Droplet PCR-based amplification, library construction and sequencing methods

Abstract

Methods for amplification, library construction and high-throughput sequencing, based on droplet PCR. The droplet PCR-based amplification method comprises: encapsulating a single cell of a sample to be tested with an oil droplet, to obtain a single cell oil droplet of the sample to be tested; and performing PCR amplification on the single cell oil droplet of the sample to be tested, the single cell of the sample to be tested being provided in the form of a cell suspension, the cell suspension comprising the single cell of the sample to be tested and a first buffer, the first buffer comprising FS, and the FS comprising Tris-HCl, NaCl and MgCl2.

Description

Droplet-based PCR amplification, library construction, and sequencing methods Technical Field

[0001] The present application belongs to the field of biotechnology, specifically, to the field of single-cell sequencing, and more specifically, to a droplet-based PCR amplification, library construction and sequencing method. Background Art

[0002] Single-cell transcriptome sequencing (scRNA-seq) is a technique that detects cellular genetic information at the single-cell level. It can be used to reveal cellular heterogeneity, developmental, and differentiation states within tissues and is widely used in biomedical clinical research and life science research. However, because this technique requires single-cell lysis, the amount of RNA extracted is relatively small.

[0003] Reverse transcription polymerase chain reaction (RT-PCR) is a conventional detection technology that can be used to target RNA samples and amplify the abundance of specific RNA samples from them. It has extremely high application value for the detection of viral RNA. In recent years, the combination of droplet high-throughput technology and RT-PCR technology has been applied to clinical testing to improve the sensitivity and throughput of detection. For example, it has been widely used in the detection of SARS-CoV-2 mutants in wastewater (Heijnen, Leo et al. "Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater." The Science of the total environment vol. 799 (2021): 149456.).

[0004] However, existing technologies primarily focus on detecting specific gene information and are not applicable to high-throughput single-cell transcriptomics. Furthermore, some existing technologies also suffer from issues such as inaccurate semipermeable membrane selectivity and difficulty in purification.

[0005] Therefore, it is currently necessary to develop a high-throughput single-cell transcriptome sequencing technology based on droplet RT-PCR to achieve high-throughput single-cell transcriptome detection.

[0006] Summary of the Invention

[0007] This application is based on the inventor's discovery and understanding of the following problems:

[0008] The inventors discovered that, on the one hand, the current in-droplet RT-PCR technology has limitations. First, due to the small size of the droplets, performing RT-PCR amplification in each droplet will result in incomplete amplification reactions (only nucleic acid substances larger than 300bp can be trapped), resulting in incomplete transcript information. Second, this technology can only detect specific genes and cannot obtain comprehensive transcript information in cells. These limitations may affect the application of this technology in clinical medicine.

[0009] On the other hand, microplate-based single-cell sequencing technologies typically use microreactors for single-cell RT-PCR amplification (e.g., 10x Genomics). However, since the numerous tiny reactors in the microplate are difficult to control, achieving high throughput is challenging. Furthermore, this approach is more expensive than droplet-based RT-PCR.

[0010] Therefore, the inventors used 5'-end droplet technology to reverse transcribe and amplify cDNA within the droplet. The PCR product was purified before library construction and sequencing. This single-step reverse transcription and amplification within the droplet not only increases throughput but also simplifies the process, shortens time, and reduces costs.

[0011] To this end, based on the above findings, in the first aspect of the present application, a droplet-based PCR amplification method is proposed. According to an embodiment of the present invention, the method includes: subjecting a single cell of the sample to be tested to an oil droplet wrapping treatment so as to obtain a single cell oil droplet of the sample to be tested; and subjecting the single cell of the sample to be tested in the oil droplet to a PCR amplification treatment; wherein the single cell of the sample to be tested is provided in the form of a cell suspension, and the cell suspension includes the single cell of the sample to be tested and a first buffer, the first buffer includes FS, and the FS includes Tris-HCl, NaCl and MgCl2. According to an embodiment of the present application, this method can comprehensively obtain the transcript information in the cell. By adding reverse transcriptase to the first buffer, RT-PCR is completed in one step, which not only improves the speed of processing samples, but also simplifies the operation steps, shortens the experimental time, and also reduces the experimental cost.

[0012] According to an embodiment of the present application, the above method may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present application, the concentration of Tris-HCl in the first buffer is 20-30 mM. In some examples of the present application, the concentration of Tris-HCl in the first buffer may optionally be 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. In some preferred examples of the present application, when the concentration of Tris-HCl in the first buffer is 25 mM, the stability and specificity of the PCR reaction are better.

