WO2021128036A1

WO2021128036A1 - Kit for constructing human single-cell tcr sequencing library and application thereof

Info

Publication number: WO2021128036A1
Application number: PCT/CN2019/128162
Authority: WO
Inventors: 张惠丹; 赵洪玉
Original assignee: 苏州绘真生物科技有限公司
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-07-01

Abstract

Provided is a kit for constructing a human single-cell TCR sequencing library and an application thereof. Also provided is a kit-based method for constructing a human single-cell TCR sequencing library, comprising: using a microfluidic chip to perform a single-cell capture and encapsulation step, and then performing steps such as RNA reverse transcription, cDNA pre-amplification, first amplification, second amplification, fragmentation, amplification, and purification. When the kit and the human single-cell TCR sequencing library are applied to the sequencing of the immune repertoire of immune cells, 500-30,000 cells can be separated in a single experiment.

Description

Kit for constructing human single cell TCR sequencing library and application thereof

Technical field

This application relates to a kit, in particular to a method for constructing a human-derived single-cell TCR (T cell receptor) sequencing library, a kit for constructing a human-derived single-cell TCR sequencing library, and a human TCR sequencing method, belonging to The field of molecular biology.

Background technique

Immune repertoire (IR) refers to the sum of all functionally diverse T cells and T cells in the circulatory system of an individual at any given time. Traditional immune repertoire research technology can often only detect the sequence information of TCR (T cell receptor) and one chain of TCR, while single-cell immune repertoire sequencing can simultaneously obtain TCR alpha and beta chains, TCR heavy and light chains, and The combined information within a cell.

The single-cell immune repertoire sequencing technology is a new technology that simultaneously performs high-throughput sequencing of the adaptive immune receptor library at the single cell level, providing a more scalable platform for immunomics research.

Some traditional single-cell sequencing technologies have their own shortcomings. For example, the flow cytometer-based sequencing method requires a large amount of samples, requires precise control, damages the cells, and requires high requirements for subsequent database construction; based on the C1 system (Fluidigm) Sequencing technology uses microvalves to separate single cells, with low throughput, only 938 cells at most, and high cost; Sequencing technology based on Microwell (microwell plate) method has low cell capture rate and high pollution; based on existing others The sequencing technology of the microfluidic platform is cumbersome and takes a long time.

At present, single-cell immune repertoire sequencing is generally performed based on the 10X Genomics platform, which can isolate 500 to 10,000 single cells at a time, and can simultaneously obtain 5'gene expression data and TCR/TCR V(D)J data. Long sequence. However, the 10X Genomics platform has limitations in the number of isolated single cells and requires high cell viability. At the same time, sequencing of single-cell immune repertoires based on 10X Genomics is expensive.

Summary of the invention

The main purpose of this application is to provide a kit for constructing a human single-cell TCR sequencing library and its application, so as to overcome the shortcomings of the prior art.

In order to achieve the foregoing invention objectives, this application adopts the following solutions:

The embodiment of the application provides a kit for constructing a human single-cell TCR sequencing library, which includes:

The microfluidic chip is used at least to capture and package human T cell single cells to generate water-in-oil reaction droplets. The water-in-oil reaction droplets include an oil phase and a cell liquid phase wrapped by the oil phase, and The water-in-oil reaction droplet contains an RNA reverse transcription component and a cell lysis reagent;

Oil and cell lysis reagent for forming the oil phase;

The cell label includes a deformable microbead and a molecular label connected to the deformable microbead, and the molecular label is used to identify a cell;

RNA reverse transcription component, comprising the reverse transcription primer shown in SEQ ID NO: 5, RNA reverse transcriptase, RNA reverse transcriptase inhibitor and RNA reverse transcription buffer;

The cDNA pre-amplification component includes the cDNA pre-amplification primer shown in SEQ ID NO: 6, DNA polymerase and amplification buffer;

The first amplification component includes the primers shown in SEQ ID NO: 1 to SEQ ID NO: 2, DNA polymerase, and amplification buffer;

The second amplification component includes the primers shown in SEQ ID NO: 3 to SEQ ID NO: 4, DNA polymerase, and amplification buffer;

Transposition fragmentation components, including transposase, transposase reaction buffer and transposition reaction stop solution;

The third amplification component includes two sequencing adapters, DNA polymerase and amplification buffer.

The embodiment of the present application also provides a method for constructing a human single-cell TCR sequencing library, which includes:

Use a microfluidic chip to capture and package human T cell single cells to generate water-in-oil reaction droplets containing single cells;

Performing lysis and reverse transcription on the cells in the water-in-oil reaction droplet to obtain a reverse transcription product;

Pre-amplifying the reverse transcription product, and then sequentially performing the first amplification and the second amplification to obtain the amplified product;

Fragmentation, amplification, and purification of the amplified product to generate a sequencing library;

Wherein, the primer composition used in the first amplification includes the primers shown in SEQ ID NO: 1 to SEQ ID NO: 2, and the primer composition used in the second amplification includes SEQ ID NO: 3 to The primer shown in SEQ ID NO: 4.

