CN113026111A - Kit for constructing human single cell TCR sequencing library and application thereof - Google Patents
Kit for constructing human single cell TCR sequencing library and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for constructing a human unicellular TCR sequencing library and application thereof. The kit-based construction method of the human single cell TCR sequencing library comprises the following steps: a step of single cell capture and packaging by using a microfluidic chip; and RNA reverse transcription, cDNA pre-amplification, primary amplification, secondary amplification, fragmentation, amplification, purification and other steps. When the kit and the human-derived single-cell TCR sequencing library are applied to immune cell immune repertoire sequencing, 500-30000 cells can be separated in a single experiment, so that the flux problem of the single-cell immune repertoire is fundamentally solved, and the kit and the human-derived single-cell TCR sequencing library have wide application prospects in the fields of tumor microenvironment, infectious diseases, rejection after organ transplantation, immunotherapy and the like.
Description
Technical Field
The invention 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 the human-derived single-cell TCR sequencing library and a human-derived TCR sequencing method, and belongs to the field of molecular biology.
Background
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. The traditional immune repertoire research technology can only detect the sequence information of one chain of TCR (T cell receptor) and TCR, while the sequencing of the single-cell immune repertoire can simultaneously obtain the combined information of alpha chain and beta chain of TCR, heavy chain and light chain of TCR and one cell.
The sequencing technology of the single-cell immune repertoire is a novel technology for simultaneously carrying out high-throughput sequencing on an adaptive immune receptor repertoire at a single-cell level, and provides a more extensive platform for the research of the immunomics.
Some traditional single cell sequencing technologies have various defects, for example, a sequencing method based on a flow cytometer has a large sample demand, needs to be accurately controlled, has damage to cells, and has a high requirement on subsequent library construction; the sequencing technology based on the C1 system (Fluidigm company) uses a micro valve to separate single cells, has low flux, can only make 938 cells at most and has high cost; the sequencing technology based on a micro well (micro plate) mode has low cell capture rate and high pollution; the sequencing technology based on other existing microfluidic platforms is complex in operation and long in time.
At present, sequencing of a single cell immune repertoire is generally carried out based on a 10X Genomics platform, 500-10000 single cells can be separated once, and 5' gene expression data and V (D) J full-length sequences of TCR/TCR can be obtained simultaneously. However, the 10 × Genomics platform has limitations on the number of isolated single cells and has high requirements on cell activity. Meanwhile, 10X Genomics-based sequencing of single-cell immune repertoires is expensive.
Disclosure of Invention
The invention mainly aims to provide a kit for constructing a human single cell TCR sequencing library and application thereof, thereby overcoming the defects of the prior art.
In order to achieve the aim of the invention, the invention adopts the following scheme:
the embodiment of the invention provides a kit for constructing a human single cell TCR sequencing library, which comprises:
the microfluidic chip is at least used for capturing human T cell single cells and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprises an oil phase and a cell liquid phase wrapped by the oil phase, and the water-in-oil reaction droplet comprises an RNA reverse transcription component and a cell lysis reagent;
an oil, a cell lysis reagent for forming the oil phase;
the cell label comprises a deformable microbead and a molecular label connected to the deformable microbead, wherein the molecular label is used for identifying cells;
an RNA reverse transcription module, which comprises a reverse transcription primer shown in SEQ ID NO. 5, RNA reverse transcriptase, an RNA reverse transcriptase inhibitor and an RNA reverse transcription buffer solution;
a cDNA pre-amplification component which comprises a cDNA pre-amplification primer shown as SEQ ID NO. 6, DNA polymerase and an amplification buffer solution;
a first amplification component which comprises primers shown by SEQ ID NO. 1-SEQ ID NO. 2, DNA polymerase and amplification buffer solution;
a second amplification component, which comprises primers shown in SEQ ID NO. 3-SEQ ID NO. 4, DNA polymerase and amplification buffer solution;
a transposable fragmentation component comprising a transposase, a transposase reaction buffer and a transposition reaction stop solution;
a third amplification module comprising two sequencing adaptors, a DNA polymerase, and an amplification buffer.
The embodiment of the invention also provides a construction method of the human single cell TCR sequencing library, which comprises the following steps:
capturing and packaging the human T cell single cells by using a microfluidic chip so as to generate water-in-oil reaction liquid drops containing the single cells;
cracking and reverse transcribing the cells in the water-in-oil reaction liquid drop to obtain a reverse transcription product;
pre-amplifying the reverse transcription product, and then sequentially performing primary amplification and secondary amplification to obtain an amplification product;
fragmenting, amplifying and purifying the amplification product to generate a sequencing library;
wherein the primer composition for the first amplification comprises primers shown by SEQ ID NO. 1-SEQ ID NO. 2, and the primer composition for the second amplification comprises primers shown by SEQ ID NO. 3-SEQ ID NO. 4.
In some embodiments of the foregoing embodiments of the invention, the microfluidic chip comprises a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single-cell sample collection port, the cell microchannel 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 intersects the cell label suspension outlet to enable the single-cell suspension output by the cell microchannel to mix with the cell label suspension output by the cell label microchannel to form a cell carrier liquid, the flow path of the cell carrier liquid intersects the cell isolation medium microchannel to enable the cell isolation medium flowing in the cell isolation medium microchannel to shear and wrap the cell carrier liquid to form a water-in-oil reaction droplet containing single cells, the water-in-oil reaction droplet containing the single cell is output from the single cell sample collection port.
