CN116622807A - Construction method of single-cell whole genome sequencing library - Google Patents
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Abstract
The application provides a construction method of a single-cell genome sequencing library, which comprises the following steps: the construction method comprises the following steps: 1) Providing a microcavity comprising whole genomic DNA of a single cell; 2) Adding a random primer carrying an index bar code and an amplification reagent into the microcavity for amplification to obtain a whole genome amplification product containing the index bar code; 3) Purifying to obtain the single-cell whole genome sequencing library. According to the application, in the single-cell whole genome amplification process, a cell specific barcode sequence is introduced, so that single-cell whole genome amplification is realized, meanwhile, a cell specific identification index barcode is marked on each single-cell whole genome amplification product, and three-generation sequencing is carried out by connecting an upper joint, so that complex genome assembly and metagenome assembly with haplotype resolution can be realized.
Description
Technical Field
The application belongs to the field of single-cell sequencing, and particularly relates to a construction method of a single-cell whole genome sequencing library.
Background
In nature, complex biological systems are composed of many different types of cells, and the biological functions of biological systems depend on synergy between single cells. Traditional research methods have focused on cell populations and, although large amounts of genetic composition or transcriptome data are available, cell heterogeneity that contributes to this complexity cannot be revealed. In recent years, single-cell sequencing technology is developed, and through a sequencing method based on high throughput, information of transcriptomes, genomes and proteomes of single cells can be analyzed on a single cell level, heterogeneity among the cells is analyzed, so that researchers can conveniently recognize and understand a complex biological system, and the mystery of life is revealed.
Current single cell sequencing techniques still rely on second generation sequencing methods. Taking single-cell whole genome sequencing as an example, firstly, cells are separated and captured in an independent space by a certain technology, such as a microfluidic technology, a flow cell sorting technology or a manual sorting technology; then, in the independent space, the single cells are cracked to obtain genome DNA of the single cells, and the cracking method can be ultrasonic crushing, enzyme cracking and the like; finally, constructing a second generation sequencing library according to a second generation sequencing library construction flow, wherein the second generation sequencing library construction flow comprises the steps of whole genome amplification, amplification product indexing, amplification product fragmentation, second generation sequencing connector addition and the like, and then performing sequencing analysis on a machine. However, in the sequencing technology of second generation sequencing based on short reads, single DNA molecules must be amplified into gene clusters composed of the same DNA, and then synchronously replicated to enhance fluorescence signal intensity, so that DNA sequences are read out, and as reads grow, the synergy of gene cluster replication decreases, resulting in degradation of base sequencing quality, limiting the length of reads (not more than 600 bp) of second generation sequencing, so that genome fragmentation operation is required to ensure sequencing quality. In addition, reads obtained from second generation sequencing are generally short, and when de novo splicing of genomes of unknown species is performed, contigs assembled from sequencing data are utilized, and because the read size is too small, larger genome repeated sequences cannot be identified, thus preventing assembly of complex genomes and analysis of haplotype genetic information; in metagenome, due to high sequencing complexity and low sequence coverage, short sequences obtained by sequencing are often not overlapped, thus causing difficulty in metagenome assembly, and thus making it difficult to index full-length genes and metabolic pathways from microbial communities.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present application is to provide a method for constructing a single cell whole genome sequencing library.
The first aspect of the application provides a construction method of a single-cell whole genome sequencing library, which comprises the following steps:
1) Providing a microcavity comprising whole genomic DNA of a single cell;
2) Adding a random primer carrying an index bar code and an amplification reagent into the microcavity for amplification to obtain a whole genome amplification product containing the index bar code;
3) Purifying to obtain the single-cell whole genome sequencing library.
Preferably, the length of the random primer carrying the index bar code is 2-99 bases.
Preferably, the random primer carrying the index barcode comprises a barcode sequence and a random primer sequence.
Preferably, the micro-chamber has a volume size of picoliters to nanoliters.
Preferably, the microcavity is selected from the group consisting of a microdroplet, a gel microsphere, a semipermeable membrane system, a liposome system, a microplate or a centrifuge tube.