In some embodiments of the foregoing embodiments of the application, the microfluidic chip includes a cell microfluidic channel, a cell isolation medium microfluidic channel, a cell label microfluidic channel, and a single cell sample collection port, the cell microfluidic channel having The cell suspension inlet and the single cell suspension outlet, the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet, and the single cell suspension outlet and the cell label suspension outlet meet so that all The single cell suspension output by the cell microchannel can be mixed with the cell label suspension output by the cell label microchannel to form a cell carrier liquid, and the flow path of the cell carrier liquid crosses the cell isolation medium microchannel , So that the cell isolation medium flowing in the microchannels of the cell isolation medium can shear and wrap the cell carrier liquid, thereby forming a water-in-oil reaction droplet containing single cells, and the single-cell water-in-oil reaction liquid The drop is output from the single cell sample collection port.

The embodiment of the present application also provides a human-source TCR sequencing method, which includes: constructing a human single-cell TCR sequencing library by using any of the foregoing methods, and then performing sequencing analysis

Compared with the prior art, this application uses microfluidic technology to design a method and kit for constructing a human-derived single-cell TCR separation and sequencing library. When it is applied to the sequencing of the immune repertoire of immune cells, a single experiment can be used. The separation of 500-30000 cells fundamentally solves the flux problem of the single-cell immune repertoire, and has broad application prospects in the fields of tumor microenvironment, infectious diseases, rejection after organ transplantation, and immunotherapy.

Description of the drawings

Fig. 1 is a process flow diagram of constructing a human-derived single-cell TCR sequencing library in a typical embodiment of the present application;

Fig. 2 is a schematic structural diagram of a microfluidic chip in a typical embodiment of the present application;

Fig. 3 is an optical photograph of a water-in-oil reaction droplet generated in a microfluidic chip in a specific implementation case of the present application;

FIG. 4 is a diagram of quality control results of a sequencing library in a specific implementation case of this application;

Figure 5 is a flow chart of biological information analysis in a specific implementation case of this application;

Figure 6 is a Clonotypes abundance map in a specific implementation case of this application;

Fig. 7 is an analysis diagram of V/J gene abundance in a specific implementation case of the present application;

Figure 8 is a V-J gene paired statistical analysis diagram in a specific implementation case of this application.

Detailed ways

One aspect of the embodiments of the present application provides a kit for constructing a human single-cell TCR sequencing library, which includes:

Oil and cell lysis reagent for forming the oil phase;

The third amplification component includes two sequencing adapters, DNA polymerase and amplification buffer. .

In some embodiments, the microfluidic chip includes a cell microfluidic channel, a cell isolation medium microfluidic channel, a cell label microfluidic channel, and a single cell sample collection port. The cell microfluidic channel has a cell suspension inlet and a single cell sample collection port. The cell suspension outlet, the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet, and the single cell suspension outlet meets the cell label suspension outlet to enable the cell microchannel to output The single cell suspension can be mixed with the cell label suspension output from the cell label microchannel to form a cell carrier liquid, and the flow path of the cell carrier liquid crosses the cell isolation medium microchannel, so that the Cell isolation medium The cell isolation medium flowing in the microchannel can shear and wrap the cell carrier fluid to form a water-in-oil reaction droplet containing single cells, and the single-cell water-in-oil reaction droplet is collected from a single cell sample口 output.

Of course, a transition section for the flow of the cell carrier liquid can also be provided between the intersection of the single cell suspension outlet and the cell label suspension outlet and the cell isolation medium microchannel, which can be named cell carrier liquid microfluidics. (Refer to reference numeral 13 in FIG. 2), the cell-carrying liquid microchannel and the cell isolation medium microchannel are intersected.

Further, the tail region of the cell microchannel is set as a single cell channel, the width of the single cell channel is equal to or slightly larger than the diameter of a single cell, and the exit of the single cell channel meets the exit of the cell label microchannel, The single cell suspension output from the cell microchannel is mixed with the cell label suspension output from the cell label microchannel to form a cell carrier liquid, and the continuous flow path of the cell carrier liquid is separated from the cell isolation medium The micro-channels cross, so that the cell-isolating medium flowing through the cell-isolating medium micro-channels can shear the continuous cell carrier fluid into discrete droplet-shaped cell phases and make each cell phase contain single cells and single cells. Cell labeling, and at the same time, the cell isolation medium is used as an oil to wrap the cell liquid phase to form the water-in-oil reaction droplets.

Further, the water-in-oil reaction droplets are used as water-in-oil microreactors, and their size can be upgraded.

Further, the microfluidic chip also includes a cell suspension sample cup, a cell separation medium sample cup, and a cell label that are respectively connected to the cell microchannel, cell isolation medium microchannel, and cell label microchannel. Add sample cup.

In some embodiments, a negative pressure power generation device is provided at the single-cell sample collection port. The negative pressure power generation device may use an air pump or the like, which can generate negative pressure in the microfluidic chip to drive fluid flow in each microfluidic channel. For example, an air pump can be used to extract air from the single-cell sample collection port, and a negative pressure of -4K to -10K Pa can be applied to the entire microfluidic chip. By adopting this negative pressure method and setting the chip as 3 channels, it does not need to be driven by power. Compared with the positive pressure drive and multi-channel (above 4 channels) in the prior art, the operation is more convenient, the time is greatly shortened, and the It is more flexible to do micro samples, and you can do 1 to 8 samples at the same time.