The embodiment of the invention also provides a human TCR sequencing method, which comprises the following steps: constructing a human single cell TCR sequencing library by any one of the methods described above, and then performing sequencing analysis
Compared with the prior art, the invention designs a construction method and a kit of a human-derived single-cell TCR separation and sequencing library by utilizing a microfluidic technology, and 500 plus 30000 cells can be separated in a single experiment when the kit is applied to immune repertoire sequencing of immune cells, so that the flux problem of the single-cell immune repertoire is fundamentally solved, and the kit has wide application prospects in the fields of tumor microenvironment, infectious diseases, rejection after organ transplantation, immunotherapy and the like.
Drawings
FIG. 1 is a flow chart of a process for constructing a TCR sequencing library of human single cells according to an exemplary embodiment of the invention;
FIG. 2 is a schematic diagram of a microfluidic chip according to an exemplary embodiment of the present invention;
FIG. 3 is an optical photograph of a water-in-oil reaction droplet generated in a microfluidic chip according to an embodiment of the present invention;
FIG. 4 is a diagram showing the results of quality control of a sequencing library according to an embodiment of the present invention;
FIG. 5 is a flow chart of a biological information analysis process according to an embodiment of the present invention;
FIG. 6 is a graph of Clonotypes abundance in one embodiment of the invention;
FIG. 7 is a graph showing the abundance analysis of V/J genes in an embodiment of the present invention;
FIG. 8 is a graph of a V-J gene paired statistical analysis in an embodiment of the present invention.
Detailed Description
One aspect of the embodiments of the present invention provides a kit for constructing a human single cell TCR sequencing library, comprising:
the microfluidic chip is at least used for capturing human T cell single cells and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprises an oil phase and a cell liquid phase wrapped by the oil phase, and the water-in-oil reaction droplet comprises an RNA reverse transcription component and a cell lysis reagent;
an oil, a cell lysis reagent for forming the oil phase;
the cell label comprises a deformable microbead and a molecular label connected to the deformable microbead, wherein the molecular label is used for identifying cells;
an RNA reverse transcription module, which comprises a reverse transcription primer shown in SEQ ID NO. 5, RNA reverse transcriptase, an RNA reverse transcriptase inhibitor and an RNA reverse transcription buffer solution;
a cDNA pre-amplification component which comprises a cDNA pre-amplification primer shown as SEQ ID NO. 6, DNA polymerase and an amplification buffer solution;
a first amplification component which comprises primers shown by SEQ ID NO. 1-SEQ ID NO. 2, DNA polymerase and amplification buffer solution;
a second amplification component, which comprises primers shown in SEQ ID NO. 3-SEQ ID NO. 4, DNA polymerase and amplification buffer solution;
a transposable fragmentation component comprising a transposase, a transposase reaction buffer and a transposition reaction stop solution;
a third amplification module comprising two sequencing adaptors, a DNA polymerase, and an amplification buffer. .
In some embodiments, the microfluidic chip comprises a cell microchannel, a cell isolation medium microchannel, a cell label microchannel and a single-cell sample collection port, the cell micro-flow channel is provided with a cell suspension inlet and a single cell suspension outlet, the cell isolation medium micro-flow channel is provided with a cell label suspension inlet and a cell label suspension outlet, and the single-cell suspension outlet is intersected with the cell label suspension outlet, so that the single-cell suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell carrier liquid, the flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, so that the cell isolation medium flowing in the cell isolation medium micro-channel can be cut and wrapped with the cell carrier liquid, thereby forming a water-in-oil reaction droplet comprising a single cell which is output by the single cell sample collection port.
Of course, a transition section for the cell carrier liquid to flow may also be provided between the intersection of the single-cell suspension outlet and the cell label suspension outlet and the cell isolation medium micro-channel, which may be named as a cell carrier liquid micro-channel (see reference numeral 13 in fig. 2), and the cell carrier liquid micro-channel intersects with the cell isolation medium micro-channel.
Furthermore, the tail region of the cell micro-channel 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, the outlet of the single-cell channel intersects with the outlet of the cell label micro-channel, so that the single-cell suspension output from the cell micro-channel and the cell label suspension output from the cell label micro-channel are mixed to form a cell carrier liquid, the continuous flow path of the cell carrier liquid intersects with the cell isolation medium micro-channel, so that the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell carrier liquid into discrete droplet-shaped cell liquid phase and enable each cell liquid phase to contain a single cell and a single cell label, and meanwhile, the cell isolation medium is used as an oil phase to wrap the cell liquid phase, so that the water-in-oil reaction liquid droplet is formed.
Further, the water-in-oil reaction droplets act as water-in-oil microreactors, which can be pico-liter in size.
Furthermore, the microfluidic chip also comprises a cell suspension sample adding cup, a cell isolation medium sample adding cup and a cell label sample adding cup which are respectively communicated with the cell micro-channel, the cell isolation medium micro-channel and the cell label micro-channel.
In some embodiments, a negative pressure motive force generation device is disposed at the single-cell sample collection port. The negative pressure power generation device can adopt an air pump and the like, and can generate negative pressure in the microfluidic chip so as to drive fluid in each micro-channel to flow. For example, air can be pumped out by an air pump at the single cell sample collecting port, and negative pressure of-4K to-10K Pa can be applied to the whole microfluidic chip. Through adopting this kind of negative pressure mode, and set up the chip and be 3 passageways, need not drive with power, compare malleation drive among the prior art, multichannel (more than 4 passageways), it is more convenient to operate, and the time also shortens greatly, can do trace sample, and the flexibility is higher, can do 1 to 8 samples simultaneously.