More preferably, when the microcavity is a microdroplet, the random primers carrying the index barcode and the amplification reaction reagents are added to the microdroplet.
Further preferably, the random primer carrying an index barcode is added to the microcavity in the form of a micro droplet or microsphere.
Further preferably, the amplification reagents are added to the microcavity in the form of microdroplets or microspheres.
Still more preferably, the method of addition employs one or both of a droplet fusion method and a droplet injection method.
More preferably, when the microcavity is a gel microsphere, the gel microsphere is reduced to a gel droplet, and the random primer carrying the index barcode and the amplification reagent are added to the gel droplet.
Further preferably, the reduction is carried out by treating with a reducing agent. Preferably, the reducing agent is selected from one or more of dithiothreitol, mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride.
Further preferably, the random primer carrying an index barcode is added to the microcavity in the form of a micro droplet or microsphere.
Further preferably, the amplification reagents are added to the microcavity in the form of microdroplets or microspheres.
Still more preferably, the method employs one or both of a droplet fusion method or a droplet injection method.
Preferably, in 1), single cells are captured in the micro-chamber, and whole genome DNA of the single cells is obtained by lysis.
More preferably, the single cell capture method is selected from one or more of limiting dilution, micromanipulation, fluorescence flow sorting, micro-droplet generation, laser microdissection or capillary extraction.
More preferably, the method of cleavage comprises one or more of denaturation, enzymatic cleavage, alkaline cleavage, heat shock and sonication.
Preferably, in 2), the amplification method is selected from one or more of PCR amplification, MDA amplification and MALDBAC amplification.
Preferably, the construction method further comprises ligating the purified product to a linker sequence.
Preferably, the amplification reagents further comprise pyrophosphatase. The applicant found that adding pyrophosphatase during amplification can increase amplification efficiency.
The construction method of the single-cell whole genome sequencing library has the following beneficial effects:
in the single-cell whole genome amplification process, the primers (with the length of 2-99 nucleotides) with index barcode sequences are used for replacing common random primers, so that cell-specific index barcodes are marked on each single-cell whole genome amplification product while single-cell whole genome amplification is realized, and then sequencing is carried out by connecting upper joints, so that complex genome assembly with haplotype resolution and metagenome assembly can be realized.
Drawings
FIG. 1 shows a flow chart of a single cell whole genome sequencing library construction method of the application.
FIG. 2 shows a schematic diagram of microdroplet coated with a single Shi Washi bacterium in example 1 of the present application.
FIG. 3 is a schematic diagram showing micro droplets containing single cell whole genome amplification products (arrows) after MDA reaction in example 1 of the present application.
FIG. 4 shows a sequencing analysis of the library constructed for third generation based example 1 of the present application.
FIG. 5 shows an analytical map of single cell whole genome sequencing based on the second generation.
Detailed Description
In the prior art, amplification and barcoding are carried out step by step, the steps are complicated, and the sequencing reading length is short. The applicant has found by accident that amplification and barcoding are synchronously carried out in an original micro-reaction system, and then a three-generation sequencing platform is utilized for sequencing analysis, so that genome assembly with high coverage rate and high accuracy at a single cell level can be realized. The present application has been completed on the basis of this finding.
The application comprises the following steps: 1) Capturing single cells in a single micro-reaction system (micro-chamber) through a micro-fluidic technology, and performing a cracking reaction (or not performing the cracking reaction); 2) Adding an amplification reaction system (comprising random primers carrying indexable bar code sequences, length of the random primers carrying indexable bar code sequences is 2-99 nucleotides and an amplification reagent) into a primary micro-reaction system (micro-chamber) by a related technology, and carrying out single-cell whole genome amplification and indexing reaction of genome amplification products in situ; 3) The construction of the single-cell whole genome sequencing library is completed by adding the connector sequence, the sequencing is carried out, and the single-cell whole genome analysis is carried out after the sequencing.
