CN114774519A - Method for constructing microbial single cell whole genome amplification and sequencing library - Google Patents
Method for constructing microbial single cell whole genome amplification and sequencing library Download PDFInfo
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Abstract
The invention provides a method for amplifying a microbial single cell whole genome, which at least comprises the following steps: (1) capturing single microbial cells in a single microchamber, obtaining a plurality of microchambers independently comprising single microbial cells: (2) and carrying out the amplification of the whole genome of the single cell of the microorganism in a micro chamber containing the single cell genome of the microorganism and an amplification reaction system to obtain an amplification product containing the whole genome of the single cell of the microorganism. The method for amplifying the whole genome of the single cell of the microorganism has the advantages of greatly compressing the volume of a reaction system, improving the relative concentration of substrates in the reaction system, isolating the outside and cross contamination, improving the amplification quality of the whole genome of the single cell of the microorganism and improving the genome sequencing coverage rate. According to the invention, the sequencing library construction method can greatly improve the sequencing efficiency and the sequencing coverage rate of the genome.
Description
Technical Field
The invention relates to the field of single cell sequencing, in particular to a method for amplifying a whole genome of a microbial single cell and constructing a sequencing library.
Background
For a long time, the microorganisms remained largely a black box due to the lack of a powerful research method. Since most of microorganisms cannot be purely cultured under the existing experimental conditions, the discovery and research of microbiomes mainly depend on metagenomic methods. The macro genomics uses microbial genome in environmental samples as research objects, essentially has no single cell resolution, cannot obtain independent data of a single species, and full mining of unknown microorganisms often needs to obtain the whole genome information of the microorganisms. Therefore, the mainstream metagenomic method is very popular when studying rare microbial species; the development of an efficient microbial group research method with microbial single cell resolution, in particular to a large-scale whole genome amplification and sequencing library construction method, has important value for promoting the research of microbiology.
In recent years, the rapid development of high-throughput single-cell sequencing technology is gradually pushing life sciences to the single-cell era. The successful establishment of single cell transcriptome sequencing, methylation sequencing and other advanced methodologies enables scientific researchers to analyze information such as gene expression, epigenetics and the like at a single cell level. Such methodologies rely primarily on high throughput microfluidic technologies (particularly micro-droplet technologies) to efficiently perform single cell sequencing library preparation. However, in the field of microbial research, single cell analysis is not widespread and the development of related methodologies is difficult. Taking the whole genome amplification and sequencing library construction as an example, because the genome of a microorganism is small (Kbp-Mbp level), high-quality amplification products cannot be obtained by using methods such as Polymerase Chain Reaction (PCR), Multiple Displacement Amplification (MDA) and the like, and high amplification bias and large amount of genome fragments are lost. In addition, there is a problem in how to prepare a sequencing library such as a single genomic marker.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method for amplifying a whole genome of a single cell of a microorganism and constructing a sequencing library.
The first aspect of the invention provides a method for amplifying the whole genome of a microbial single cell, which at least comprises the following steps:
(1) capturing single microbial cells in a single microchamber, obtaining a plurality of microchambers independently comprising single microbial cells:
(2) and carrying out the amplification of the whole genome of the single cell of the microorganism in a micro chamber containing the single cell genome of the microorganism and an amplification reaction system to obtain an amplification product containing the whole genome of the single cell of the microorganism.
The second aspect of the invention provides a sequencing library construction method of a whole genome of a single cell of a microorganism, which comprises the following steps:
A. fragmenting an amplification product in a microchamber containing a microbial unicell whole genome amplification product obtained by the microbial unicell whole genome amplification method to obtain a microchamber containing the fragmented amplification product;
B. and C, connecting the fragmented amplification products in the micro chambers obtained in the step A with the barcode fragments in a single micro chamber, and obtaining a sequencing library of the whole genome of the single microbial cell, wherein the whole genome of the single microbial cell is in the micro chambers, and each micro chamber has the whole genome of the single microbial cell.