Further, the structure of the microfluidic chip in the foregoing embodiment can be seen from fig. 2, and includes a cell suspension sample cup 1, a cell label sample cup 2, and a cell isolation medium sample cup 4, where the cell suspension sample cup 1, the cell label sample cup 2, and the cell isolation medium sample cup 4 are respectively communicated with a cell micro flow channel 11, a cell label micro flow channel 12, and a cell isolation medium micro flow channel 14, and the microfluidic chip is further provided with a single-cell sample collection port 3. Wherein, the cell micro-flow channel 11 and the cell label micro-flow channel 12 are crossed with each other and then crossed with the cell isolation medium micro-flow channel 14, and further communicated with the single cell sample collection port 3.
In the above embodiment of the present invention, the cell microchannel of the microfluidic chip is a flow channel for forming a cell suspension of one component of the cell liquid phase, the cell label channel is a flow channel for forming a cell label suspension of another component of the cell liquid phase, the cell isolation medium microchannel is a flow channel for a component of the oil phase, all the components flow at a certain speed along with the flow channel thereof under the condition that pressure is applied to the chip, and the cell suspension and the cell label suspension are mixed to form cell carrier liquid which is cut by a cell isolation medium as an oil phase to form physical isolation, through controlling the pressure and flow resistance design, the cell isolation medium cuts single cells and cell labels, the separation of the single cells and gel microbeads is realized, and each water-in-oil reaction liquid drop is ensured to be used as a micro-reaction system and comprises one cell and one cell label.
In some embodiments, the oil phase comprises an oil and a cell lysis reagent and the cellular liquid phase comprises an RNA reverse transcription module.
In some embodiments, the volume ratio of oil to cell lysis reagent is 100: 1-500: 1, for example, may be 100: 1. 200: 1. 300, and (2) 300: 1. 500: 1, etc., but are not limited thereto.
Further, the molecular tag further comprises a barcode for identifying the cell.
Further, the barcode may have a length of 4 to 30nt, but is not limited to the above base sequence and the certain sequence combination.
Further, the barcode may comprise 3 constant base sequences but is not limited to 3.
Further, the barcode may comprise 3nt of riboG bases, but is not limited to 3 nt.
Further, the total length of the molecular tag may vary from 50nt to 200nt, and is not limited thereto.
In some embodiments, the molecular tag may be chemically and/or physically attached to the microsphere. For example, the molecular tag and the bead may be linked by a covalent bond, chemical polymerization, antigen-antibody binding, enzyme-catalyzed linking reaction, and the like, without being limited thereto.
In some embodiments, the molecular tag is capable of being physically and/or chemically separated from the deformable microbead. Further, the physical and/or chemical action includes, but is not limited to, light irradiation, specific enzyme cleavage, and the like.
For example, the oligonucleotide strand as the molecular tag may be labeled as ultraviolet-sensitive, light-sensitive, or may be specifically enzyme-cleaved, and is not limited thereto. In the invention, ultraviolet illumination and other modes are preferably adopted to separate the molecular label from the deformable microbead, which is not only very convenient, but also can not introduce other chemical substances into a micro-reaction system, thereby avoiding potential pollution risk.
In some embodiments, the deformable beads may be of an organic material or an inorganic-organic composite material, and may be, for example, polyacrylamide gel beads, agarose coated magnetic beads, silica beads, inert material-made beads, and the like, without being limited thereto. Preferably, the deformable beads can be gel beads (such as hydrogel beads), which is beneficial to further improving the efficiency of the microfluidic chip for carrying out the liquid drop packaging of the water-in-oil reaction, and greatly improving the cell flux in cooperation with the microfluidic chip. The aforementioned beads can be commercially available or can be self-made in accordance with known literature. Further, in the present invention, it is preferable to use porous polyacrylamide beads, which carry far more primers than other beads because they have a specific surface area much larger than that of other beads, for example, hard beads such as resin or magnetic beads. When the polyacrylamide microbeads are synthesized, the concentration of the used acrylamide monomer can be 1% -10%.
In some embodiments, the diameter of the beads may be from 10 μ M to 200 μ M.
In the foregoing embodiment of the present invention, with the microfluidic chip, under the condition of the same concentration of cells, the single-port double-encapsulation rate is 1/2 with two ports, different cell sizes can be encapsulated by adjusting the pressure (for example, using the pressure generated by the negative pressure power generation device as the power source), especially when the negative pressure power generation device is used as the power source, the encapsulation of cells can be rapidly and efficiently completed, and when the gel beads are used to form cell labels, the flow rate of the suspension has impact force, and is controllable, so that the encapsulation rate of 90% or more can be achieved.
And, in the foregoing embodiments of the present invention, a water-in-oil microreactor with a pico-upgrade can be realized by a microfluidic chip technology, and compared with the prior art in which separation is performed in a 96-well plate, a larger number of cells can be detected under the condition of the same number of reverse transcription components, so that the detection cost of a single cell is greatly reduced, and separation and detection of up to 30000 cells can be realized by increasing molecular tags loaded on gel beads.
In the foregoing embodiments of the present invention, the single-cell lysis module may be selected from cell lysis reagents known to those skilled in the art, including any protease and protein denaturing reagents and lysis buffer systems known to those skilled in the art suitable for cell lysis.