Further, the structure of a microfluidic chip in the foregoing embodiment can be referred to as shown in Figure 2, including a cell suspension sample cup 1, a cell label sample cup 2, a cell isolation medium sample cup 4, and the cell suspension The liquid sample addition cup 1, the cell label sample cup 2, the cell isolation medium sample cup 4 are respectively connected with the cell microchannel 11, the cell label microchannel 12, and the cell isolation medium microchannel 14, and the microfluidic control The chip is also provided with a single-cell sample collection port 3. Wherein, after the cell microchannel 11 and the cell label microchannel 12 intersect each other, they also intersect with the cell isolation medium microchannel 14 and further communicate with the single cell sample collection port 3.

In the above embodiments of the present application, the cell microchannel of the microfluidic chip is a flow channel for forming a cell suspension of one component of the cell liquid phase, and the cell labeling channel is another channel for forming the cell liquid phase. The flow channel of the cell label suspension of the components, the cell isolation medium micro flow channel is the flow channel of the oil phase components. All the components flow at a certain speed along with the flow channel when the pressure on the chip is increased. And the cell carrier liquid formed by mixing the cell suspension and the cell label suspension is cut by the cell isolation medium as the oil phase to form a physical isolation. By controlling the pressure and flow resistance design, the cell isolation medium can cut individual cells and cell tags. , To achieve the separation of single cells and gel beads, ensuring that each water-in-oil reaction droplet serves as a micro-reaction system and contains a cell and a cell label.

In some embodiments, the oil phase includes oil and a cell lysis reagent, and the cell liquid phase includes an RNA reverse transcription component.

In some embodiments, the volume ratio of the oil to the cell lysis reagent is 100:1 to 500:1, such as 100:1, 200:1, 300:1, 500:1, etc., but is not limited thereto.

Further, the molecular tag also includes a barcode for identifying cells.

Furthermore, the length of the barcode can be 4-30 nt, but it is not limited to the base sequence and certain sequence combination.

Furthermore, the barcode may include 3 but not limited to 3 constant base sequences.

Furthermore, the barcode may include 3 nt riboG bases but not limited to 3 nt.

Furthermore, the total length of the molecular tag may range from 50 nt to 200 nt, and is not limited thereto.

In some embodiments, the molecular tag may be chemically and/or physically attached to the microspheres. For example, the molecular tag and the microbead can be connected by covalent bonding, chemical polymerization, antigen-antibody binding, enzyme-catalyzed connection reaction, etc., and it is not limited thereto.

In some embodiments, the molecular tag can be separated from the deformable microbead under physical and/or chemical action. Further, the physical and/or chemical action includes illumination, specific enzyme digestion, etc., and is not limited thereto.

For example, the oligonucleic acid strand as the molecular tag may be labeled as ultraviolet-sensitive, light-sensitive, or capable of being cleaved with specific enzymes, and is not limited thereto. In this application, it is preferred to use ultraviolet light to remove the molecular label from the deformable beads. It is not only very convenient, but also does not introduce other chemical substances into the microreaction system, thereby avoiding potential pollution risks. In addition, free The molecular tag captures the target product more efficiently, and can eliminate the problem of low capture efficiency caused by the steric hindrance of the microbead when the primer fixed on the microbead captures the target product.

In some embodiments, the deformable beads can be made of organic materials or inorganic-organic composite materials, for example, polyacrylamide gel beads, agarose gel beads, agarose-coated magnetic beads, Silicon beads, microbeads made of inert materials, etc. are not limited thereto. Preferably, the deformable microbeads can be gel microbeads (for example, hydrogel microspheres), which are beneficial to further improve the efficiency of the microfluidic chip for water-in-oil reaction droplet packaging, as well as synergistic microfluidics. Control the chip and greatly increase the cell throughput. The aforementioned microbeads can be purchased from the market or self-made according to known literature. Furthermore, porous polyacrylamide microbeads are preferably used in this application, because they have a much larger specific surface area than other microbeads, such as hard microbeads such as resin or magnetic microbeads, so they carry far more primers than others. Microbeads. When synthesizing the polyacrylamide microbeads, the concentration of acrylamide monomer used can be 1%-10%.

In some embodiments, the diameter of the microbeads may be 10 μM-200 μM.

In the foregoing embodiment of the present application, using the microfluidic chip, the single-port double-packing rate is 1/2 of the double-port under the same concentration of cells, and different cell sizes can be adjusted by pressure (for example, with negative pressure). The pressure generated by the power generation device is used as the power) to realize the wrapping. Especially when the negative pressure power generation device is used as the power source, the cell wrapping can be completed quickly and efficiently, and when the gel beads are used to form the cell label, the suspension The liquid flow rate has an impact force, the flow rate is controllable, and the wrapping rate can be over 90%.

And, in the foregoing embodiments of the present application, the water-in-oil microreactor that can be upgraded by the microfluidic chip technology is compared with the separation in a 96-well plate in the prior art. In the case of the reverse transcription component, a larger number of cells can be detected, which greatly reduces the detection cost of a single cell, and by increasing the molecular label loaded on the gel beads, it can also achieve up to 30,000 cells Separation and detection.