As shown in FIG. 1, the application provides a construction method of a single-cell whole genome sequencing library, which comprises the following steps:
1) Providing a microcavity comprising whole genomic DNA of a single cell;
2) Adding a random primer carrying an index bar code and an amplification reagent into the microcavity for amplification to obtain a whole genome amplification product containing the index bar code;
3) Purifying to obtain the single-cell whole genome sequencing library.
According to the method, the whole genome DNA is amplified, meanwhile, the index barcode sequence is introduced, the amplification and the barcode indexing are synchronously carried out, on one hand, the library building step is simplified, the amplified genome amplification product is not required to be fragmented, and the sequence can be directly sequenced on a machine by adding a sequence connector adapted to a three-generation sequence platform; on the other hand, the sequencing library constructed by the method has longer reading length, which is far higher than that of the second generation sequencing technology, and the long reading length library can orient, sort, interval or connect the contigs in the genome splicing, so that the quality of the genome splicing is improved, and the method has unique advantages in the aspect of analyzing long repeated areas. Therefore, when the method is used for analyzing metagenome, the defect of short second-generation sequencing reading section can be overcome, and genome assembly with high coverage rate and high accuracy and single cell level can be realized.
In the method of the present application, in step 1), the microcavity is selected from the group consisting of a microdroplet, a gel microsphere, a semipermeable membrane system, a liposome system, a microplate, and a centrifuge tube; the micro-chamber has a volume size of picoliters to nanoliters.
In the method of the present application, in step 1), single cells may be captured in the microcavity, and the whole genome DNA of the single cells may be obtained by lysis. Single cells can be captured in separate microcavities (picoliter to nanoliter levels) by utilizing various techniques in preparation for subsequent experiments. To meet the requirements of single cell experiments, single cells must first be captured by a series of means. The method for capturing single cells is selected from one or more of limiting dilution method, micromanipulation method, fluorescence flow type sorting method, micro-droplet technology, laser micro-cutting technology or directly picking single cells by capillary.
In certain embodiments, single cells are captured in a microcavity (picoliter to nanoliter scale) by microdroplet technology in preparation for subsequent experiments. Methods of trapping single cells within a microcavity include, but are not limited to, any of the following 1) -2):
1) When the microcavity is a micro-droplet, preparing single-cell suspension, and capturing single cells in the micro-droplet (the volume size is in the order of picoliter to nanoliter) by utilizing a high-flux micro-droplet technology;
2) When the microcavity is a gel microsphere, a single cell suspension is prepared, single cells are captured in a hydrogel-containing droplet by using a high-throughput micro droplet technique, and the hydrogel-containing droplet is coagulated into a hydrogel microsphere containing single cells under conditions of proper condition coagulation. Optionally, the hydrogel reagent comprises an acrylamide monomer and ammonium persulfate; such conditions suitable for coagulation include, but are not limited to, UV irradiation, temperature changes, or pH environment changes, etc. In one embodiment, the hydrogel microspheres are selected from polyacrylamide hydrogel microspheres. In one embodiment, the injection of hydrogel reagents may be performed by a microfluidic drop generating chip.
After capturing the single cells in the microdroplet, a single cell lysis operation is performed in order to perform the subsequent nucleic acid amplification step. The single cell lysis procedure is selected from any one of the following I) -V), but is not limited to the following methods:
i) The denaturation reaction, i.e., the denaturation temperature similar to that of a PCR reaction for single cells, releases intracellular DNA.
II) enzymatic cleavage, i.e.adding a cleaving enzyme to a single cell, and performing a corresponding chemical reaction to cleave the cell and release intracellular DNA. The lyase may be, but is not limited to: labiase lyase, lysostaphin, egg protein derived lysozyme, human derived lysozyme, or digestive peptidase.
III) alkaline lysis, i.e.adding alkali to single cells, under specific alkaline conditions (pH > 7 and pH < 14), and combining with a suitable temperature to rupture the cells and release intracellular DNA. The base may be, but is not limited to: KOH or NaOH, etc.