In the foregoing embodiments of the present invention, the RNA reverse transcription module comprises an RNA reverse transcription primer and RNA reverse transcriptase, RNA reverse transcriptase inhibitor, RNA reverse transcription buffer, and the like, which are well known to those skilled in the art. Wherein, the sequence of the RNA reverse transcription primer is shown as SEQ ID NO. 5.
For example, the reverse transcriptase can include M-MLV reverse transcriptase, which is an RNA template dependent DNA polymerase.
For example, the reverse transcription buffer may comprise Tris-HCl, KCl, MgCl as main component2、DTT、Mn2+Ions and water, wherein the content of each component can be as follows: concentration range of Tris-HCl 50-500mM (pH 7.0-9.0), concentration range of KCl 50-500mM, MgCl2The concentration range of (A) is 10mM-25mM, and the concentration range of DTT is 10mM-100 mM. A
According to the invention, a template conversion mode is adopted to carry out reverse transcription reaction in a water-in-oil reaction liquid drop, and a transposase library building mode is combined outside the liquid drop, so that the whole reverse transcription process takes less than 8 hours, and in contrast, the time consumption of an in vitro transcription mode adopted in the prior art is generally more than 30 hours. In addition, the reverse transcription reaction mode adopted by the invention can avoid cross contamination caused by reverse transcription outside the liquid drop, effectively reduce double-package rate and reduce false positive results. And, it is also beneficial to simplify the subsequent library building operation, for example, there is no need to adopt a terminal-plus-a library building method which is complicated and time-consuming in operation.
The demulsification treatment in the invention preferably adopts physical demulsification modes such as ultrasound and the like, so as to avoid the influence of chemical components such as PFO existing in the chemical demulsification mode on subsequent reactions.
In the previous examples of the invention, the cDNA pre-amplification module comprises cDNA pre-amplification primers and DNA polymerase and amplification buffer well known to those skilled in the art. For example, the amplification buffer, i.e., the cDNA pre-amplification reaction solution, may comprise, as essential components: KCl, NH4Cl、NaCl、Tris、MgCl2Betaine, DMSO, water, and the like. For example, the DNA polymerase, i.e., cDNA preamplifying enzyme, may be selected from Taq DNA polymerase, Hot Start Taq polymerase, high Fidelity enzyme, and the like.
Wherein, the sequence of the cDNA preamplification primer is shown as SEQ ID NO. 6.
In the previous embodiments of the present invention, the first amplification module comprises the primers shown in SEQ ID NO 1-2 and DNA polymerase and amplification buffer well known to those skilled in the art.
In the previous embodiments of the present invention, the second amplification module comprises primers shown in SEQ ID NO 3-4 and DNA polymerase and amplification buffer well known to those skilled in the art.
Furthermore, the first amplification module and the second amplification module both comprise a mixture of a plurality of primers, and the use concentration of any one primer is preferably 0.1-0.5 [ mu ] mol/L.
In the preceding embodiments of the invention, the DNA polymerase may be any thermostable DNA polymerase known to those skilled in the art, for example: LA-Taq, rTaq, Phusion, Deep Vent (exo-), Gold 360, Platinum Taq, KAPA 2G Robust, and the like, without being limited thereto.
In the foregoing embodiments of the present invention, the transposition fragmentation component may comprise a transposase, a transposase reaction buffer, and a transposition reaction stop solution, which are well known to those skilled in the art. For example, the transposase may be Tn5 or the like, but is not limited thereto.
In another aspect, the embodiments of the present invention provide a method for constructing a sequencing library of human single-cell TCRs. The method comprises the steps of wrapping single cells and cell labels by using a microfluidic chip, capturing the single cells by the cell labels of the unique type, further performing the cracking of the single cells, loading the unique cell labels, performing RNA reverse transcription and cDNA pre-amplification to realize the enrichment of low-concentration cDNA, then performing TCR specificity amplification, realizing TCR specificity enrichment, then performing TCR specificity amplification to avoid insufficient specificity of the first enrichment, performing second specificity enrichment, performing transposase fragmentation, amplification, purification and the like.
In detail, referring to fig. 1, in some embodiments of the present invention, the construction method may include:
capturing and packaging the human T cell single cells by using a microfluidic chip so as to generate water-in-oil reaction liquid drops containing the single cells;
cracking and reverse transcribing the single cells in the water-in-oil reaction liquid drop to obtain a reverse transcription product;
pre-amplifying the reverse transcription product, and then sequentially performing primary amplification and secondary amplification to obtain an amplification product;
fragmenting, amplifying and purifying the amplification product to generate a sequencing library;
wherein the primer composition for the first amplification comprises primers shown by SEQ ID NO. 1-SEQ ID NO. 2, and the primer composition for the second amplification comprises primers shown by SEQ ID NO. 3-SEQ ID NO. 4. The combination of the plurality of primers used in the first amplification module and the second amplification module is specifically selected from a plurality of primers and combinations thereof, which ensures very high amplification efficiency.
In some embodiments, the method of construction specifically comprises:
providing any one of the kits of the preceding embodiments;
respectively injecting the cell suspension, the cell label suspension and the cell isolation medium into the microfluidic chip, mixing the cell suspension and the cell label suspension after flowing through the cell microchannel and the cell label microchannel respectively to form cell carrier liquid, and shearing and wrapping the cell carrier liquid by the cell isolation medium flowing in the cell isolation medium microchannel so as to form water-in-oil reaction liquid drops containing single cells and single cell labels;
and collecting water-in-oil reaction droplets containing single cells from the single cell sample collection port.