In the foregoing embodiments of the present application, the single-cell lysis component can use cell lysis reagents well known to those skilled in the art, which include any protease and protein denaturing reagents and lysis reagents that are well-known to those skilled in the art and are suitable for cell lysis. Buffer system.

In the foregoing embodiments of the present application, the RNA reverse transcription component includes RNA reverse transcription primers and RNA reverse transcriptase, RNA reverse transcriptase inhibitors, and RNA reverse transcription buffers that are well known to those skilled in the art Wait. Wherein, the sequence of the RNA reverse transcription primer is shown in SEQ ID NO: 5.

For example, the reverse transcriptase may include M-MLV reverse transcriptase, which is an RNA template-dependent DNA polymerase.

For example, the main components of the reverse transcription buffer may be Tris-HCl, KCl, MgCl ₂ , DTT, Mn ²⁺ ions, and water, and the content of each component may be: the concentration range of Tris-HCl is 50-500 mM ( pH of 7.0-9.0), KCl concentration in the range of 50-500mM, MgCl concentration range of 10mM-25mM, DTT concentration range of 10mM-100mM _2. ,

In this application, the reverse transcription reaction is performed in the water-in-oil reaction droplet by using the template conversion method, and the transposase is combined with the transposase outside the droplet to build the library. The overall reverse transcription process takes less than 8 hours, which is in sharp contrast. However, the in vitro transcription method used in the prior art generally takes more than 30 hours. In addition, the reverse transcription reaction method adopted in this application can also avoid cross-contamination caused by reverse transcription outside the droplet, effectively reduce the double-packing rate, and reduce false positive results. In addition, it is also beneficial to simplify subsequent library building operations, for example, there is no need to use the complicated and time-consuming terminal addition A library building method.

The demulsification treatment in this application preferably adopts physical demulsification methods such as ultrasound to avoid the influence of chemical components such as PFO existing in the chemical demulsification method on subsequent reactions.

In the foregoing embodiments of the present application, the cDNA pre-amplification component includes a cDNA pre-amplification primer, a DNA polymerase and an amplification buffer well known to those skilled in the art. For example, the main components of the amplification buffer, that is, the cDNA pre-amplification reaction solution may include: KCl, NH ₄ Cl, NaCl, Tris, MgCl ₂ , betaine, DMSO, water, and the like. For example, the DNA polymerase, ie, cDNA preamplification enzyme, can be selected from Taq DNA polymerase, hot-start Taq polymerase, high-fidelity enzymes and the like.

Wherein, the sequence of the cDNA pre-amplification primer is shown in SEQ ID NO: 6.

In the foregoing embodiments of the present application, the first amplification component includes primers shown in SEQ ID NO: 1 to SEQ ID NO: 2 and a DNA polymerase and amplification buffer well known to those skilled in the art.

In the foregoing embodiments of the present application, the second amplification component includes primers shown in SEQ ID NO: 3 to SEQ ID NO: 4, and a DNA polymerase and amplification buffer well-known to those skilled in the art.

Further, the first amplification component and the second amplification component both contain a mixture of multiple primers, and the use concentration of any one of the primers is preferably 0.1-0.5 μmol/L.

In the foregoing embodiments of the present application, the DNA polymerase can be any thermostable DNA polymerase known to those skilled in the art, such as: LA-Taq, rTaq, Phusion, Deep Vent, Deep Vent (exo-) , Gold 360, Platinum Taq, KAPA 2G Robust, etc., but not limited to this.

In the foregoing embodiments of the present application, the transposable fragmentation component may include a transposase, a transposase reaction buffer, and a transposition reaction terminator that are well known to those skilled in the art. For example, the transposase can be selected from Tn5 and the like, and is not limited thereto.

Another aspect of the embodiments of the present application provides a method for constructing a human single-cell TCR sequencing library. In a nutshell, this method includes the use of microfluidic chips to package single cells and cell labels, so that the single cells are captured by a unique type of cell label, and then the lysis of a single cell, the loading of the unique cell label, and RNA reaction are performed. Transcription and cDNA pre-amplification to achieve the enrichment of low-concentration cDNA, and then through the TCR-specific primary amplification to achieve the specific enrichment of the TCR, and then through the TCR-specific secondary amplification to avoid the first enrichment The specificity of the collection is not enough, the second specific enrichment is performed, and then the transposase fragmentation, amplification, purification and other steps are performed.

In detail, referring to FIG. 1, in some embodiments of the present application, the construction method may include:

Performing lysis and reverse transcription on the single cell in the water-in-oil reaction droplet to obtain a reverse transcription product;

Wherein, the primer composition used in the first amplification includes the primers shown in SEQ ID NO: 1 to SEQ ID NO: 2, and the primer composition used in the second amplification includes SEQ ID NO: 3 to The primer shown in SEQ ID NO: 4. The combination of multiple primers used in the first amplification component and the second amplification component is specially selected from a large number of primers and combinations thereof, which ensures a very high amplification efficiency.

In some embodiments, the construction method specifically includes:

Provide any one of the kits in the foregoing embodiments;

The cell suspension, cell label suspension, and cell isolation medium are respectively injected into the microfluidic chip, and the cell suspension and cell label suspension are mixed with each other after flowing through the cell microchannel and the cell label microchannel to form a cell carrier. The cell carrier fluid is sheared and wrapped by the cell isolation medium flowing in the cell isolation medium microchannel to form a water-in-oil reaction droplet containing single cells and single cell labels;

And, collecting the water-in-oil reaction droplet containing the single cell from the single cell sample collection port.