As mentioned above, the method for constructing the microbial single cell whole genome amplification and sequencing library has the following beneficial effects:
a. the invention can provide a new important method for exploring microorganism dark substances.
b. The amplification method of the whole genome of the single cell of the microorganism greatly compresses the volume of a reaction system, improves the relative concentration of substrates in the reaction system, isolates the outside and cross contamination, improves the amplification quality of the whole genome of the single cell of the microorganism and improves the genome sequencing coverage rate.
c. In the sequencing library construction method, the steps of indexing the amplified product of the whole genome of the single cell of the microorganism are completed by fusing the amplified product fragment of the whole genome of the single cell of the microorganism and microspheres or microdroplets carrying barcode sequences in an independent micro-chamber and under the action of PCR or ligase (ligase); the method can greatly improve the sequencing efficiency and the sequencing coverage rate of the genome.
Drawings
FIG. 1: the invention discloses a flow chart of a method for constructing a sequencing library of a single cell whole genome of a microorganism.
FIG. 2 is a schematic diagram: micro-droplets of either a single E.coli or a single B.subtilis were packed (arrowed).
FIG. 3: after the MDA reaction, micro-droplets containing single-bacterium whole genome amplification products (green fluorescence indicates that the whole genome amplification products of single escherichia coli or bacillus subtilis are contained in the micro-droplets).
FIG. 4 is a schematic view of: polyacrylamide hydrogel microspheres containing single-bacterium whole genome amplification products.
FIG. 5: polyacrylamide hydrogel microspheres containing single-bacterium whole genome amplification product fragments.
FIG. 6: bioanalyzer analysis of unsorted magnetic bead libraries versus magnetic bead sorted libraries.
FIG. 7: distribution of index sequences and corresponding numbers of reads.
FIG. 8: and (3) comparing the sequencing data with reference to escherichia coli and bacillus subtilis genomes.
Detailed Description
The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any number between the two endpoints are optional unless otherwise specified in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in connection with the examples may be used in the practice of the invention, as will be recognized by those skilled in the art after having the benefit of the description of the invention and understanding the nature of the present application.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
The amplification method of the microbial single cell whole genome of one embodiment of the invention at least comprises the following steps:
(1) capturing single microbial cells in a single microchamber, obtaining a plurality of microchambers independently comprising single microbial cells:
(2) and carrying out amplification on the whole microbial unicell genome in a micro chamber containing the microbial unicell genome and an amplification reaction system to obtain an amplification product containing the whole microbial unicell genome.
Amplification in the microchamber can greatly compress the volume of the reaction system and simultaneously can improve the relative concentration of the substrate in the reaction system. For example, the whole genome amplification of a single cell in a 20. mu.l system in a PCR tube is compared with the whole genome amplification in a single micro-droplet, and the volume of the droplet is about nl, so that the volume of the reaction can be greatly compressed, and the relative concentration of the genome of a single microorganism cell in the PCR tube is different from that in the single droplet.
In the present invention, single cells of the target microorganism can be captured in a separate microchamber (picoliter to nanoliter scale) for subsequent experiments by using various techniques.
Alternatively, the micro-chamber may be in a form selected from, but not limited to, a micro-droplet, a microsphere, a semi-permeable membrane system, a liposome system, a micro-well plate, or a centrifuge tube.
The volume of the micro-chamber is in the order of picoliters to nanoliters.
In one embodiment, the micro-chamber contains a micro-chamber matrix, which may be selected from, but not limited to, polyacrylamide, polyethylene glycol, or agarose.
Alternatively, step (1) may be performed in a microfluidic droplet generation chip. Specifically, oil, the microbial single cell lysate and the microbial single cell suspension are pumped into a flow channel of the microfluidic droplet generation chip to be mixed to obtain the micro-droplets containing the microbial single cell whole genome.
The volume of the micro-chamber is in the order of picoliters to nanoliters.
In the step (2), the mode for forming the micro-chamber containing the microbial single cell genome and the amplification reaction system is selected from any one of the following modes:
I. carrying out in-situ lysis on the microbial single cells contained in the single micro-chamber to obtain a micro-chamber containing the genome of the microbial single cells, adding an amplification system into the micro-chamber containing the genome of the microbial single cells to form the micro-chamber containing the genome of the microbial single cells and an amplification reaction system;
and II, adding an amplification reaction system into the micro-chamber containing the microbial single cells to form the micro-chamber containing the microbial single cells and the amplification reaction system, and carrying out in-situ lysis and amplification on the microbial single cells in the micro-chamber to form the micro-chamber containing the microbial single cell genome and the amplification reaction system.