[0014] According to an embodiment of the present application, the concentration of NaCl in the first buffer is 25-75mM. In some examples of the present application, the concentration of NaCl in the first buffer is optionally 25mM, 30mM, 35mM, 40mM, 41mM, 44mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 55mM, 53mM, 55mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 65mM, 70mM or 75mM. In some preferred examples of the present application, when the concentration of NaCl in the first buffer is 50mM, non-specific binding of primers can be avoided.

[0015] According to an embodiment of the present application, the concentration of MgCl2 in the first buffer is 3 to 8 mM. In some examples of the present application, the concentration of MgCl2 in the first buffer can optionally be 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. In some preferred examples of the present application, when the concentration of MgCl2 in the first buffer is 5 mM, the specificity and amplification efficiency of the PCR product can be increased.

[0016] According to an embodiment of the present application, the first buffer further includes betaine, dNTP, dCTP, trihydroxypropylphosphine, a nuclease inhibitor, an S2 primer (cDNA amplification primer) and KAPA high-fidelity DNA polymerase. The inventors ensured that the reverse transcription reaction was fully carried out by adding dCTP to the first buffer.

[0017] According to an embodiment of the present application, the final concentration of betaine in the first buffer is 0.5 to 2 M. In some examples of the present application, the final concentration of betaine in the first buffer is optionally 0.5 M, 1 M, 1.5 M, or 2 M. In some preferred examples of the present application, the final concentration of betaine in the first buffer is 1 M.

[0018] According to an embodiment of the present application, the final concentration of the dNTPs in the first buffer is 0.2 to 2 mM. In some examples of the present application, the final concentration of the dNTPs in the first buffer is optionally 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, or 2 mM. In some preferred examples of the present application, the final concentration of the dNTPs in the first buffer is optionally 1 mM.

[0019] According to embodiments of the present application, the final concentration of dCTP in the first buffer is 1 to 5 mM. In some examples of the present application, the final concentration of dCTP in the first buffer is optionally 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM. In some preferred examples of the present application, the final concentration of dCTP in the first buffer is optionally 5 mM.

[0020] According to embodiments of the present application, the final concentration of the KAPA high-fidelity DNA polymerase in the first buffer is 0.01 to 0.03 U / μL. In some examples of the present application, the final concentration of the KAPA high-fidelity DNA polymerase in the first buffer can optionally be 0.01 U / μL, 0.02 U / μL, or 0.03 U / μL. In some preferred embodiments of the present application, the final concentration of the KAPA high-fidelity DNA polymerase in the first buffer is 0.02 U / μL.

[0021] According to an embodiment of the present application, the final concentration of trihydroxypropylphosphine in the first buffer is 0.01 to 0.03 M. In some examples of the present application, the final concentration of trihydroxypropylphosphine in the first buffer is optionally 0.01 M, 0.02 M, or 0.03 M. In some preferred examples of the present application, the final concentration of trihydroxypropylphosphine in the first buffer is 0.01 M.

[0022] According to an embodiment of the present application, the final concentration of the nuclease inhibitor in the first buffer is 1 to 3 U / μL. In some examples of the present application, the final concentration of the nuclease inhibitor in the first buffer can optionally be 1 U / μL, 1 U / μL, or 3 U / μL. In some preferred examples of the present application, the final concentration of the nuclease inhibitor in the first buffer is 1 U / μL.

[0023] According to an embodiment of the present application, the final concentration of the S2 primer in the first buffer is 0.6 to 1.2 μM. In some examples of the present application, the final concentration of the S2 primer in the first buffer is optionally 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, or 1.2 μM. In some preferred examples of the present application, the final concentration of the S2 primer in the first buffer is 0.9 μM.

[0024] According to an embodiment of the present application, the first buffer further includes: SSIV reverse transcriptase; the final concentration of the SSIV reverse transcriptase in the first buffer is 3 to 5 U / μL. In some examples of the present application, the final concentration of the SSIV reverse transcriptase in the first buffer can optionally be 3 U / μL, 3.5 U / μL, 4 U / μL, 4.5 U / μL, or 5 U / μL. In some preferred examples of the present application, the final concentration of the SSIV reverse transcriptase in the first buffer is 4.5 U / μL. According to an embodiment of the present application, adding SSIV reverse transcriptase to the first buffer can perform an RT-PCR reaction in a single step within the droplet, reducing the number of operation steps and saving time and cost.