IV) heat shock, i.e. repeated freeze thawing of single cells, causes swelling of the cells, resulting in disruption of the cellular structure and release of intracellular DNA. The method comprises the steps of carrying out a first treatment on the surface of the
V) ultrasonic treatment, namely, ultrasonic heating method is utilized to cause single cell to break and release intracellular DNA.
And (3) carrying out in-situ lysis on the single cells, and after releasing intracellular DNA, amplifying the whole genome DNA of the single cells and indexing the amplified products.
In the method of the present application, the amplification reagent comprises DNA polymerase, dNTPs or Mg 2+ Any one or a combination of at least two of these. The DNA polymerase is a long fragment high-fidelity DNA polymerase, for example, phi29 DNA polymerase (phi 29DNA polymerase). The amplification reagent may further comprise pyrophosphatase. In addition to the DNA polymerase, pyrophosphatase (PPase) may be added during amplification to hydrolyze pyrophosphoric acid (PPi) produced in the amplification reaction, thereby improving the efficiency and yield of the amplification reaction.
In the method of the present application, in step 2), the length of the random primer carrying the index barcode is 2 to 99 bases. The random primer carrying an index barcode comprises a barcode sequence and a random primer sequence. The barcode sequence is compatible with a third generation sequencing system; the random primer sequence comprises a3 'end primer synthesis phosphorylation label from a 3' end to a 5 'end, and a first base and a second base of the 3' end of the random primer sequence are phosphorylation labels. The barcode sequence comprises an immobilized structure as a barcode sequence and a variable structure as a barcode sequence, preferably the barcode sequence comprises an alternating arrangement of immobilized structures and variable structures, the immobilized structures being used to anchor the variable structure of the barcode sequence and extract the variable structure of the barcode sequence for single cell analysis in a subsequent analysis, for example, the immobilized structure of the barcode sequence is selected from TGCC, CCGCT, GTCT and GTCT, etc.; the variable structure of the barcode sequence is a UMI sequence (NNNNNNN), where the number of N in the UMI sequence depends on the number of single cells to be analyzed, so as to ensure that one barcode sequence corresponds to only 1 single cell, and if the number of single cells is large, the number of N increases, for example, may be NNNNNNN, NNNNNNNN, NNNNNNNNN …, etc. In one example, using Shi Washi bacteria as an example, when constructing a single-cell whole genome sequencing library, the index barcode is TGCC-NNNNNNN-CCGCT-NNNNNNN-GTCT-nnnnn-CTCG, wherein the fixed structure of the barcode sequence is selected from TGCC, CCGCT, GTCT and CTCG, and the variable structure of the barcode sequence is selected from NNNNNNN; the primer is NNNN N, wherein the penultimate and penultimate N require phosphorylation; the sequence of the random primer carrying the index bar code is TGCC-NNNNNNNNN-CCGCT-NNNNN-GTCT-NNNNNNN-CTCG-NNNNN. The barcode sequence is compatible with a third generation sequencing system, which may be, for example, any one of single molecule live DNA sequencing, heliscope single molecule sequencing, live DNA sequencing based on fluorescence resonance energy transfer, nanopore single molecule sequencing or ion flow semiconductor sequencing, preferably nanopore single molecule sequencing.
In the method of the application, in step 2), single cells are subjected to in-situ whole genome amplification, and an amplification reaction system is required to be added into a primary microcavity. The method of adding the amplification reaction system to the microcavity is selected from any one of the following A1) to A3), but is not limited to the following experimental methods:
a1 When the microcavity where the whole genome DNA of the single cell is located is a micro-droplet, the collected micro-droplet containing the whole genome DNA of the single cell, the micro-droplet containing the random primer of the index barcode or the micro-droplet containing the random primer of the index barcode sequence, and the micro-droplet containing the amplification reaction reagent (comprising DNA polymerase and required buffer solution) are fused together by a droplet fusion method, the primer containing the index barcode is released from the micro-droplet containing the random primer of the index barcode or the micro-droplet containing the random primer of the index barcode sequence, and the whole genome amplification is carried out at a proper temperature for 0.01 to 999 hours. Methods of shedding barcodes from microspheres containing barcode sequences are well known in the art and may employ, but are not limited to, chemical bond (e.g., disulfide bond) cleavage and uv cleavage methods, for example, DTT (dithiothreitol) may be added to cause disulfide bond breaking reagents to cause the index barcodes to be shed into microdroplets for reaction.