The operation of lysing the single cells of the microorganism may be selected from any of the following methods, but is not limited to the following methods:
1) the enzyme cracking method is to add lyase into the unicell of the microorganism to carry out corresponding chemical reaction to crack the cell and release the intracellular DNA. The lytic enzyme may be, but is not limited to: labiase lyase, lysostaphin, hen egg protein-derived lysozyme, human-derived lysozyme, or digestive peptidase.
2) The alkaline lysis method is that alkaline is added into the microbial unicell, and the unicell is ruptured and intracellular DNA is released under specific alkaline conditions (pH is more than 7 and pH is less than or equal to 14) and at proper temperature. The base may be, but is not limited to: KOH or NaOH, etc.
3) The heat shock method is that microbial unicells are repeatedly frozen and thawed to cause swelling of the cells, so that cell structures are broken, and intracellular DNA is released;
4) ultrasonic treatment, i.e., ultrasonic heating, causes the cells to be disrupted, releasing intracellular DNA.
In the step (2), the microbial single cells are subjected to in-situ whole genome amplification, and an amplification reaction system (reaction reagent) needs to be fused with a microcavity containing the whole genome of the microbial single cells.
Optionally, the amplification reaction system is enclosed in a micro chamber.
In one embodiment, the method for fusing the amplification reaction system to the micro-chamber containing the whole genome of a single cell of a microorganism may be selected from any one of the following methods, but is not limited to the following experimental methods:
injecting the amplification reaction system into a micro chamber containing the whole genome of the single cell of the microorganism or fusing the amplification reaction system with the micro chamber containing the whole genome of the single cell of the microorganism.
In one embodiment, the amplification reaction system may be injected into or fused with the microdroplet via a microfluidic droplet generation chip.
In one embodiment, the amplification is performed at a suitable temperature for 0.01 to 999 hours. After amplification, a high-quality microbial unicell whole genome amplification product which can be used for downstream experiments can be obtained.
In one embodiment, in step (2), the amplification method may be selected from any one of the following methods but is not limited to the following amplification methods:
1) PCR (polymerase chain reaction) for amplifying single cells of microorganisms by using the polymerase chain reaction.
2) Mda (multiple displacement amplification) that binds to a DNA template using random primer hexamers and polymerase and amplifies.
The polymerase may be Φ 29DNA polymerase.
3) MALBAC (Multiple Annealing and Looking-Based Amplification Cycles) performs Multiple Annealing circular Amplification on the whole genome of a single cell of a microorganism.
4) LIANTI (Linear amplification of a single cell transposon insertion), linear amplification of the whole genome of a single cell of a microorganism by insertion of a transposon.
For the PCR reaction, the cell can be cracked without the modes of an enzymatic cracking method, an alkaline cracking method, a heat shock method, an ultrasonic wave treatment method and the like, the denaturation temperature of the PCR reaction is equivalent to that in-situ cracking is carried out in a micro chamber containing the single cell of the microorganism, and the cell can be cracked to release the DNA in the cell, meanwhile, the primer of the PCR reaction can be designed to amplify a specific segment in the single cell of the microorganism, including but not limited to the amplification of the 16S full length or the variable region of the microorganism, or be designed to be a plurality of pairs of random primers to realize the whole gene amplification in the genome of the single cell of the microorganism.
As shown in fig. 1, the method for constructing a sequencing library of a whole genome of a single cell of a microorganism according to an embodiment of the present invention includes the following steps:
A. fragmenting an amplification product in a microchamber containing a microbial unicell whole genome amplification product obtained by the microbial unicell whole genome amplification method to obtain a microchamber containing the fragmented amplification product;
B. and C, connecting the fragmented amplification products in the micro chambers obtained in the step A with the barcode fragments in a single micro chamber, and obtaining a sequencing library of the whole genome of the single microbial cell, wherein the whole genome of the single microbial cell is in the micro chambers, and each micro chamber has the whole genome of the single microbial cell.
In the step (2), the amplification primer is selected from a random primer or a region-specific primer.