[0025] According to an embodiment of the present application, the first buffer further includes: dithiothreitol (DTT); the final concentration of the dithiothreitol in the first buffer is 0.01 to 0.03 M. In some examples of the present application, the final concentration of the dithiothreitol in the first buffer is optionally 0.01 M, 0.02 M or 0.03 M. In some preferred examples of the present application, the final concentration of the dithiothreitol in the first buffer is 0.01 M. According to an embodiment of the present application, in RT-PCR, DTT is mainly used to reduce the disulfide bonds in the RNA molecules to convert them into single sulfide bonds, thereby making the RNA molecules easier to unravel and facilitating the action of reverse transcriptase. DTT can also enable thermostable reverse transcriptase to maintain activity at high temperatures and prevent the RNA template from being degraded or forming complex secondary structures, thereby increasing the efficiency and accuracy of the cDNA synthesis process.

[0026] According to an embodiment of the present application, the first buffer further includes: Poly dT; the final concentration of the Poly dT in the first buffer is 1 to 4 μM. In some examples of the present application, the final concentration of the Poly dT in the first buffer can optionally be 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, or 4 μM. In some preferred examples of the present application, the final concentration of the Poly dT in the first buffer is 2.5 μM. According to an embodiment of the present application, in RT-PCR, Poly dT is mainly used to capture mRNA molecules, which are used as templates for reverse transcription reactions, thereby transcribing them into cDNA.

[0027] According to an embodiment of the present application, the oil droplet encapsulation process is performed in the following manner: a single cell suspension of the sample to be tested, a microbead suspension, and droplet-forming oil are mixed and processed.

[0028] Exemplarily, a vaporization component is used to vaporize the single cell suspension and rapidly push it into the droplet-forming oil to form PCR droplets.

[0029] According to an embodiment of the present application, the microbead suspension includes microbeads and a second buffer, the second buffer includes FS, and the FS includes Tris-HCl, NaCl and MgCl2.

[0030] According to an embodiment of the present application, the concentration of Tris-HCl in the first buffer is 20-30 mM. In some examples of the present application, the concentration of Tris-HCl in the first buffer may optionally be 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. In some preferred examples of the present application, when the concentration of Tris-HCl in the first buffer is 25 mM, the stability and specificity of the PCR reaction are better.

[0031] According to an embodiment of the present application, the concentration of NaCl in the first buffer is 40-60 mM. In some examples of the present application, the concentration of NaCl in the first buffer is optionally 40 mM, 41 mM, 44 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 55 mM, 53 mM, 55 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM or 60 mM. In some preferred examples of the present application, when the concentration of NaCl in the first buffer is 50 mM, non-specific binding of primers can be avoided.

[0032] According to an embodiment of the present application, the concentration of MgCl2 in the first buffer is 3 to 8 mM. In some examples of the present application, the concentration of MgCl2 in the first buffer can optionally be 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. In some preferred examples of the present application, when the concentration of MgCl2 in the first buffer is 5 mM, the specificity and amplification efficiency of the PCR product can be increased.

[0033] According to an embodiment of the present application, the second buffer further comprises: Triton X-100, digitonin and ficoll.

[0034] According to an embodiment of the present application, the concentration of Triton X-100 in the second buffer is 0.1% to 0.3% (w / v). In some examples of the present application, the concentration of Triton X-100 in the second buffer is optionally 0.1% (w / v), 0.2% (w / v), or 0.3% (w / v). In some preferred examples of the present application, the concentration of Triton X-100 in the second buffer is 0.1% (w / v).

[0035] According to an embodiment of the present application, the concentration of the digitonin in the second buffer is 0.001 to 0.003% (w / v). In some examples of the present application, the concentration of the digitonin in the second buffer is optionally 0.01% (w / v), 0.015% (w / v), 0.02% (w / v), 0.025% (w / v) or 0.03% (w / v). In some preferred examples of the present application, the concentration of the digitonin in the second buffer is 0.025% (w / v).

[0036] According to an embodiment of the present application, the final concentration of the ficoll aqueous solution in the second buffer is 2 to 10% (w / v). In some examples of the present application, the final concentration of the ficoll aqueous solution in the second buffer is optionally 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v) or 10% (w / v). In some preferred examples of the present application, the final concentration of the ficoll aqueous solution in the second buffer is 4% (w / v). According to an embodiment of the present application, the addition of Ficol can make it difficult for cells and magnetic beads to sink during the droplet running process, thereby preventing clogging of the microfluidic channel.