In some embodiments, the construction method specifically includes: and the negative pressure power generation device drives the cell suspension, the cell label suspension, the cell carrier liquid and the cell isolation medium to continuously flow in the microfluidic chip, and the formed water-in-oil reaction liquid drop is output from the single cell sample collection port.
In some embodiments, the water-in-oil reaction droplet further comprises a single-cell lysis component comprising a lytic enzyme for lysing a single cell and a lysis buffer, and an RNA reverse transcription component.
Wherein, the compositions of the single-cell lysis component and the RNA reverse transcription component can be as described above, and are not described in detail here.
In some embodiments, the water-in-oil reaction droplet further comprises a single cell tag. The cell tag may comprise a molecular tag that is fixedly attached to a solid support such as a microsphere. For the cell signature, see above for details.
In some embodiments, the method of construction may further comprise: and after the reverse transcription is finished, performing demulsification treatment on the water-in-oil reaction liquid drop, then performing purification treatment, and performing pre-amplification on a purified reverse transcription product.
In some embodiments, the method of construction specifically comprises: and selecting a transposase library construction mode, randomly breaking the second amplification product by using Tn5 to construct a library, and adding the library to construct a joint.
More specifically, the fragmentation treatment can be performed using Tn5 transposase, and the resulting fragmentation products can be amplified, wherein one of the sequencing adapter primers used can be as shown in SEQ ID NO 7, and the sequence of the other can be as described below.
In some embodiments, the construction method may further include a step of pre-treating the "sample to be tested" or the "sample to be tested". However, the method provided by the embodiment of the present invention has low requirements for pretreatment, for example, the method can perform primary enrichment according to the physical characteristics of the human T cells or perform primary enrichment according to the biological characteristics of the human T cells, and the obtained sample containing the human T cells can be used for the subsequent steps. In the method of the invention, this preliminary enrichment allows the sample to still contain a certain amount of cells other than human T cells.
In the present specification, a "test sample" or "test sample" refers to a substance comprising T cells of human origin, which may be from an individual (e.g., human blood, biological tissue, etc.), or may be of other origin, such as some processed or unprocessed laboratory material. In addition, in the present specification, the detection of a "sample" or "specimen" is not only related to a diagnostic purpose, but may also be related to other non-diagnostic purposes. The invention allows the inclusion of a certain amount of other cell types in the sample, without affecting the assay result.
Another aspect of the embodiments of the present invention also provides a method for sequencing a human TCR, which may include:
constructing a human-derived single cell TCR sequencing library by using the method; and
sequencing analysis was performed.
Further, the sequencing analysis may also be performed in a manner well known to those skilled in the art. Reference may be made, for example, to FIG. 4, which may include a base analysis, a standard analysis, and a high-level analysis. For example, sequencing analysis of the TCR CDR3 receptor library of human T cells can be performed, and the like.
The method and the kit for constructing the human-derived single-cell TCR sequencing library provided by the embodiment of the invention can be applied to sequencing of an immune repertoire of immune cells, 500-30000 cells can be separated in a single experiment, the flux problem of the single-cell immune repertoire is fundamentally solved, and the method and the kit also have the advantages of simplicity and convenience in operation, low cost (less than one third of the sequencing mode based on platforms such as dispeq, 10X and the like), high accuracy of the sequencing result and the like, and have wide application prospects in multiple fields.
The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, various reagents used in the following examples are well known to those skilled in the art and available from commercial sources and the like. However, the experimental methods in the following examples, in which specific conditions are not specified, are generally performed under conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or under the conditions recommended by the manufacturers.
A kit for constructing a human single cell TCR sequencing library used in the following examples comprises reagents and consumables required for construction of a single cell immune repertoire library, such as: the microfluidic chip has the functions of single cell separation and water-in-oil reaction droplet generation; oil l required for generating the reaction droplets; cell tags (including hydrogel microbeads and molecular tags attached to the hydrogel microbeads); RNA reverse transcriptase; RNA reverse transcription buffer solution; nuclease-free water; taq polymerase reaction solution; TCR primer mixture I (SEQ ID NO: 1-SEQ ID NO:2, wherein the concentration of each primer is 0.1-0.5. mu.M); TCR primer mixture II (SEQ ID NO: 3-SEQ ID NO:4, wherein each primer concentration is 0.1-0.5. mu.M); a polyT oligonucleotide containing a constant sequence (SEQ ID NO:5, wherein TACACGACGCTCTTCAGA is a constant region); cDNA amplification reaction buffer solution; a cDNA amplification primer mixture; a cDNA amplification enzyme; transposase reaction buffer; a transposase; a transposition reaction stop solution; magnetic beads for double-stranded DNA purification; adaptors for library amplification, and the like, without limitation. The aforementioned reagents are commercially available unless otherwise specified. For example, the purified magnetic beads used in the following examples may preferably be Ampure XP beads from Beckman, SPRI beads from Beckman, and the like, and are not limited thereto.
1. Preparation of the experiment
1.1 oil phase (cell isolation Medium) (details in Table 1)
TABLE 1 oil phase Components
1.2 cell suspensions (see Table 2 for details)
TABLE 2 cell suspension Components
Note: after the cell phase cells are detected by using a fluorescence cell analyzer for concentration and activity, the required volume is calculated according to the experiment requirement.