A2 When the micro-chamber where the whole genome DNA of the single cell is located is a gel microsphere, reducing the gel microsphere into gel droplets, injecting an amplification system into the gel droplets by a droplet injection method, and performing whole genome amplification at a proper temperature for 0.01-999 hours. The reduction method is a method of adding a reducing agent selected from one or more of dithiothreitol, mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride, but is not limited to the method of adding a reducing agent. Wherein the amplification system comprises amplification reagents and the random primer carrying an indexed barcode.
A3 When the micro-chamber where the whole genome DNA of the single cell is located is a micro-droplet, injecting the amplification system into the micro-droplet containing the whole genome DNA of the single cell by a droplet injection method, and carrying out whole genome amplification for 0.01-999 hours at a proper temperature. Wherein the amplification system comprises amplification reagents and the random primer carrying an indexed barcode.
In certain embodiments, when the microcavity is a microdroplet, random primers carrying an index barcode and amplification reagents are added to the microdroplet. Preferably, the random primer carrying the index bar code is added into the microcavity in the form of a micro droplet or microsphere; the amplification reagents are added to the microcavity in the form of microdroplets or microspheres. In one specific example, the microcavity in which the single-cell genomic DNA is located is a microdroplet, and the random primer and amplification reagent carrying the index barcode are added into the microdroplet in which the single-cell genomic DNA is located in the form of a microsphere and a microdroplet, respectively.
Methods of amplification include, but are not limited to, the following amplification methods:
1) PCR (Polymerase chain reaction), whole genome DNA amplification of single cells was performed by polymerase chain reaction.
2) MDA (Multiple displacement amplification), random primer hexamers and polymerase are used to bind to DNA templates and perform single cell whole genome DNA amplification. Such polymerases include, but are not limited to, Φ29DNA polymerase.
3) MALDBAC (Multiple Annealing and Looping-Based Amplification Cycles) performs multiple annealing loop amplification of genomic DNA of single cells.
4) Pyrophosphatase may be added to the amplification reaction.
In the method, single-cell whole genome DNA, random primers carrying index bar codes and amplification reagents are subjected to amplification reaction, and then purified to obtain the single-cell whole genome sequencing library. Library purification methods include, but are not limited to, methods using commercial purification kits, and the like.
In certain embodiments, the purification further comprises the step of ligating the purified product to a linker sequence. 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 completed, the sequencing library is subjected to size selection and library purification by methods including but not limited to: magnetic beads, gel electrophoresis, and the like. After library construction and sequencing, a metagenomic analysis can be performed that recognizes individual genomes.
In one embodiment, the microcavity is formed in a microfluidic drop generating chip.
The microfluidic droplet generation chip is used for realizing fusion of micro droplets or microspheres. And pumping a preparation required by the experiment to an inlet of a microfluidic droplet generation chip, converging and forming a laminar flow, and converging with an oil phase to form monodisperse droplets.
The microfluidic drop generating chip is of the prior art and can be constructed, for example, according to https:// mccarrolla. Org/dropseq/construction (Macosko E, basu A, satija R, et al Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets [ J ]. Cell,2015,161 (5): 1202-1214).
Adding an upper joint sequence, sequencing by a machine, and analyzing sequencing data after sequencing is completed.
In one embodiment, a microdroplet comprising single-cell whole genome DNA is formed, a microdroplet comprising an amplification reagent is formed, a microsphere comprising a random primer carrying an index barcode sequence is formed, then the microdroplet comprising an amplification reagent and the microsphere comprising a random primer carrying an index barcode sequence are fused into the microdroplet comprising single-cell whole genome DNA by a droplet fusion method, amplified, the whole genome amplification product comprising an index barcode is obtained, purified, and a linker is attached, and sequencing is performed.