[0037] In a second aspect of the present application, a method for constructing a sequencing library is provided. According to an embodiment of the present application, the method comprises: performing a PCR amplification reaction on a test sample based on the method described in the first aspect of the present invention to obtain an amplified product, wherein the amplified product constitutes the sequencing library. According to an embodiment of the present application, constructing a sequencing library using the aforementioned in-droplet RT-PCR method can simplify the operational steps, shorten library construction time, and save costs.

[0038] According to an embodiment of the present application, the method further comprises breaking the emulsion and fragmenting the amplified product; and ligating the fragmented product to a sequencing adapter. According to an embodiment of the present application, the emulsion is broken to release the amplified PCR product. The PCR product is fragmented and then ligated to a sequencing adapter to form a sequencing library.

[0039] According to embodiments of the present application, the demulsification treatment is performed by mixing the amplification product with a surfactant (e.g., PFO or sodium octane sulfonate) to obtain a demulsified product. In one example of the present application, the amplification product is mixed with PFO (Sigma-Aldrich, Catalog No.: 370533-25G), and the mixed product is centrifuged at 1000 g for 2 minutes to obtain a demulsified product. In some examples of the present application, a surfactant is added to demulsify the oily droplets.

[0040] It should be noted that the surfactant can be freely selected according to experimental requirements and is not specifically limited in this application.

[0041] According to an embodiment of the present application, the fragmentation treatment is performed in the presence of a fragmentation enzyme.

[0042] In a third aspect of the present application, a sequencing library is provided. According to embodiments of the present application, the sequencing library is obtained by the method described in the second aspect of the present application. According to embodiments of the present application, a sequencing library can be rapidly constructed based on the method described in the second aspect of the present application.

[0043] In a fourth aspect, the present application provides a method for sequencing a target nucleic acid molecule. According to an embodiment of the present application, the method comprises: constructing a sequencing library for the target sequence according to the method described in the third aspect of the present application; sequencing the sequencing library to obtain sequencing results; and determining the nucleic acid sequence of the target nucleic acid molecule based on the sequencing results. According to an embodiment of the present application, constructing a library for high-throughput sequencing based on the method described herein reduces manual steps, shortens time, and reduces costs.

[0044] In a fifth aspect, this application proposes a droplet PCR amplification kit. According to embodiments of this application, the kit comprises: the first and second buffers defined in the method described in the first aspect of the invention; and a droplet generation oil. According to embodiments of this application, the kit enables rapid PCR amplification, is easy to operate, and is low-cost. Both reverse transcription and PCR amplification reactions can be performed within the droplets, making it more efficient than previous amplification methods.

[0045] According to an embodiment of the present application, the above-mentioned kit may further include at least one of the following additional technical features:

[0046] According to an embodiment of the present application, the kit further includes at least one of a digestive enzyme and an instruction manual.

[0047] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0049] FIG1 is a schematic diagram of a sequencing quality inspection report result of the 1-1 experimental group according to an embodiment of the present invention;

[0050] FIG2 is a schematic diagram of the sequencing quality inspection report results of the 1-2 experimental group according to an embodiment of the present invention;

[0051] FIG3 is a schematic diagram of the sequencing quality inspection report results of the 2-1 experimental group according to an embodiment of the present invention;

[0052] FIG4 is a schematic diagram of a sequencing quality inspection report result of the 2-2 experimental group according to an embodiment of the present invention;

[0053] FIG5 is a schematic diagram showing the fragment distribution of PCR amplification product 2100 when sodium ions are present in the 5xFS buffer of experimental group 1-1 according to an embodiment of the present invention; wherein the 5xFS composition is: 125 mM Tris-HCl, 250 mM NaCl, 25 mM MgCl2;

[0054] FIG6 is a schematic diagram showing the fragment distribution of PCR amplification product 2100 when potassium ions are present in 5xFS buffer in experimental group 2-1 according to an embodiment of the present invention; wherein the 5XFS composition is: 125 mM Tris-HCl, 250 mM NaCl, 25 mM MgCl2;

[0055] FIG7 is a schematic diagram of the fragment distribution of PCR amplification products 2100 tested at different salt ion concentrations according to an embodiment of the present invention; RP1-4 represents the fragment distribution of PCR amplification products 2100 of experimental groups 1-4; RP2-4 represents the fragment distribution of PCR amplification products 2100 of experimental groups 2-4; RP3-4 represents the fragment distribution of PCR amplification products 2100 of experimental groups 3-4; and RP4-4 represents the fragment distribution of PCR amplification products 2100 of experimental groups 4-4.