In some embodiments, the construction method specifically includes: driving the cell suspension, cell label suspension, cell carrier fluid, and cell isolation medium to flow continuously in the microfluidic chip through a negative pressure power generating device, and make the formed The water-in-oil reaction droplets are output from the single-cell sample collection port.

In some embodiments, the water-in-oil reaction droplet further includes a single cell lysis component and an RNA reverse transcription component, and the single cell lysis component includes a lysis enzyme and a lysis buffer for lysing single cells.

Wherein, the composition of the single cell lysis module and the RNA reverse transcription module can be as described above, and will not be repeated here.

In some embodiments, the water-in-oil reaction droplet further comprises a single cell label. The cell label may include a molecular label fixedly connected to a solid carrier such as a microsphere. For the cell label, see above for details.

In some embodiments, the construction method may further include: after the reverse transcription is completed, demulsification treatment is performed on the water-in-oil reaction droplets, followed by purification treatment, and then the purified reaction The transcription product is pre-amplified.

In some embodiments, the construction method specifically includes: selecting a transposase library building method, using Tn5 to randomly interrupt the second amplification product to build a library, and adding the library to construct an adapter.

More specifically, Tn5 transposase can be used to perform the fragmentation treatment, and then the obtained fragmentation product can be amplified. One of the sequencing adapter primers used can be as shown in SEQ ID NO: 7, and the other The sequence of those can be found below.

In some embodiments, the construction method may further include a step of pre-processing the "sample to be tested" or the "sample to be tested". However, the application of the method provided in the examples of this application requires relatively low pre-treatment requirements. For example, preliminary enrichment can be performed based on the physical characteristics of human T cells, or based on the biological characteristics of human T cells. The obtained sample containing human T cells can be used in subsequent steps. In the method of the present application, this preliminary enrichment allows the sample to still contain a certain amount of cells other than human T cells.

In this specification, "sample to be tested" or "sample to be tested" refers to a substance containing human-derived T cells, which can be derived from an individual (such as human blood, biological tissue, etc.) or from other sources, such as Some processed or unprocessed laboratory materials. In addition, in this specification, the detection of "sample" or "sample" does not only involve diagnostic purposes, but may also involve other non-diagnostic purposes. This application allows the sample to contain a certain amount of other types of cells, which has no effect on the test results.

Another aspect of the embodiments of the present application also provides a human TCR sequencing method, which may include:

Use the aforementioned method to construct a human-derived single-cell TCR sequencing library; and

Perform sequencing analysis.

Further, the sequencing analysis can also be performed in a manner well known to those skilled in the art. For example, refer to FIG. 4, which may include basic analysis, standard analysis, and advanced analysis. For example, sequencing analysis of the TCR CDR3 receptor library of human T cells can be performed, and so on.

The human-derived single-cell TCR sequencing library construction method and kit provided in the foregoing embodiments of this application can be applied to the sequencing of immune repertoires of immune cells. A single experiment can separate 500-30000 cells, which fundamentally solves the single-cell immune repertoire The library has the advantages of simple operation, low cost (less than one-third of the sequencing methods based on dropseq, 10X and other platforms), and high accuracy of sequencing results. It has broad application prospects in many fields.

The application will be further elaborated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not to limit the scope of the application. Unless otherwise specified, the various reagents used in the following examples are well known to those skilled in the art and can be obtained through market purchases and other channels. However, the experimental methods that do not specify specific conditions in the following examples are usually based on conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in manufacturing The conditions suggested by the manufacturer.

The following example uses a kit for constructing a human single-cell TCR sequencing library containing reagents and consumables required for constructing a single-cell immune repertoire library, for example: single cell separation and water-in-oil reaction droplet generation Functional microfluidic chip; oil required to generate the reaction droplets; cell tags (including hydrogel beads and molecular tags attached to the hydrogel beads); RNA reverse transcriptase; RNA reverse Transcription buffer; nuclease-free water; Taq polymerase reaction solution; TCR primer mix I (SEQ ID NO: 1 to SEQ ID NO: 2, wherein the concentration of each primer is 0.1-0.5 μM); TCR primer mix II (SEQ ID NO: 3 to SEQ ID NO: 4, where the concentration of each primer is 0.1-0.5 μM); a polyT oligonucleotide containing a constant sequence (SEQ ID NO: 5, where TACACGACGCTCTTCAGA is the constant region); cDNA Amplification reaction buffer; cDNA amplification primer mixture; cDNA amplification enzyme; transposase reaction buffer; transposase; transposition reaction terminator; magnetic beads for double-stranded DNA purification; for library amplification Joints, etc., and not limited to this. Unless otherwise specified, all the aforementioned reagents can be purchased from the market. For example, the purified magnetic beads used in the following embodiments may preferably be Ampure XP beads of Beckman Company, SPRI beads of Beckman Company, etc., and are not limited thereto.

1. Experiment preparation

1.1 Oil phase (cell isolation medium) (see Table 1 for details)

Table 1 Oil phase components

1.2 Cell suspension (see Table 2 for details)

Table 2 Cell suspension components

Note: After the concentration and activity of the cell phase cells are detected by the fluorescence cell analyzer, the required volume can be calculated according to the experimental needs.