When fused, the microdroplet comprising single cell whole genome DNA, the microdroplet comprising amplification reagents, the microsphere comprising random primers carrying an index barcode sequence are fused in a volume ratio of 1:1:1.
During fusion, the interface of the liquid drops is broken by using methods such as an electric field, a magnetic field, a temperature field, surface acoustic waves or laser focusing, so that fusion occurs.
Other advantages and effects of the present application will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present application with reference to specific examples. The application may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present application.
Before the embodiments of the application are explained in further detail, it is to be understood that the application is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the application is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the application; in the description and claims of the application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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 application belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present application may be used to practice the present application as would be apparent to one of skill in the art having possession of the prior art and having possession of the present application. Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present application employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.
Example 1
In example 1, a single-cell whole genome sequencing library was constructed for Shi Washi bacteria. Comprises the following steps:
1.1, separating and capturing single cells by adopting a micro-droplet generation method, and lysing the single cells to obtain the whole genome DNA
1.1.1 preparation of Shi Washi bacterial suspension
Culturing Shi Washi bacteria (Shewanella oneidensis) in LB (Luria-Bertani) culture solution, measuring light absorption value at 600nm, and respectively calculating Shi Washi bacteria number (OD 600 = 1, corresponding bacteria amount 10) by using relationship between bacteria concentration and light absorption value 9 CFU/mL), shi Washi bacterial liquid is obtained.
Centrifuging Shi Washi bacteria solution for 5min to remove supernatant, adding PBS buffer solution, washing, centrifuging again to remove supernatant, dissolving in sterile PBS solution (commercially available, irradiating with ultraviolet for half an hour before use), and diluting to 2.4X10 7 CFU/mL (i.e. 2.4X10) 7 microbial single cell/mL) to give Shi Washi bacterial suspension.
1.1.2 preparation of lysate
The lysate used was provided by the REPLI-g Single Cell Kit (Qiagen, germany) kit.
1.1.3 micro-droplet formats for obtaining whole genome DNA
Customizing a microfluidic droplet generation chip, inputting Shi Washi bacterial suspension obtained in the step 1.1.1 and lysate obtained in the step 1.1.2 into corresponding channels on the chip, generating micro droplets coated with single Shi Washi bacteria by using a high-flux micro droplet generation method, and collecting the micro droplets into a 1.5mL sterile centrifuge tube.
The collected microdroplets were subjected to denaturing culture at 65℃for 10min to effect bacterial lysis and sufficient release of intracellular DNA, at which time the lysed microdroplets contained single Shi Washi single cell whole genome DNA.
FIG. 2 shows the micro-droplet obtained in this example, which contains a single Shi Washi cell whole genome DNA.
1.2 index amplification of Whole genomic DNA from Single cells
1.2.1 obtaining MDA reagent
According to the operation of a commercially available single cell whole genome amplification kit (MDA), MDA reaction reagents were obtained by referring to the formulation of Table 1, and droplets containing MDA reaction reagents were formed by a droplet generation chip.
TABLE 1 MDA reaction reagent proportioning table
| Component (A) | Volume of |
| Non-ribozyme water | 45.6μL |
| Phi29MAX DNA polymerase | 24μL |
| Phi29MAX DNA polymerase buffer (without random primer) | 348μL |
| EvaGreen(10×) | 36μL |
| Stop solution | 48μL |
1.2.2 obtaining random primers carrying an index barcode
Designing the index barcode sequence to obtain random primer carrying the index barcode sequence, TGCC-NNNNNNN-CCGCT-NNNNNNN-GTCT-NNNNNNN-CTCG-NNNN*N*N. Underlined bold barcode sequences; the non-bolded and underlined fixed sequences for subsequent analysis of the anchoring barcode sequences and extraction of the barcode sequences for single cell analysis; only the random primers used for the MDA reaction are underlined, where the penultimate and penultimate N require phosphorylation.