[0056] FIG8 is a schematic diagram of droplet generation according to an embodiment of the present invention. DETAILED DESCRIPTION

[0057] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0058] Prior art related to this application:

[0059] Currently, droplet RT-PCR is used to detect specific genes in single cells. This method uses a specific material to form a semi-permeable membrane-like droplet. The semi-permeable membrane of the droplet has selective permeability, which allows cells to be encapsulated in the droplet and then lysed to release specific nucleic acid substances. The liquid is then replaced and the lysate is removed before the RT-PCR reaction is performed (Leonaviciene, Greta, and Linas Mazutis. "RNA cytometry of single-cells using semi-permeable microcapsules." Nucleic acids research vol. 51, 1(2023): e2. doi: 10.1093 / nar / gkac918). This method has the advantage of removing the inhibitory effect of the lysate on the enzyme, thereby not affecting subsequent reactions. However, this approach also has significant drawbacks. First, the semipermeable membrane can only retain nucleic acids larger than 300 bp, while the primers used for high-throughput labeling are all smaller than 300 bp. Therefore, these primers will freely shuttle through the membrane, preventing droplet labeling. Second, the droplets forming the semipermeable membrane are not easily broken, making it impossible to purify the entire cellular transcriptome. Currently, its primary application is in detecting specific genetic information, and it cannot be applied to high-throughput single-cell transcriptomics.

[0060] Currently, RT-PCR can be applied to single-cell transcriptome sequencing, which is basically based on single-tube technology. Since single-tube technology can use high-temperature lysis, the cells in the well plate are heated and lysed before adding the RT-PCR reaction reagent, thus solving the problem of high incompatibility between lysis and RT. For example, smart-seq3 and Flash-seq are both based on well plates (Hagemann-Jensen, M., Ziegenhain, C., Chen, P., D., Hendriks, GJ, Larsson, AJM, Faridani, OR, & Sandberg, R. (2020). Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nature biotechnology, 38(6), 708-714.; Hahaut, V., Pavlinic, D., Carbone, W., Schuierer, S., Balmer, P., Quinodoz, M., Renner, M., Roma, G., Cowan, CS, & Picelli, S. (2022). Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq. Nature biotechnology, 40(10), 1447-1451.). Although they have simplified the process of single-cell library construction to a certain extent, FLASH-seq has shortened the entire library construction process to 4.5h. Although there has been a significant improvement in time cost, plate-based technology is difficult to achieve high throughput.

[0061] Droplet PCR-based amplification method

[0062] Therefore, to address the shortcomings of the aforementioned prior art, the present application has developed a droplet PCR-based amplification method. The method comprises: encapsulating a single cell of a sample to be tested in an oil droplet to obtain a single cell oil droplet of the sample to be tested; and amplifying the single cell oil droplet of the sample to be tested by PCR. The single cell to be tested is provided in the form of a cell suspension, wherein the cell suspension comprises the single cell to be tested and a first buffer solution, wherein the first buffer solution comprises FS, which comprises Tris-HCl, NaCl, and MgCl2.

[0063] According to an embodiment of the application, reverse transcriptase, DTT (dithiothreitol) and Poly dT can be added to the first buffer to achieve a one-step droplet reaction of reverse transcription and PCR amplification. Not only can high throughput be achieved, but the experimental steps are also greatly simplified, shortening the reaction time. It is also possible to save experimental costs. Compared with the droplet RT-PCR (ddRT-PCR) and the single-cell RT-PCR transcriptome technology based on the well plate used in clinical detection in the past, the method described in the present application can obtain the complete transcript information of a single cell, and can quickly achieve high throughput, which can provide a research basis for the application of future automated library construction. Therefore, the present application has obvious advantages and application value for the development of future single-cell high-throughput transcriptome sequencing technology.

[0064] Specifically, for ease of understanding, the above method and technical solution are explained and illustrated in detail below.

[0065] 1. Prepare single-cell suspension: Digest the tissue (grind or use digestive enzymes), wash in PBS (containing 0.04% BSA), filter through a cell sieve, and resuspend in the first cell buffer (formula see Table 1) to obtain a single-cell suspension;

[0066] 2. Prepare microbeads: Resuspend the index carrier (short DNA sequence used to label the sample) in the prepared bead buffer (microbead buffer, preparation method see Table 3), mix thoroughly, then resuspend the cell beads (cell microbeads), mix thoroughly;

[0067] It should be noted that the microbeads were already linked to barcode 1, barcode 2, UMI, and TSO primer (rGrGrG) sequences during preparation.

