sequencing method based on the digital microfluidic technology include bacterial staining, single-bacterium separation, cell lysis, whole genome amplification, library construction and on-machine sequencing. The invention takes escherichia coli as a subject to carry out bacterial single genome sequencing, and the method comprises the following specific steps:
1. preparing a colibacillus sample into a bacterial suspension, adding fluorescent dye FM 1-43 with the final concentration of 5 mu g/mL into the bacterial suspension, and incubating for 30min on ice.
2. The lysis reagent A is lysozyme Ready-Lyse lysozyme 1600U/microliter, 800mM Dithiothreitol (DTT) and 4mM Ethylene Diamine Tetraacetic Acid (EDTA); the lysis reagent B is 400mM KOH,100mM DTT,10mM EDTA; the neutralization solution is 1M Tris-HCl. 300nL of the stained bacterial liquid, 300nL of lysis reagent A (lysozyme, DTT, EDTA mixed solution), 450nL of lysis reagent B (KOH and DTT mixed solution), 450nL of neutralization solution (HCl solution), 3000nL of MDA amplification reagent (random primer, equiphi29 polymerase, dNTP, reaction Buffer, DTT mixed solution, prepared according to Table 1) were added to different liquid storage tanks on the DMF chip.
TABLE 1 amplification mix preparation
| Reagent | Final concentration | Volume of | ||
| 1 | Equiphi29 DNA polymerase (10U/. Mu.L) | 1U/μL | 1.2 |
|
| 2 | 10 XEquiphi 29 |
1× | 1.2μL | |
| 3 | dNTP(10mM) | 0.4mM | 0.48 |
|
| 4 | Random primer (500. Mu.M) | | 1μL | |
| 5 | Dithiothreitol(DTT,100mM) | 10mM | 1.2μL | |
| 6 | H 2 O | 4.92μL |
3. The electrode driving circuit is controlled by a controller and a circuit control module, the on-off control of the electrodes is carried out according to a preset sequence, so that a bacterial suspension liquid drop (30 nL) is generated from the electrode unit of the bacterial liquid storage area, the liquid drop is moved to a hydrophilic point area by control, the cell suspension liquid drop passes through a hydrophilic point, a small drop is left at the hydrophilic point under the action of surface tension, the small drop possibly contains different numbers of bacteria, and the small drop repeatedly passes through the hydrophilic point until only contains a single bacterium, so that the single bacterium is captured. The original cell suspension liquid drop is moved to a waste liquid area to be removed by controlling an electrode driving circuit.
4. According to the method of step 3, a droplet (30 nL) of lysis reagent A is generated from the electrode unit of the liquid storage area of the lysis reagent 1, the droplet is moved to the electrode where the hydrophilic site is located to be mixed with single bacteria, the droplet is moved back and forth on the electrode to fully mix the reagents, and the mixture is incubated for 20min at normal temperature.
5. According to the method of step 3, a droplet (45 nL) of lysis reagent B is generated from the electrode unit of the liquid storage area of the lysis reagent 2, the droplet is moved to the electrode where the hydrophilic site is located and mixed with the droplet, the droplet is moved back and forth on the electrode to fully mix the reagents, and the mixture is incubated for 20min at normal temperature.
6. According to the method of step 3, a drop of neutralising solution (45 nL) is generated from the electrode unit in the reservoir of the neutralising solution, this drop is moved to the electrode where the hydrophilic site is located and mixed with the drop, the drop is moved back and forth over the electrode to mix the reagents thoroughly, and lysis is terminated.
7. According to the method of step 3, 3 MDA amplification droplets (3X 250 nL) are generated from the electrode unit of the MDA amplification solution storage area, the electrode where the droplets move to the hydrophilic site is mixed with the droplets, the droplets are moved back and forth on the electrode to fully mix the reagents, and the DNA is amplified after incubation for 6h at 45 ℃.
8. The amplification reaction is stopped at 75 ℃ for 15min, the whole process is completely closed, and pollution is not easy to introduce.
9. Recovering the amplified droplets into a test tube, and using a purification kit
The amplification product was purified using DNA Clean Beads (Vazyme biotech co., ltd.).
10. The purified product was quantified using the Equalbit dsDNA HS Assay Kit.
11. Use of TruePrep library construction kit TM DNA Library Prep Kit V2 for
And (5) carrying out library establishment on the amplification products.
12. Finally, the Illumina NovaSeq 6000 sequencing platform is used for carrying out 2 × 150bp double-end sequencing. 3. As a result:
we compared the results of sequencing of a single bacterial genome in DMF with sequencing of a single bacterial genome in a test Tube (Tube) and sequencing of a group bacterial (Bulk) genome. The results are shown in table 1, the read comparison rate and the contamination rate of the three are basically consistent, while the comparison rate of DMF is the highest, and the contamination rate is less than 5%, which indicates that DMF is excellent in preventing contamination. In the aspect of the most key index genome integrity, DMF is far higher than Tube and is close to Bulk, the average integrity of the genome is about 90%, and the genome of individual single bacterium is close to 100%, which shows that the small-volume reaction system of DMF can actually reduce the amplification deviation, thereby improving the genome integrity. In terms of the key indicators of genome assembly, maximum Contig and N50, DMF is much higher than Tube, individually comparable to Bulk. On the whole, the single bacterial genome sequencing result based on the digital microfluidic technology DMF established by the invention is obviously superior to the single bacterial genome sequencing result of a test tube, and the method has obvious advantages in single cell genome sequencing.