In one embodiment, the method comprises obtaining a fragmented amplification product in a microchamber comprising: enabling the micro-cavity of the amplification product of the whole genome of the single cell of the microorganism to contain hydrogel components, and solidifying under the condition of proper solidification to obtain the hydrogel micro-cavity containing the amplification product of the whole genome of the single cell of the microorganism; and fusing the hydrogel micro-chamber containing the microbial single cell whole genome amplification product with a fragmentation reaction system to obtain the hydrogel micro-chamber containing the fragmented amplification product.
The hydrogel is a gel with a three-dimensional network structure, and the aggregation state of the gel is not complete solid or complete liquid. The behavior of a solid is that it can maintain a certain shape and volume under certain conditions, and the behavior of a liquid is that a solute can diffuse or permeate from the hydrogel. Therefore, the fragmented amplification products can be loaded in the grid of the hydrogel, and a liquid-state amplification system or a fragmentation reaction system can penetrate into the hydrogel microspheres to react with the fragmented amplification products.
Optionally, the hydrogel agent comprises acrylamide monomer and ammonium persulfate.
In one embodiment, the conditions suitable for solidification include, but are not limited to, UV irradiation, a change in temperature or a change in pH environment, and the like.
The injection of the hydrogel reagent can be performed in any one of the steps (1) to (3), and the hydrogel microspheres containing the fragmented amplification products can be obtained.
In one embodiment, the hydrogel microspheres are selected from polyacrylamide hydrogel microspheres.
In one embodiment, the injection of the hydrogel reagent may be performed by a microfluidic droplet generation chip.
In one embodiment, in step a, the fragmentation method is selected from any one of, but not limited to, the following methods:
1) and (3) an ultrasonic method, wherein ultrasonic waves are utilized to break the amplification product of the single cell genome of the microorganism.
2) And (3) a centrifugal shearing method, wherein shearing force is generated by centrifugation to fragment the amplified product of the single cell genome of the microorganism.
3) Enzymatic cleavage methods, including but not limited to the following methods:
a. shearing the amplified product of the microbial unicell genome by using dsDNA fragmentation enzyme, and fragmenting the amplified product;
b. a transposase is used, which is inserted into the amplification product of the microbial unicellular genome and fragmented.
Transposases include, but are not limited to, Tn5 transposase. Tn5 transposases are currently commercially available and kits containing Tn5 transposases (including but not limited to Nextera XT DNA Library Preparation Kit, TruePrepTM DNA Library Kit V2 for Illumina) can be used
Etc.).
Preferably, in step B, the micro-chamber containing the fragmented amplification products and the microspheres containing the barcode sequences, the amplification reaction system, the ligase reaction system, or the hybridization reaction system are co-embedded in the same micro-chamber, so that the barcodes on the microspheres containing the barcode sequences are detached, and the amplification or ligation or hybridization reaction is performed to ligate the fragmented amplification products to the barcode fragments.
The barcode sequences attached to each barcode microsphere are different. Enabling fragmentation amplification products in the same micro-droplet to be from the same single cell and connected with the same bar code; that is, the fragmented amplification products contained in different microdroplets are all from different single cells and are linked to different barcodes, allowing for label discrimination during sequencing.
Methods for shedding barcodes from microspheres containing barcode sequences are known in the art and include, but are not limited to, chemical bond (e.g., disulfide bond) cleavage and ultraviolet light cleavage, for example, DTT (dithiothreitol) can be added to cause a disulfide cleavage reagent to shed barcodes into the microdroplets for reaction.
Indexing can be accomplished without electrode fusion.
The ligation method may be PCR, ligase ligation, hybridization or the like.
The sequencing library can be added with an adaptor which can be identified by a sequencing platform, and the step of adding the adaptor can be carried out after the indexing step is completed, or can be simultaneously added to the genome fragment in the indexing process.
Linker sequences include, but are not limited to, P5 and P7 linker sequences common to Illumina sequencing platforms, autonomously designed linker sequences, and the like.
After the addition of the linker sequence is complete, the sequencing library is size-selected and library purified by methods including, but not limited to: magnetic beads, gel electrophoresis, etc., and library purification methods include, but are not limited to, methods using commercial purification kits, etc.