1.3 Cell label suspension (see Table 3 for details)

Table 3 Cell label phase components

成分ingredient	体积(μL)Volume (μL)
细胞标签 Cell label	100100

The gel beads constituting the cell label can be selected from polyacrylamide gel beads, agarose gel beads, etc. with a diameter of 10 μM-200 μM, and the molecular label attached to the gel beads is a UV-sensitive oligonucleotide The chain, which can be 50nt-200nt in length, contains a molecular barcode with a length of about 4-30nt, the molecular barcode can contain 3 or more constant base sequences, and the molecular barcode can contain 3nt or more riboG bases base. For example, the molecular tag sequence structure can be: 5'-TACACGACGCTCTTCCGATCT- (4-30 nt base barcode sequence) GTGATTGCTTGTGAC (other sequences, constant sequence)-(4-30 nt base barcode sequence) CGACTCACACTACACGC (also other Sequence, constant sequence)-(4-30nt base barcode sequence)-NNNNNNNNN-rGrGrG-3'. The segment shown in NNNNNNNNN may be a random sequence.

2. Experimental operation and results display

2.1 Cell preparation

2.1.1 According to the needs of the number of samples to be processed, take an appropriate amount of PBS solution and put it in a water bath half an hour in advance to preheat, and take an appropriate amount of PBS solution half an hour in advance and put it on ice for pre-cooling.

2.1.2 For freshly cultured cells/separated tissues: Collect freshly cultured cells/tissues, digest by enzyme to obtain single cells, and centrifuge at 300g for 5min at room temperature.

For the cells frozen in the cell cryopreservation solution: preheat the water bath to 37°C in advance, remove the frozen cells, thaw the frozen cells in the 37°C water bath within 1 min, then collect the cells into the centrifuge tube, and centrifuge at 300g at room temperature 5min.

2.1.3 Water-in-oil: Inject the cell suspension and cell label suspension into the cell sample cup and cell label sample cup respectively, and make the cell suspension and cell label suspension flow through the cell microchannel and cell label respectively. After the micro-channels are mixed with each other to form a cell carrier fluid;

Inject the cell isolation medium into the cell isolation medium sample cup, and make the cell isolation medium contact the cell carrier fluid after flowing through the cell isolation medium microchannels, and separate the cell carrier fluid from continuous liquid phase dispersion to dispersion through shearing action The liquid phase forms water-in-oil reaction droplets containing single cells.

The water-in-oil step can be implemented based on the microfluidic chip shown in FIG. 2, wherein the process of generating water-in-oil reaction droplets is shown in FIG. 3.

2.1.4 Collect the resulting water-in-oil reverse transcription system into a 0.2mL centrifuge tube, add it to the PCR machine and run the reaction at 42°C for 90 minutes;

2.1.5 Add PFO to the reverse transcription product of the above 0.2mL centrifuge tube to demulsify, centrifuge to separate the water and oil, and absorb the water phase;

2.1.6 Add 0.6X Beckman Ampure XP Beads purification to the water phase;

2.17 The purified product is subjected to PCR pre-amplification according to the following conditions

试剂 Reagent		体积volume
2×cDNA扩增反应液2×cDNA amplification reaction solution	25μL25μL
纯化后反转录产物Reverse transcription product after purification	24μL24μL
cDNA预扩增引物(20uM)cDNA pre-amplification primer (20uM)	1μL1μL

Among them, the sequence of the cDNA pre-amplification primer is shown in SEQ ID NO: 6

2.18 After amplification, the product is purified with 0.8X bead; the purified product is amplified with TCR primer mixture I

试剂 Reagent		体积volume
2×Taq聚合酶反应液2×Taq polymerase reaction solution	25μL25μL
TCR引物混合物ITCR primer mix I	1μL1μL
纯化后的反转录产物Purified reverse transcription product	24μL24μL

Number of amplification cycles:

The range of X is 9-14.

2.19 The amplified product of TCR primer mixture I was purified with 0.8X beads, and the purified product was amplified with TCR primer mixture II

试剂 Reagent		体积volume
2×Taq聚合酶反应液2×Taq polymerase reaction solution	25μL25μL
TCR引物混合物IITCR primer mix II	1μL1μL

Purified reverse transcription product

24μL

Amplification cycle parameters:

The value range of Y is 9-14.

2.20 Use 0.5X-0.8X beads to screen the amplified products of TCR primer mix II, and build the library with the obtained screening products

2.21 Use transposase Tn5 to interrupt library construction

5X转座酶反应缓冲液5X Transposase Reaction Buffer	10μL10μL
50ngDNA50ngDNA	XμLXμL
转座酶Transposase	5μL5μL
ddH2OddH2O	YμLYμL
TotalTotal	50μL50μL

The value of X can be adjusted according to the concentration of the amplified product, and the value of Y can be adjusted according to the amount of the amplified product added.

2.22 Transposase interruption temperature conditions

温度temperature	时间time
55℃55°C	热盖Hot cover
55℃55°C	5min5min
4℃4℃	HoldHold

2.23 Terminate the fragmentation reaction

After the reaction is over, add 10 μL of 1% SDS solution, mix by pipetting, and incubate at room temperature for 5 minutes.