1.3 cell-tag suspension (see Table 3 for details)
TABLE 3 cellular signature phase Components
| Composition (I) | Volume (μ L) |
| |
100 |
The gel beads constituting the cell tags may be selected from polyacrylamide gel beads, agarose gel beads, etc. having a diameter of 10 μ M to 200 μ M, and the molecular tags attached to the gel beads are ultraviolet-sensitive oligonucleotide strands, which may have a length of 50nt to 200nt, and which include molecular barcodes having a length of about 4 to 30nt, which may include 3 segments or more of constant base sequences, and which may include 3nt or more of riboG bases. For example, the molecular tag sequence structure may be: 5 '-TACACGACGCTCTTCCGATCT- (4-30nt base barcode sequence) GTGATTGCTTGTGAC (other sequences, constant sequence) - (4-30nt base barcode sequence are also possible) CGACTCACACTACACGC (other sequences, constant sequence) - (4-30nt base barcode sequence are also possible) -NNNNNNNNN-rGrGrG-3'. The fragments shown at NNNNNNNNN may be random sequences.
Second, experimental operation and result display
2.1 cell preparation
2.1.1 taking a proper amount of PBS solution according to the requirement of the number of processed samples, putting the PBS solution into a water bath kettle for preheating half an hour in advance, taking a proper amount of PBS solution, putting the PBS solution on ice for precooling half an hour in advance.
2.1.2 for freshly cultured cells/isolated tissue: collecting fresh cultured cells/tissues, performing enzyme digestion to obtain single cells, and centrifuging at normal temperature of 300g for 5 min.
For cells cryopreserved in cell cryopreserved medium: preheating the water bath to 37 deg.C, taking out the frozen cells, melting the frozen cells in 37 deg.C water bath for 1min, collecting the cells, centrifuging at 300g for 5 min.
2.1.3 Water-in-oil: respectively injecting the cell suspension and the cell label suspension into a cell sample adding cup and a cell label sample adding cup, and mixing the cell suspension and the cell label suspension to form cell carrier liquid after the cell suspension and the cell label suspension respectively flow through a cell micro-channel and a cell label micro-channel;
injecting a cell isolation medium into the cell isolation medium sample adding cup, enabling the cell isolation medium to be in contact with the cell carrier liquid after flowing through the cell isolation medium micro-channel, and dispersing and isolating the cell carrier liquid from a continuous liquid phase into a dispersed liquid phase through shearing action to form water-in-oil reaction liquid drops containing single cells.
This water-in-oil step can be carried out on the basis of a microfluidic chip as shown in fig. 2, wherein the process of generating water-in-oil reaction droplets is shown in fig. 3.
2.1.4 collecting the generated water-in-oil reverse transcription system into a 0.2mL centrifuge tube, adding a PCR instrument, and reacting at 42 ℃ for 90 min;
2.1.5 adding PFO into the reverse transcription product of the centrifuge tube of 0.2mL for demulsification, centrifuging to separate water and oil, and absorbing a water phase;
2.1.6 adding Beckman Ampure XP Beads of 0.6X into the water phase for purification;
2.17 purification of the product PCR Pre-amplification was performed according to the following conditions
| Reagent | Volume of |
| 2X cDNA amplification reaction solution | 25μL |
| Purified reverse transcription product | 24μL |
| cDNA Pre-amplification primer (20uM) | 1μL |
Wherein the sequence of the cDNA pre-amplification primer is shown as SEQ ID NO. 6
2.18 the amplified product was purified with 0.8X bead; amplification of the purified product with TCR primer mixture I
| Reagent | Volume of |
| 2 XTaq polymerase reaction solution | 25μL |
| TCR primer mixture I | 1μL |
| Purified reverse transcription product | 24μL |
Number of amplification cycles:
wherein the value range of X is 9-14.
2.19 TCR primer mixture I amplification product using 0.8 Xbads purification, purification of products using TCR primer mixture II amplification
| Reagent | Volume of |
| 2 XTaq polymerase reaction solution | 25μL |
| TCR primer mixture II | 1μL |
| Purified reverse transcription product | 24μL |
Amplification cycle parameters:
wherein the value range of Y is 9-14.
2.20 amplification products of TCR primer mixture II fragment screening with 0.5X-0.8X beads, library construction of the screening products
2.21 disruption of the library with transposase Tn5
| 5 Xtransposase reaction buffer | 10μL |
| 50ng DNA | XμL |
| Transposase | 5μL |
| ddH2O | YμL |
| Total | 50μL |
Wherein, the value of X can be adjusted according to the concentration of the amplification product, and Y can be adjusted according to the addition of the amplification product.
2.22 transposase disruption temperature conditions
| Temperature of | Time |
| 55℃ | Hot lid |
| 55℃ | 5min |
| 4℃ | Hold |
2.23 termination of the fragmentation reaction
After the reaction, 10. mu.L of 1% SDS solution was added, mixed by aspiration and incubated at room temperature for 5 min.
2.24 PCR amplification Using library amplification primers, respectively
Wherein, the joint 1 is a universal joint, and the sequence thereof is as follows:
wherein, the joint 1 is a universal joint, and the sequence thereof is as follows:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGT
where NNNNNNNN is a barcode, it may be a random sequence.