Index barcode synthesis was performed on polystyrene microspheres 55 microns in diameter using a nucleic acid synthesizer: in the synthesis process, each round, one base is synthesized on the polystyrene microsphere until the Nth base is synthesized on the Nth round, and the synthesis process of the DNA coding microsphere is completed.
1.2.3 MDA amplification
And (3) fusing the microdroplet obtained in the step 1.1.3 and coated with single Shi Washi bacteria single cell genome DNA with the microdroplet containing MDA reaction reagent obtained in the step 1.2.1 and the microsphere containing the random primer carrying the index bar code obtained in the step 1.2.2 according to the volume ratio of 1:1 under the action of an electric field to generate new microdroplet (containing single bacteria genome DNA, MDA reaction reagent, index bar code and random primer), and collecting the new microdroplet into a 1.5mL sterile centrifuge tube.
Since the lysed bacterial microdroplet obtained in step 1.1.3 contains Dithiothreitol (DTT), which can dissociate the random primer from the microsphere, breaking the disulfide bond connecting the indexing barcode DNA to the microsphere, and dissociating the barcode DNA into the reaction solution, no DTT is required to be added in the reaction.
Collecting new micro-droplets, placing the micro-droplets at 30 ℃ for reaction for 8 hours, and then reacting the micro-droplets at 65 ℃ for 15 minutes to terminate the amplification reaction, thus obtaining the micro-droplets carrying the Shi Washi single-cell whole genome amplification product of the index barcode sequence.
EvaGreen staining results are shown in FIG. 3. As can be seen from FIG. 3, the amplified genomic clusters exhibited green fluorescence.
1.3 purification
After the amplification reaction was completed, 20. Mu.L of perfluorohexyl ethyl alcohol (perfluor-octanol) was added to the microdroplet of the Shi Washi-cell whole genome amplification product containing the index barcode sequence obtained in step 1.2, and the mixture was blown and homogenized by a pipette, centrifuged for 20 seconds by a table centrifuge, and the supernatant was removed and transferred to a new 1.5mL sterile centrifuge tube, and purified by a DNA purification kit clean up and concentrator kit (Zymo research D4001T) to obtain a purified amplification product.
1.4 linker and sequencing
Using Nanopore ligation kit (SQK-LSK 109), the amplified product purified in step 1.3 was added with the adaptors required for three-generation sequencing according to the experimental procedure provided by the supplier, and after the reaction was completed, three-generation sequencing was performed using the Nanopore Minion platform.
1.5 sequencing data analysis
Sequencing data were analyzed and the sequencing results are shown in FIG. 4.
As can be seen from FIG. 4, the construction method of the single-cell whole genome sequencing library can achieve up to 68.7% of Shi Washi bacteria genome coverage and has high sequence coverage.
The single cell whole genome sequencing step based on the second generation comprises the following steps: the capturing and cracking method of Shi Washi bacteria is the same as 1.1. Microdroplets with a single Shi Washi bacterial genome encapsulated were obtained, 1:1 fusion of microdroplets containing a single Shi Washi bacterial genome and microdroplets with MDA amplification reagents (MDA reagents in REPLI-g Single Cell Kit) encapsulated by high throughput microdroplet technology, collected into sterile centrifuge tubes, and reacted at 30 ℃ for 8 hours. The microdroplet 1:1 containing the single Shi Washi bacterial whole genome amplification product pair microdroplet and the microdroplet 1:1 containing Tn5 transposase transposable reagent (Nextera XT DNA Library Preparation Kit) are fused again by using a high throughput microdroplet technology and received into a sterile centrifuge tube for reaction for 10 minutes at 55 ℃. And (3) carrying out PCR reaction in a PCR instrument by utilizing a high-throughput micro-droplet technology to pair and fuse micro-droplets containing genome fragments, a PCR amplification reaction system and microspheres carrying barcode sequences in a ratio of 1:1:1. After the reaction is finished, breaking emulsion of the liquid drops, screening fragments by using AMPure magnetic beads, screening genome fragments with the sizes of 200-600bp, and performing quality control on the screened genome fragments, and then performing second-generation sequencing based on a second-generation sequencing platform. After the sequencing is completed, data analysis is performed, and the sequencing result is shown in fig. 5.