2.24 PCR amplification with library amplification primers

试剂Reagent	体积/μLVolume/μL
2×PCRMix2×PCRMix	25μL25μL
片段化产物Fragmentation product	15μL15μL
接头1(10uM)Connector 1 (10uM)	5μL5μL
接头2(10uM)Connector 2 (10uM)	5μL5μL
TotalTotal	50μL50μL

Among them, linker 1 is a universal linker, and its sequence is as follows:

Linker 2 contains the index used for library splitting, and its sequence is as follows:

Where NNNNNNNN is a barcode, which can be a random sequence.

2.25 Library construction thermal cycling parameters

2.26 Library amplification products are screened with 0.6X-0.75X beads. The quality control results are shown in Figure 4.

3. Sequencing analysis

The sequencing process can be performed on platforms such as Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, and Illumina Miseq, and the corresponding operation methods and experimental conditions are well known to those skilled in the art.

The specific analysis process is shown in Fig. 5, which includes first obtaining clean data through quality control for the original sequencing data, and then performing data comparison, screening, and annotation analysis. To count the quality control information, comparison information and annotation information, first compare the clean data with the known V(D)J reference library, and keep the paired reads that have at least 20bp compared to the segment. Then use UMI for screening. If 400 paired reads support the UMI, it is a valid UMI, and the valid UMI is reserved. Perform Contig splicing for each cell, annotate Barcode and filter out non-cell sequences, perform annotation analysis on Contig, screen the annotated contig, and further splice the filtered Contig to obtain the Consensus sequence supported by the sample VDJ (consensus sequence) ), perform Clonotype typing according to the VDJ amino acid sequence of the sample consensus sequence obtained by splicing.

More specifically, the TCR uses the amino acid sequence of the CDR3 region to perform cloning subtype typing, and its effective Barcode is the subtype typing abundance, which is used for abundance analysis. The results can be viewed and exported through Loupe V(D)J Browser. After Clonotype typing of the CDR3 amino acid sequence in TRA(TCRα)/TRB(TCRβ), the number of each subtype was counted for abundance analysis. Draw the Clonotype abundance map shown in Figure 6, where the abscissa is each Clonotype subtype, and the ordinate is the proportion of this subtype in all types.

Please refer to Figure 7, CDR3V/J region genes can reflect the characteristics of TCR Clonotype, so V/J genes can also be analyzed for abundance and V-Jpaired. Data statistics can be used for abundance analysis through TRA/TRB and TRA+TRB respectively.

Please refer to Figure 8. The statistics of VJ gene paired can be used for further immunological research. For example, by viewing the VJ gene pair with high abundance (high cell support) of a certain immune period sample or comparing two different immune periods High abundance of VJ gene pairs to obtain specific expression of immune genes. The results of the V/J gene heat map analysis are as follows, where the abscissa is Vgene, the ordinate is Jgene, and the abscissa is V/Jpaired. The deeper the color, the higher the abundance.

In addition, using the same cell sample as the embodiment of the present application, and in a manner known to those skilled in the art, a comparative sequencing test is performed based on the 10X Genomics platform, and the obtained sequencing result is compared with the sequencing result of the embodiment of the present application. It can be found that using the methods of the embodiments of the present application, the obtained sequencing results are accurate, highly sensitive, and the efficiency and cost are far superior to the sequencing solution based on the 10X Genomics platform.

All documents mentioned in this application are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

A kit for constructing a human single-cell TCR sequencing library, which is characterized in that it comprises:

The microfluidic chip is used at least to capture and package human T cell single cells to generate water-in-oil reaction droplets. The water-in-oil reaction droplets include an oil phase and a cell liquid phase wrapped by the oil phase, and The water-in-oil reaction droplet contains an RNA reverse transcription component and a cell lysis reagent;

Oil and cell lysis reagent for forming the oil phase;

The cell label includes a deformable microbead and a molecular label connected to the deformable microbead, and the molecular label is used to identify a cell;

RNA reverse transcription component, comprising reverse transcription primer shown in SEQ ID NO: 5, RNA reverse transcriptase, RNA reverse transcriptase inhibitor and RNA reverse transcription buffer;

The cDNA pre-amplification component includes SEQ ID NO: 6 cDNA pre-amplification primer, DNA polymerase and amplification buffer;

The first amplification component includes primers shown in SEQ ID NO: 1 to SEQ ID NO: 2, DNA polymerase, and amplification buffer;

The second amplification component includes the primers shown in SEQ ID NO: 3 to SEQ ID NO: 4, DNA polymerase, and amplification buffer;

Transposition fragmentation components, including transposase, transposase reaction buffer and transposition reaction stop solution;