[0068] 3. Droplet generation: Mix the droplet generation oil, cell suspension (prepared in step 1), and cell microbeads (prepared in step 2) and transfer them to a PCR tube (an eight-tube strip can also be used). Cover with mineral oil and perform the PCR reaction on the machine.

[0069] It should be noted that during droplet running, mRNA is captured by poly dT after cell lysis. Reducing agents (such as DTT) release oligos (DNA primers) on the surface of the magnetic beads into the droplets. Under the action of RT enzyme (reverse transcriptase), reverse transcription is performed using mRNA as a template to synthesize the first cDNA strand. CCC is added to the end of the first strand. The primers on the magnetic beads hybridize with the CCC and are captured by the labeled oligos, completing the template conversion. The strands are then melted at 95°C, and a complementary second cDNA strand is synthesized using the first cDNA strand as a template. PCR reaction is then performed using PCR primers at both ends to amplify the target fragment.

[0070] Sequencing library construction method

[0071] In a second aspect of the present application, a method for constructing a sequencing library is proposed. According to an embodiment of the present application, a PCR amplification reaction is performed on a sample to be tested based on the above-mentioned droplet PCR amplification method to obtain an amplification product, which constitutes the sequencing library.

[0072] It should be noted that after the PCR amplification, the amplified cell oil droplets are demulsified to release the amplified cDNA. The amplified cDNA is purified (to remove impurities such as PCR primers, salts, and buffers), and the purified product is fragmented (generally using a fragmentation enzyme, but other methods can also be used). Adaptor ligation and single-strand circularization are then performed to complete the construction of the sequencing library.

[0073] Sequencing library

[0074] In a third aspect of the present application, a sequencing library is proposed. According to an embodiment of the present application, the sequencing library is constructed and obtained by the above method.

[0075] Method for sequencing target nucleic acid molecules

[0076] In a fourth aspect of the present application, a method for sequencing a target nucleic acid molecule is proposed. According to an embodiment of the present application, the method is to perform high-throughput sequencing on the sequencing library constructed above on a sequencing platform.

[0077] It should be noted that the sequencing platform is not particularly limited and can be any common first-generation, second-generation or third-generation sequencing platform.

[0078] Droplet PCR Amplification Kit

[0079] In a fifth aspect of the present application, a droplet PCR amplification kit is provided. According to an embodiment of the present application, the kit comprises: a digestive enzyme; the first buffer and the second buffer of the first aspect; and droplet generation oil.

[0080] According to the embodiments of the present application, the kit is more convenient and quick in scientific research or medical applications, and can perform a one-step RT-PCR reaction, reducing manual operations, lowering the risk of sample contamination, and having a faster amplification speed than traditional PCR.

[0081] The present invention will be further explained below with reference to specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0082] Example 1: High-throughput single-cell transcriptome sequencing process based on droplet RT-PCR

[0083] 1. Preparation of single cell suspension

[0084] 1.1 Prepare single-cell suspensions of human 293T cell lines and mouse 3T3 cell lines after recovery, wash 1-2 times with PBS (containing 0.04% BSA), and filter through a 40 μm pore size cell sieve;

[0085] 1.2 Use a cell counting plate or a cell counter to detect the concentration of cells (or cell nuclei);

[0086] 1.3 Depending on the cell concentration, aspirate approximately 22,000 cells (or cell nuclei), centrifuge at 300-500g, 4°C, for 5 min to collect the cell pellet, and add 100 μL Cell Resuspension Buffer (cell resuspension buffer, preparation method see Table 1) to resuspend the cells (or cell nuclei).

[0087] Table 1: Cell resuspension buffer configuration

[0088] Note: Preparation of 5X FS: 125mM Tris-HCl, 250mM NaCl, 25mM MgCl2.

[0089] The manufacturers and product numbers of the reagents used to prepare the above buffer solutions are shown in Table 2.

[0090] Table 2:

[0091] 2. Microbead Preparation

[0092] 2.1 Pipette 350,000 magnetic beads and 2.5 million index carriers (short DNA sequences used to label samples) into two 0.2 mL PCR tubes, place on a magnetic stand and let stand for 2 minutes, then remove the supernatant.

[0093] 2.2 Remove the PCR tube from the magnetic stand and resuspend the index carrier in the prepared bead buffer (see Table 3 for preparation). Mix thoroughly and then resuspend the cell beads. Mix thoroughly.