After library construction and sequencing, metagenomics analysis can be performed that can identify individual genomes.
In one embodiment, the construction process of the sequencing library construction method of the whole genome of the single cell of the microorganism is carried out in a microfluidic droplet generation chip.
The microfluidic droplet generation chip is used for realizing the fusion of micro droplets or microspheres. The preparation required by the experiment is pumped to the inlet of the microfluidic droplet generation chip, converged and formed into laminar flow, and then converged with the oil phase, so that monodisperse micro-droplets can be formed.
The microfluidic droplet generation chip is of the prior art, e.g. according tohttps://mccarrolllab.org/ dropseq/Construction was performed (Macosko E, Basu A, Satija R, et al. highly Parallel Genome-wide Expression Profiling of Industrial Cells Using nanoliters drops [ J]Cell,2015,161(5): 1202-1214). The innovation point of the method is that after the method is obtained by the technical personnel in the field, the microfluidic droplet generation chip can be used for operation without creative labor.
Example 1 preparation of microbial Single-cell Whole genome amplification and sequencing library of Mixed samples of Escherichia coli and Bacillus subtilis Escherichia coli (Escherichia coli) and Bacillus subtilis (Bacillus subtilis) were respectively cultured in LB (Luria-Bertani) culture solution, the absorbance at 600nm was measured, and the number of Escherichia coli and Bacillus subtilis (OD600 ═ 1, corresponding bacteria) was calculated from the relationship between the bacterial concentration and absorbanceAmount of 109CFU/ml). Centrifuging Escherichia coli solution and Bacillus subtilis solution at 2000g for 5min respectively to remove supernatant, adding PBS buffer solution, cleaning, centrifuging again to remove supernatant, dissolving in sterile 1xPBS solution (pH 7.2) under ultraviolet irradiation for half an hour before use, and diluting to 2.4 × 107CFU/ml (i.e., 2.4X 10)7microbial single cell/ml). And (3) uniformly mixing the newly prepared escherichia coli liquid and bacillus subtilis liquid in a ratio of 1:1(v/v) to obtain the mixed bacterial suspension required by the experiment.
Preparing a lysate: the lysate used was the lysate provided in the REPLI-g Single Cell Kit (Qiagen, Germany) Kit.
And (3) inputting the mixed bacterial suspension and the lysate into a microfluidic droplet generation chip to generate micro-droplets wrapped by single escherichia coli or single bacillus subtilis, and collecting the micro-droplets into a 1.5ml sterile centrifuge tube. The single bacterial micro-droplets collected, which contain the DNA of a single Escherichia coli or a single Bacillus subtilis, are incubated at 65 ℃ for 10 minutes to achieve bacterial lysis and sufficient release of intracellular DNA.
Mixing micro-droplets containing single escherichia coli or single bacillus subtilis DNA with micro-droplets containing MDA reaction reagents (the formula is shown in table 1) (a micro-fluidic droplet generation chip is used in advance to generate micro-droplets containing MDA reflecting the reality), fusing 1:1 under the action of an electric field to generate new micro-droplets (containing single bacterial DNA and MDA reaction reagents), collecting the new micro-droplets into a 1.5ml sterile centrifuge tube, reacting for 8 hours at 30 ℃, and reacting for 15 minutes at 65 ℃ to terminate the amplification reaction, thus obtaining micro-droplets containing single bacterial whole genome amplification products (as shown in figure 3).
TABLE 1 MDA reaction reagent compounding list
And fusing the micro-droplets containing the single-bacterium whole genome amplification product with polyacrylamide hydrogel micro-droplets (which are generated by a micro-fluidic droplet generation chip in advance, wherein the concentration of acrylamide monomers is 30% w/v, and the concentration of ammonium persulfate is 3.3% w/v) in a ratio of 1:1 under the action of an electric field, collecting the newly generated micro-droplets in a 1.5ml sterile centrifuge tube, and standing at room temperature overnight to form the gel, thereby obtaining the hydrogel microspheres containing the single-bacterium whole genome amplification product (as shown in figure 4). And (3) performing demulsification and cleaning on the collected hydrogel microspheres to perform fragmentation operation of single-bacterium whole genome amplification yield increase.