The third amplification component includes two sequencing adapters, DNA polymerase and amplification buffer.
The kit according to claim 1, wherein the microfluidic chip comprises a cell microfluidic channel, a cell isolation medium microfluidic channel, a cell label microfluidic channel, and a single cell sample collection port, and the cell microfluidic chip The channel has a cell suspension inlet and a single cell suspension outlet, the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet, and the single cell suspension outlet meets the cell label suspension outlet, The single cell suspension output by the cell microchannel can be mixed with the cell label suspension output by the cell label microchannel to form a cell carrier liquid, and the flow path of the cell carrier liquid is the same as the cell isolation medium microflow The channels cross, so that the cell isolation medium flowing in the cell isolation medium microchannels can shear and wrap the cell carrier liquid, thereby forming a water-in-oil reaction droplet containing single cells, and the single-cell-containing water-in-oil reaction droplet The reaction droplets are output from the single-cell sample collection port.
The kit according to claim 2, wherein the tail region of the cell microchannel is set as a single-cell channel, the width of the single-cell channel is equal to or slightly larger than the single-cell diameter, and the single-cell channel is The outlet meets the outlet of the cell label microchannel, so that the single cell suspension output from the cell microchannel and the gel microbead molecular label suspension output from the gel microbead molecular label microchannel are mixed to form The cell carrier fluid, the continuous flow path of the cell carrier fluid intersects the cell isolation medium microchannels, so that the cell isolation medium flowing through the cell isolation medium microchannels can shear the continuous cell carrier fluid into discrete cells Droplet-shaped cell liquid phase and make each cell liquid phase contain single cell and single cell label, and at the same time make the cell isolation medium as an oil to wrap around the cell liquid phase, thereby forming the water-in-oil reaction droplet .
The kit according to claim 2 or 3, wherein the microfluidic chip further comprises a cell suspension in communication with the cell microchannel, the cell isolation medium microchannel, and the cell label microchannel, respectively Sample cup, cell isolation medium sample cup, cell label sample cup.
The kit according to claim 3 or 4, wherein a negative pressure power generation device is provided at the single cell sample collection port, and the negative pressure power generation device is used to generate negative pressure in the microfluidic chip , So as to drive the fluid flow in each micro channel.
The kit according to claim 1, wherein the oil phase contains oil and a cell lysis reagent, and the cell liquid phase contains an RNA reverse transcription component; preferably, the volume ratio of the oil to the cell lysis reagent is It is 100:1 to 500:1.
The kit according to claim 1, wherein the molecular tag can be separated from the deformable microbeads under physical and/or chemical action, and the physical and/or chemical action includes light or specific enzymes. And/or, the molecular tag includes an oligonucleic acid strand; and/or, the molecular tag includes a barcode for identifying cells, the length of the barcode is 4-30 nt; preferably, the barcode includes 3 segments The above constant base sequence; more preferably, the barcode contains riboG bases of 3 nt and above; and/or the length of the molecular tag is 50 nt to 200 nt; and/or the diameter of the deformable beads It is 10 μm to 200 μm; and/or, the deformable microbeads include porous polyacrylamide microbeads.
The kit according to claim 1, wherein the concentration of any one of the primers in the first amplification component and the second amplification component is 0.1-0.5 μmol/L.
A method for constructing a human single-cell TCR sequencing library, which is characterized in that it comprises:

Use a microfluidic chip to capture and package human T cell single cells to generate water-in-oil reaction droplets containing single cells;

Performing lysis and reverse transcription on the cells in the water-in-oil reaction droplet to obtain a reverse transcription product;

Pre-amplifying the reverse transcription product, and then sequentially performing the first amplification and the second amplification to obtain the amplified product;

Fragmentation, amplification, and purification of the amplified product to generate a sequencing library;

Wherein, the primer composition used in the first amplification includes the primers shown in SEQ ID NO: 1 to SEQ ID NO: 2, and the primer composition used in the second amplification includes SEQ ID NO: 3 to The primer shown in SEQ ID NO: 4.
The construction method according to claim 9, characterized in that it specifically comprises:

Provide the kit of any one of claims 1-8;

The cell suspension, cell label suspension, and cell isolation medium are respectively injected into the microfluidic chip, and the cell suspension and cell label suspension are mixed with each other after flowing through the cell microchannel and the cell label microchannel to form a cell carrier. The cell carrier fluid is sheared and wrapped by the cell isolation medium flowing in the cell isolation medium microchannel to form a water-in-oil reaction droplet containing single cells and single cell labels;

And, collecting the water-in-oil reaction droplet containing the single cell from the single cell sample collection port.
The construction method according to claim 10, characterized in that it specifically comprises: driving the cell suspension, cell label suspension, cell carrier liquid, and cell isolation medium to flow continuously in the microfluidic chip through a negative pressure power generating device, and making The formed water-in-oil reaction droplets are output from the single-cell sample collection port.
The construction method according to claim 9, further comprising: irradiating the water-in-oil reaction droplets with ultraviolet light, so that the molecular tags therein are released from the deformable beads.
The construction method according to claim 9, characterized in that it further comprises: after the completion of the reverse transcription, demulsifying the water-in-oil reaction droplets, then purifying, and then demulsifying the purified water-in-oil reaction droplets. The reverse transcription product is pre-amplified; preferably, the water-in-oil reaction droplets are demulsified by a physical method; more preferably, the physical method includes an ultrasonic treatment method.
The construction method according to claim 9, characterized in that it specifically comprises: using Tn5 transposase to perform the fragmentation treatment, and then amplify the obtained fragmentation product.
A human TCR sequencing method, which is characterized by comprising: constructing a human single-cell TCR sequencing library using the method of any one of claims 9-14, and then performing sequencing analysis.