2.25 library construction thermal cycling parameters
2.26 library amplification products were fragment screened with 0.6X-0.75X beads. The control results are shown in FIG. 4.
3. Sequencing analysis
The sequencing process can be performed on Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, Illumina Miseq and other platforms, and corresponding operation methods and experimental conditions are well known to those skilled in the art.
The specific analysis flow is shown in fig. 5, and comprises that clear data is obtained by quality control on sequencing raw data, and then data analysis such as comparison, screening, annotation and the like is performed. Counting quality control information, comparison information and annotation information, firstly comparing clean data with a known V (D) J reference library, and reserving at least 20bp of paired reads which are compared to the section. And then screening by using UMI, and if the UMI is supported by 400 paired reads, determining the UMI as valid UMI and reserving the valid UMI. Performing Contig splicing on each cell, annotating Barcode, screening to remove non-cell sequences, performing annotation analysis on the Contig, screening annotated Contig, further splicing the filtered Contig to obtain a Consensus sequence (a Consensus sequence) supported by the sample VDJ, and performing Clonotype typing according to the VDJ amino acid sequence of the spliced sample Consensus sequence.
More specifically, the amino acid sequence of the CDR3 region is used for clone subtype typing of the TCR, and the effective Barcode of the TCR is the subtype typing abundance for abundance analysis. The result can be exported by looking through Loupe V (D) J Browser. After Clonotype typing of the CDR3 amino acid sequence in TRA (TCR α)/TRB (TCR β), the number of each subtype was counted for abundance analysis. FIG. 6 is a graph showing abundance of clonotypes, in which each Clonotype subtype is plotted on the abscissa and the proportion of the subtype in all the subtypes is plotted on the ordinate.
Referring to FIG. 7, the CDR 3V/J region gene reflects the characteristics of TCR Clonotype, so the abundance and V-J paired analysis of V/J gene can be performed. And carrying out data statistics through TRA/TRB and TRA + TRB respectively to carry out abundance analysis.
Referring to FIG. 8, subsequent immunological studies can be further performed by counting V-J gene pairs, for example, by looking at V-J gene pairs that are abundant (high cell support) in a sample from one immunization period or comparing V-J gene pairs that are abundant in two different immunization periods to obtain specifically expressed immune genes. The results of the thermographic analysis of the V/J genes are as follows, wherein the abscissa represents Vgene, the ordinate represents Jgene, the abscissa represents V/J paired, and the deeper the color, the higher the abundance.
In addition, by using the cell sample same as that in the embodiment of the invention and comparing the obtained sequencing result with the sequencing result of the embodiment of the invention by performing a comparative sequencing test based on the 10X Genomics platform according to a manner known by a person skilled in the art, the method of the embodiment of the invention can find that the obtained sequencing result is accurate, the sensitivity is high, and the efficiency, the cost and the like are far better than those of the sequencing scheme based on the 10X Genomics platform.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Claims (15)
1. A kit for constructing a sequencing library of human single cell TCRs comprising:
the microfluidic chip is at least used for capturing human T cell single cells and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprises an oil phase and a cell liquid phase wrapped by the oil phase, and the water-in-oil reaction droplet comprises an RNA reverse transcription component and a cell lysis reagent;
an oil, a cell lysis reagent for forming the oil phase;
the cell label comprises a deformable microbead and a molecular label connected to the deformable microbead, wherein the molecular label is used for identifying cells;
an RNA reverse transcription component, which comprises a reverse transcription primer shown in SEQ ID NO. 5, RNA reverse transcriptase, an RNA reverse transcriptase inhibitor and an RNA reverse transcription buffer solution;
a cDNA pre-amplification component which comprises a cDNA pre-amplification primer shown as SEQ ID NO. 6, DNA polymerase and an amplification buffer solution;
the first amplification component comprises primers shown in SEQ ID NO. 1-SEQ ID NO. 2, DNA polymerase and amplification buffer solution;
the second amplification component comprises primers shown in SEQ ID NO. 3-SEQ ID NO. 4, DNA polymerase and amplification buffer solution;
a transposable fragmentation component comprising a transposase, a transposase reaction buffer and a transposition reaction stop solution;
a third amplification module comprising two sequencing adaptors, a DNA polymerase, and an amplification buffer.
2. The kit of claim 1, wherein: the micro-fluidic chip comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port, the cell micro-flow channel is provided with a cell suspension inlet and a single cell suspension outlet, the cell isolation medium micro-flow channel is provided with a cell label suspension inlet and a cell label suspension outlet, and the single-cell suspension outlet is intersected with the cell label suspension outlet, so that the single-cell suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell carrier liquid, the flow path of the cell carrier liquid is intersected with the cell isolation medium micro-channel, so that the cell isolation medium flowing in the cell isolation medium micro-channel can be cut and wrapped with the cell carrier liquid, thereby forming a water-in-oil reaction droplet comprising a single cell which is output by the single cell sample collection port.
3. The kit of claim 2, wherein: the tail region of the cell micro-flow channel 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, the outlet of the single cell channel is intersected with the outlet of the cell label micro-channel, so that the single cell suspension output from the cell micro-channel is mixed with the gel microbead molecular label suspension output from the gel microbead molecular label micro-channel to form cell carrier liquid, the continuous flow path of the cell carrier fluid intersects the cell isolation media microchannel such that the cell isolation media flowing through the cell isolation media microchannel is capable of shearing the continuous cell carrier fluid into discrete droplet-like cell liquid phases and causing each cell liquid phase to contain a single cell and a single cell label, while the cell isolation medium is encapsulated as an oil phase against the cell liquid phase, thereby forming the water-in-oil reaction droplets.