Adding the polyacrylamide hydrogel microspheres containing the single-bacterium whole genome amplification product into a fragmentation reaction system (shown in table 2), and reacting at 55 ℃ for 2 hours to obtain the polyacrylamide hydrogel microspheres containing the single-bacterium whole genome amplification product fragments (shown in figure 5).
TABLE 2 fragmentation reaction System
The hydrogel microspheres containing the single-bacterium whole genome amplification product fragments, microbeads containing barcode sequences and micro-droplets of PCR reaction reagents are fused in a ratio of 1:1 under the action of an electric field, DTT reagents are added to enable the barcode sequences to fall off from the microbeads containing the barcode sequences into the micro-droplets, and the indexing step of the single-bacterium whole genome amplification product fragments is completed through PCR reaction (a reaction system is shown in table 3, and reaction conditions are shown in table 4). In this indexing step, the genome fragments have been added with P5 and P7 linkers recognizable by the Illumina sequencing platform, and thus, the sequencing library construction has been completed.
The preparation method of the micro-beads of the barcode sequences comprises the following steps: the surface of the microspheres was modified by an Applied Biosystems,3900DNA synthesizer to synthesize the desired barcode sequences.
TABLE 3 indexed reaction systems
| Components | Volume of |
| 2％SDS | 2μl |
| Enhancer(4x) | 25μl |
| Stabilizer(5x) | 20μl |
| EvaGreen(20x) | 10μl |
| Primer FL127 | 6μl |
| Barcode-15-3-66 | 6μl |
| TAE | 20μl |
| Ribozyme-free water | 9μl |
TABLE 4 indexed PCR reaction conditions
And (3) carrying out size screening and purification on the constructed sequencing library by utilizing VAHTS DNA Clean Beads (Novozam), screening and purifying the library with the size within the range of 200-600bp according to an experimental process provided by a manufacturer, carrying out quality control on the sequencing library by utilizing a Bioanalyzer (figure 6), and carrying out second-generation sequencing on the purified sequencing library after error is determined. The library was paired-end 150bp sequenced by Decode (Shanghai) biomedical science and technology, Inc. (hereinafter "Decode") using the DNBSEQ-T7 platform.
After sequencing is completed, the original data fastq file obtained from the decoded DNBSEQ-T7 platform is subjected to quality control to remove data with sequencing quality lower than Q20, and then the sequencing data after quality control are grouped by using a bbtools program according to an index sequence. Theoretically, each index sequence represents a single cell. The distribution of index sequences and corresponding numbers of reads is shown in fig. 7. After grouping, sequencing data of each group were aligned with reference to E.coli and B.subtilis genomes using bowtie2 v2.4.4, respectively, and the alignment results of kraken were visualized with krona.
Alignment results are shown in fig. 8, which is a graph of the results in fig. 8 showing information that there is and only one bacterial genome inside each droplet. The result shows that the sequencing data obtained by the method has low loss of genome fragments and high accuracy.
The above examples are intended to illustrate the disclosed embodiments of the present invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (10)
1. A method for amplifying the whole genome of a microbial single cell at least comprises the following steps:
(1) capturing single microbial cells in a single microchamber, obtaining a plurality of microchambers independently comprising single microbial cells:
(2) and carrying out the amplification of the whole genome of the single cell of the microorganism in a micro chamber containing the single cell genome of the microorganism and an amplification reaction system to obtain an amplification product containing the whole genome of the single cell of the microorganism.
2. The method for amplifying the whole genome of a single cell of a microorganism according to claim 1, wherein in the step (2), the manner of forming the microchamber comprising the genome of the single cell of the microorganism and the amplification reaction system is selected from any one of the following:
I. carrying out in-situ lysis on the microbial single cells contained in the single micro-chamber to obtain a micro-chamber containing the genome of the microbial single cells, and adding an amplification system into the micro-chamber containing the genome of the microbial single cells to form the micro-chamber containing the genome of the microbial single cells and an amplification reaction system;
and II, adding an amplification reaction system into the micro-chamber containing the microbial single cells to form the micro-chamber containing the microbial single cells and the amplification reaction system, and carrying out in-situ lysis and amplification on the microbial single cells in the micro-chamber to form the micro-chamber containing the microbial single cell genome and the amplification reaction system.