[0094] Table 3:

[0095] 3. Droplet generation (Figure 8)

[0096] 3.1 Tear off the protective film on the chip surface and place it in the chip slot area of ​​the droplet generator;

[0097] 3.2 Insert the A end of the connecting tube on the collection cover (the connecting tube that contacts the bottom of the collection tube) into the outlet hole of the chip;

[0098] 3.3 Place the 30ml syringe in the holder and adjust the plunger to the initial position of 17ml. Use a flat needle to connect the syringe to the end of the connecting tube B on the collection tube cap (the connecting tube that does not touch the bottom of the collection tube).

[0099] 3.4 Add 100 μL of droplet generation oil to the collection tube, tighten the collection cap, and place the collection tube vertically on the fixed stand.

[0100] 3.5 Use a pipette to gently pipette and mix the cells. Add 100 μL of cell suspension to the cells well of the chip, making sure the pipette tip touches the bottom of the well.

[0101] 3.6 Use a pipette to gently pipette and mix the beads. Add 100 μL of beads to the beads well of the chip, making sure the pipette tip touches the bottom of the well.

[0102] 3.7 Immediately add 350 μl of droplet generation oil to the Oil well of the chip;

[0103] 3.8 Quickly pull the push rod of the syringe to the slot position and lock the push rod in the slot;

[0104] 3.9 After droplets are generated, immediately loosen the collection cap on the collection tube, pull out the connecting tube from the chip outlet hole, stretch the connecting tube vertically to allow the droplets in the tube to flow into the collection tube, and cut off the connecting tube on the collection tube;

[0105] 3.10 Transfer the droplets to an eight-tube strip, ensuring the droplet surface does not exceed 100 μL. Cover the droplet surface with 50-100 μL of mineral oil. Cover the eight-tube strip with the cap and perform RT-PCR according to the following protocol (Table 4).

[0106] Table 4:

[0107] Note: PBMC (human peripheral blood mononuclear cells) / cell nucleus: 20 cycles; cell lines: 15 cycles.

[0108] 4. Demulsification

[0109] 4.1 After RT (reverse transcription) is completed, transfer the droplets to a new low-absorption 1.5 mL centrifuge tube. Slowly aspirate from the bottom, excluding the top layer of mineral oil. Insert the pipette tip to the bottom and slowly aspirate the oil generated by the droplets and discard it, leaving the middle emulsion layer.

[0110] 4.2 Add 50 μL of PFO (perfluorooctane sulfonic acid) to the droplets, mix thoroughly by inversion, and centrifuge at 1000 g for 2 min.

[0111] 4.3 Slowly pipette the aqueous supernatant (upper middle layer) into a new 1.5 mL centrifuge tube and measure the volume of the supernatant.

[0112] 5. Purification of amplified product fragments

[0113] 5.1 Take out the Novozyme DNA purification beads (or Ampure XP beads) in advance, equilibrate them at room temperature for at least 30 minutes, and shake thoroughly before use;

[0114] 5.2 Calculate the number of magnetic beads to be used according to the measured volume;

[0115] 5.3 Use a pipette to draw 0.6X Novozyme DNA purification beads into the PCR product and gently pipette at least 10 times to mix thoroughly. The last time, make sure all the liquid and magnetic beads in the pipette tip are pipetted into the PCR tube;

[0116] 5.4 Incubate at room temperature for 10 minutes;

[0117] 5.5 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the liquid becomes clear;

[0118] 5.6 Aspirate the supernatant into a new 1.5 ml tube and save it for the next step of oligo product purification.

[0119] 5.7 Keep the centrifuge tube on the magnetic rack and add 1 ml of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0120] 5.8 Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0121] 5.9 Keep the centrifuge tube fixed on the magnetic rack, open the tube cover, and dry it at room temperature until the surface of the magnetic beads is free of reflections and cracks;

[0122] 5.10 Remove the centrifuge tube from the magnetic stand, add 48 μl Nuclease-Free Water to elute the cDNA, and gently pipette at least 10 times to mix thoroughly.

[0123] 5.11 Incubate at room temperature for 5 minutes;

[0124] 5.12 Centrifuge briefly, place the centrifuge tube on a magnetic rack, and let it stand for 2-5 minutes until the liquid becomes clear. Transfer 46 μl of the supernatant to a new eight-tube strip.

[0125] 5.13 Take 1 μL of cDNA and use the Qubit dsDNA HS Kit to determine the concentration.