4. The kit according to claim 2 or 3, characterized in that: the microfluidic chip also comprises a cell suspension sample adding cup, a cell isolation medium sample adding cup and a cell label sample adding cup which are respectively communicated with the cell micro-channel, the cell isolation medium micro-channel and the cell label micro-channel.
5. The kit according to claim 3 or 4, characterized in that: and a negative pressure power generation device is arranged at the single cell sample collecting port and is used for generating negative pressure in the microfluidic chip so as to drive fluid in each micro-channel to flow.
6. The kit of claim 1, wherein: the oil phase comprises an oil and a cell lysis reagent, and the cell liquid phase comprises an RNA reverse transcription module; preferably, the volume ratio of the oil to the cell lysis reagent is 100: 1-500: 1.
7. the kit of claim 1, wherein: the molecular label can be separated from the deformable microbead under physical and/or chemical action, wherein the physical and/or chemical action comprises illumination or specific enzyme digestion; and/or, the molecular tag comprises an oligonucleotide strand; and/or, the molecular label comprises a barcode for identifying the cell, the barcode is 4-30nt in length; preferably, the barcode comprises 3 or more constant base sequences; more preferably, the barcode comprises 3nt and above riboG bases; and/or the length of the molecular label is 50nt-200 nt; and/or the diameter of the deformable microbeads is 10-200 mu m; and/or, the deformable beads comprise porous polyacrylamide beads.
8. The kit of claim 1, wherein: the use concentration of any one primer in the first amplification assembly and the second amplification assembly is 0.1-0.5 mu mol/L.
9. A method for constructing a sequencing library of a human single cell TCR is characterized by comprising the following steps:
capturing and packaging the human T cell single cells by using a microfluidic chip so as to generate water-in-oil reaction liquid drops containing the single cells;
cracking and reverse transcribing the cells in the water-in-oil reaction liquid drop to obtain a reverse transcription product;
pre-amplifying the reverse transcription product, and then sequentially performing primary amplification and secondary amplification to obtain an amplification product;
fragmenting, amplifying and purifying the amplification product to generate a sequencing library;
wherein the primer composition for the first amplification comprises primers shown by SEQ ID NO. 1-SEQ ID NO. 2, and the primer composition for the second amplification comprises primers shown by SEQ ID NO. 3-SEQ ID NO. 4.
10. The construction method according to claim 9, characterized by specifically comprising:
providing the kit of any one of claims 1-8;
respectively injecting the cell suspension, the cell label suspension and the cell isolation medium into the microfluidic chip, mixing the cell suspension and the cell label suspension after flowing through the cell microchannel and the cell label microchannel respectively to form cell carrier liquid, and shearing and wrapping the cell carrier liquid by the cell isolation medium flowing in the cell isolation medium microchannel so as to form water-in-oil reaction liquid drops containing single cells and single cell labels;
and collecting water-in-oil reaction droplets containing single cells from the single cell sample collection port.
11. The construction method according to claim 10, characterized by specifically comprising: and the negative pressure power generation device drives the cell suspension, the cell label suspension, the cell carrier liquid and the cell isolation medium to continuously flow in the microfluidic chip, and the formed water-in-oil reaction liquid drop is output from the single cell sample collection port.
12. The building method according to claim 9, characterized by further comprising: and irradiating the water-in-oil reaction liquid drop by using ultraviolet light, so that the molecular label in the water-in-oil reaction liquid drop is released from the deformable microbead.
13. The building method according to claim 9, characterized by further comprising: after the reverse transcription is finished, performing demulsification treatment on the water-in-oil reaction liquid drop, then performing purification treatment, and performing pre-amplification on a purified reverse transcription product; preferably, the water-in-oil reaction liquid drop is subjected to demulsification treatment in a physical mode; more preferably, the physical means comprises sonication means.
14. The construction method according to claim 9, characterized by specifically comprising: the fragmentation treatment was performed using Tn5 transposase, followed by amplification of the resulting fragmentation product.
15. A method of sequencing a human TCR, comprising: a TCR sequencing library of human single cells constructed using the method of any one of claims 9 to 14 followed by sequencing analysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201911353107.3A CN113026111A (en) | 2019-12-25 | 2019-12-25 | Kit for constructing human single cell TCR sequencing library and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201911353107.3A CN113026111A (en) | 2019-12-25 | 2019-12-25 | Kit for constructing human single cell TCR sequencing library and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528617A (en) * | 2021-07-28 | 2021-10-22 | 新格元(南京)生物科技有限公司 | High-throughput single immune cell surface receptor nucleic acid sequence detection method |
| CN116640855A (en) * | 2023-04-13 | 2023-08-25 | 华南农业大学 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
| CN116640855B (en) * | 2023-04-13 | 2024-10-15 | 华南农业大学 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
-
2019
- 2019-12-25 CN CN201911353107.3A patent/CN113026111A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528617A (en) * | 2021-07-28 | 2021-10-22 | 新格元(南京)生物科技有限公司 | High-throughput single immune cell surface receptor nucleic acid sequence detection method |
| CN116640855A (en) * | 2023-04-13 | 2023-08-25 | 华南农业大学 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
| CN116640855B (en) * | 2023-04-13 | 2024-10-15 | 华南农业大学 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
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