3. The method for amplifying the single cell of the microorganism according to claim 2, wherein the lysis method is selected from the group consisting of enzymatic lysis, alkaline lysis, heat shock lysis and ultrasonic lysis.
4. The method for amplifying the whole genome of a single cell of a microorganism according to claim 1, further comprising one or more of the following characteristics:
1) the micro chamber is in the form of micro liquid drop, microsphere, semi-permeable membrane system, liposome system, micro porous plate or centrifugal tube;
2) the volume of the micro chamber is from picoliter to nanoliter magnitude;
3) the micro-chamber contains a micro-chamber matrix, and the micro-chamber matrix is selected from polyacrylamide, polyethylene glycol or agarose;
4) in step (2), the amplification method is selected from PCR, MDA, MALBAC or LIANTI.
5) In the step (2), the amplification primer is selected from a random primer or a region-specific primer.
5. A method for constructing a sequencing library of a microbial single cell whole genome comprises the following steps:
A. fragmenting amplification products in a microchamber containing amplification products of the whole genome of the single cell of the microorganism obtained by the amplification method of the whole genome of the single cell of the microorganism of any one of claims 1 to 4 to obtain a microchamber containing the fragmented amplification products;
B. and C, connecting the fragmented amplification products in the micro chambers obtained in the step A with the barcode fragments in a single micro chamber, and obtaining a sequencing library of the whole genome of the single microbial cell, wherein the whole genome of the single microbial cell is in the micro chambers, and each micro chamber has the whole genome of the single microbial cell.
6. The method for constructing the sequencing library of the whole genome of a single cell of a microorganism according to claim 5, further comprising one or more of the following features:
1) the micro chamber is in the form of micro liquid drop, microsphere, semi-permeable membrane system, liposome system, micro porous plate or centrifugal tube;
2) the volume of the micro chamber is from picoliter to nanoliter magnitude;
2) the micro-chamber is internally provided with a micro-chamber matrix, and the micro-chamber matrix is selected from polyacrylamide, polyethylene glycol or agarose.
7. The method for constructing the sequencing library of the whole genome of a single cell of a microorganism according to claim 5, wherein the microchamber containing the fragmented amplification product is obtained by the following method: enabling the micro-cavity of the amplification product of the whole genome of the single cell of the microorganism to contain hydrogel components, and solidifying under the condition of proper solidification to obtain the hydrogel micro-cavity containing the amplification product of the whole genome of the single cell of the microorganism; and fusing the hydrogel micro-chamber containing the microbial single cell whole genome amplification product with a fragmentation reaction system to obtain the hydrogel micro-chamber containing the fragmented amplification product.
8. The method for constructing the sequencing library of the whole genome of a single cell of a microorganism according to claim 5, wherein in the step A, the fragmentation method is selected from any one of the following methods: ultrasonic breaking, centrifugal shearing or enzyme digestion.
9. The method for constructing the sequencing library of the whole genome of a single cell of the microorganism according to claim 8, wherein the enzyme used in the enzymatic cleavage is selected from dsDNA fragmentation enzyme and transposase.
10. The method of claim 5, wherein in step B, the micro-chamber containing the fragmented amplification products and the microspheres containing the barcode sequences, the amplification reaction system, the ligase reaction system, or the hybridization reaction system are co-embedded in the same micro-chamber, so that the barcodes on the microspheres containing the barcode sequences are detached, and the amplification, ligation, or hybridization reactions are performed, so that the fragmented amplification products and the barcode fragments are ligated.
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| CN116656780A (en) * | 2023-05-26 | 2023-08-29 | 上海科技大学 | Single-cell whole genome sequencing library-building method |
| CN116694736A (en) * | 2023-07-18 | 2023-09-05 | 墨卓生物科技(浙江)有限公司 | Multiple displacement amplification reaction liquid, amplification method and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116656780A (en) * | 2023-05-26 | 2023-08-29 | 上海科技大学 | Single-cell whole genome sequencing library-building method |
| CN116694736A (en) * | 2023-07-18 | 2023-09-05 | 墨卓生物科技(浙江)有限公司 | Multiple displacement amplification reaction liquid, amplification method and kit |
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