TABLE 2 sequencing results and evaluation of the genome of a single bacterium
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The digital microfluidic chip is characterized by comprising a bacteria capturing and cracking unit, a liquid drop generating channel, a liquid storage pool unit and an electrode interface; the number of the liquid storage tank units is not less than 5, and a cracking reagent A, a cracking reagent B, a neutralization solution, an amplification reagent and a bacterial solution are respectively stored in the liquid storage tank units; wherein,
the cracking reagent A comprises: 800-2400U/microliter lysozyme, 400-1200 mM dithiothreitol and 2-6 mM ethylene diamine tetraacetic acid;
lysis reagent B comprises: 300-500 mM KOH, 80-120 mM dithiothreitol and 5-15 mM ethylene diamine tetraacetic acid;
the neutralizing liquid comprises: 0.8-1.2M Tris-HCl solution;
the amplification reagents include: random primers, DNA polymerase, dNTP, amplification buffer and dithiothreitol.
2. The digital microfluidic chip according to claim 1, wherein the DNA polymerase in the amplification reagents comprises: 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, further comprising a fluorescent dye, a filling oil and a purification reagent;
the fluorescent dye is selected from PI, DAPI, SYTO-9, FM 1-43FX, FM 4-64, FM 4-64FX, bacLigh background colors, alexa Fluor 647NHS ester, cellTracker TM Red CMTPX or FM 1-43;
the filling oil is selected from mineral oil, fluorine oil, silane oil and dimethyl silicone oil;
the purification reagent is selected from a column purification reagent or a magnetic bead purification reagent.
4. The digital microfluidic chip according to any one of claims 1 to 3, wherein the volume ratio of the lysis reagent A to the lysis reagent B to the neutralization solution is 1 (1-1.5) to 1 (1-1.5); the volume ratio of the amplification reagent to the mixed solution of the lysis reagent A, the lysis reagent B and the neutralization solution is 1.
5. The digital microfluidic chip according to claim 1, wherein the bacteria capturing and lysing unit comprises: the device comprises an upper polar plate substrate, an ITO coating arranged on the lower surface of the upper polar plate substrate, and an upper hydrophobic layer arranged on the lower surface of the ITO coating; the device comprises a lower polar plate substrate, a medium layer arranged on the upper surface of the lower polar plate substrate, an electrode embedded in the medium layer, a lower hydrophobic layer arranged on the upper surface of the medium layer, and a support body supported between the lower hydrophobic layer and the upper hydrophobic layer, wherein a liquid drop generation channel is formed among the upper hydrophobic layer, the lower hydrophobic layer and the support body, a hydrophilic groove is embedded in the lower hydrophobic layer, and an upper notch of the hydrophilic groove is communicated with the liquid drop generation channel; the port of the liquid drop generating channel is communicated with the liquid storage pool unit; the electrode interface is connected with the electrode.
6. The digital microfluidic chip according to claim 5, wherein the hydrophilic groove is a hollow cylindrical structure with a diameter of 150-300 μm and is spaced from each reservoir by more than 2 electrodes.
7. The digital microfluidic chip according to claim 5, wherein the size of the liquid reservoir is 1mm x 1mm, and the ratio of the diameter of the liquid reservoir to the diameter of the channel is (5-8): 1.
8. a digital microfluidic device comprising the digital microfluidic chip of any one of claims 1 to 7, an integrated circuit, and an imaging system; the integrated circuit is connected with an electrode interface of the digital microfluidic chip.
9. The method for sequencing the genome of a single bacterium is characterized by comprising the digital microfluidic chip of any one of claims 1 to 7 and/or the digital microfluidic device of claim 8 for performing separation and capture, nucleic acid amplification, library construction and genome sequencing of the single bacterium.
10. The method according to claim 9, comprising in particular:
after dyeing, placing the bacterial liquid in a liquid storage tank of a digital micro-fluidic chip, sealing the chip by filling oil, and then controlling the on-off of an electrode by an integrated circuit, so that the bacterial liquid moves to a hydrophilic groove through a droplet generation channel, and separating and capturing single bacteria;
controlling the electrode to be powered on and powered off, enabling the cracking reagent A, the cracking reagent B, the neutralization solution and the amplification reagent to enter the hydrophilic groove through the liquid drop generation channel in sequence, and then carrying out amplification;
and (4) purifying the amplification product, constructing a library, and sequencing.
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