As can be seen from FIG. 5, the genome coverage of Shi Washi bacteria was 39.4% based on the whole genome sequencing of the second generation single cells.
The technology can be used for sequencing whole single-cell genome, and after indexing amplification is carried out on the whole single-cell genome, the whole single-cell genome is directly connected with a sequencing joint without breaking, so that sequencing is carried out, and sequencing and library building steps are simplified. In the second generation sequencing library construction, the genome DNA is fragmented, and the short sequence obtained by sequencing cannot be used for analysis of repeated sequences in the subsequent genome assembly, so that the assembly of a complex genome and the analysis of haplotype genetic gene information are hindered; in metagenome, due to high sequencing complexity and low sequence coverage, short sequences obtained by sequencing are often not overlapped, thus causing difficulty in metagenome assembly, and thus making it difficult to index full-length genes and metabolic pathways from microbial communities. In the technology, a cell specific barcode sequence is introduced in the single-cell whole genome amplification process, so that the single-cell whole genome amplification is realized, meanwhile, a cell specific index barcode is marked on each single-cell whole genome amplification product, and the sequencing is carried out by connecting an upper joint, so that the complex genome assembly and metagenome assembly of haplotype resolution can be realized.
The above examples are provided to illustrate the disclosed embodiments of the application and are not to be construed as limiting the application. In addition, many modifications and variations of the methods and compositions of the application set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the application. While the application has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the application should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the application which are obvious to those skilled in the art are intended to be within the scope of the present application.
Claims (10)
1. The construction method of the single-cell whole genome sequencing library is characterized by comprising the following steps of:
1) Providing a microcavity comprising whole genomic DNA of a single cell;
2) Adding a random primer carrying an index bar code and an amplification reagent into the microcavity for amplification to obtain a whole genome amplification product containing the index bar code;
3) Purifying to obtain the single-cell whole genome sequencing library.
2. The construction method according to claim 1, comprising at least one of the following technical features:
a1 The length of the primer carrying the index bar code is 2-99 bases;
a2 The primer carrying the index barcode comprises a barcode sequence and a random primer sequence;
a3 The microcavity is selected from a microdroplet, a gel microsphere, a semipermeable membrane system, a liposome system, a microplate or a centrifuge tube;
a4 The micro-chamber has a volume size of picoliters to nanoliters.
3. The construction method according to claim 2, characterized by comprising any one of the following technical features:
b1 When the micro chamber is a micro droplet, adding a random primer carrying an index bar code and an amplification reaction reagent into the micro droplet;
b2 When the microcavity is a gel microsphere, reducing the gel microsphere into a gel droplet, and adding random primers carrying an index barcode and an amplification reagent into the gel droplet.
4. The method of claim 3, wherein the random primer carrying an index barcode is added to the microcavity in the form of a micro-droplet or microsphere;
and/or the amplification reagents are added to the microcavity in the form of microdroplets or microspheres.
5. The method of claim 4, wherein the method used in the addition is one or both of a droplet fusion method and a droplet injection method.
6. A method of construction according to claim 3, wherein the reduction is carried out using a reducing agent; preferably, the reducing agent is selected from one or more of dithiothreitol, mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride.
7. The construction method according to claim 1, wherein in 2), the amplification method is selected from one or more of PCR amplification, MDA amplification and MALBAC amplification;
and/or, the amplification reagents further comprise pyrophosphatase.
8. The method of claim 1, wherein 1) single cells are captured in the microcavity and lysed to obtain whole genome DNA of the single cells.
9. The method of claim 8, wherein the method of cleavage comprises one or more of denaturation, enzymatic cleavage, alkaline cleavage, heat shock and sonication;
and/or the single cell capturing method is selected from one or more of limiting dilution method, micromanipulation method, fluorescence flow sorting method, micro-droplet generation method, laser micro-cutting method or capillary tube picking method.
10. The method of claim 1, further comprising ligating the purified product to a linker sequence.
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