[0126] 6. Oligo product purification

[0127] 6.1 Add 0.8X Novozyme DNA beads to the supernatant retained in this step and incubate for 5 minutes;

[0128] 6.2 After a brief centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the liquid becomes clear. Discard the supernatant.

[0129] 6.3 Keep the centrifuge tube on the magnetic rack and add 1 ml of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0130] 6.4 Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0131] 6.5 Keep the centrifuge tube fixed on the magnetic rack, open the tube cover, and dry it at room temperature until the surface of the magnetic beads is free of reflection and cracks;

[0132] 6.6 Remove the centrifuge tube from the magnetic stand, add 23 μl of Nuclease-Free Water to elute the cDNA, and gently pipette at least 10 times to mix thoroughly.

[0133] 6.7 Incubate at room temperature for 5 minutes;

[0134] 6.8 Centrifuge briefly, place the centrifuge tube on a magnetic rack, and let it stand for 2-5 minutes until the liquid becomes clear. Transfer 21 μl of the supernatant to a new PCR tube and prepare for oligo library amplification.

[0135] 7. Oligo secondary amplification

[0136] Prepare cDNA amplification PCR mix according to Table 5.

[0137] Table 5:

[0138] Add 54 μl of the oligo amplification PCR mix to 46 μl of the cDNA purified product from the previous step to perform the reaction. The reaction program settings are shown in Table 6.

[0139] Table 6: PCR program

[0140] 7. Construction of cDNA 5' end library

[0141] 7.1 Prepare the interruption reaction system on ice according to Table 7

[0142] Table 7:

[0143] 7.2 Based on the cDNA product concentration, take 100 ng of the purified cDNA product from step 5 and place it in a new 0.2 mL PCR tube. Add NF Water to the volume to 45 μL and place the PCR tube on ice.

[0144] 7.3 Use a pipette to draw 15 μL of the prepared disruption reaction system into the PCR tube prepared in step 7.2. Vortex three times for 3 seconds each time and briefly centrifuge to collect the reaction system at the bottom of the tube.

[0145] 7.4 When the PCR instrument temperature drops to 4°C, place the PCR tubes described in step 7.2 on the PCR instrument, skip the 4°C reaction, and immediately proceed to the 30°C reaction (see Table 8 for the reaction program);

[0146] Table 8:

[0147] After the reaction is completed, centrifuge briefly to collect the reaction system to the bottom of the tube, and place the PCR tube on ice.

[0148] 8. Connector connection

[0149] 8.1 Prepare the adapter ligation reaction system on ice according to Table 9;

[0150] Table 9:

[0151] 8.2 Use a pipette to slowly pipette 40 μL of the prepared adapter ligation reaction mixture into the PCR tube prepared in step 7.4. Vortex to mix thoroughly. Centrifuge briefly to collect the reaction mixture at the bottom of the tube.

[0152] 8.3 Place the PCR tubes described in step 8.2 on a PCR instrument and perform the reaction according to the conditions in Table 10;

[0153] Table 10:

[0154] After the reaction is completed, centrifuge briefly to collect the reaction system to the bottom of the tube;

[0155] 8.4 Take out the DNA Clean Beads in advance and equilibrate them at room temperature for at least 30 minutes. Vortex mix thoroughly before use.

[0156] 9. Purification of adapter ligation products and fragment screening

[0157] 9.1 Pipette 100 μL of DNA Clean Beads into the adapter-ligated product from step 8.3 and gently pipette up and down at least 10 times to mix thoroughly. The last time, ensure that all the liquid and beads in the pipette tip are pumped into the PCR tube.

[0158] 9.2 Incubate at room temperature for 5 minutes;

[0159] 9.3 After a brief centrifugation, place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the liquid becomes clear. Discard the supernatant.

[0160] 9.4 Keep the PCR tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube wall. Let it stand for 30 seconds and discard the supernatant.

[0161] 9.5 Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge it immediately. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0162] 9.6 Keep the PCR tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is free of reflections and cracks;

[0163] 9.7 Remove the PCR tube from the magnetic stand, add 102 μL of NF Water, and pipette gently aspirate at least 10 times to mix thoroughly.

[0164] 9.8 Incubate at room temperature for 5 minutes;

[0165] 9.9 Briefly centrifuge, place the PCR tube on a magnetic rack, and let it stand for 2-5 minutes until the liquid becomes clear. Transfer 100 μL of the supernatant to a new 0.2 mL PCR tube;

[0166] 9.10 Pipette 55 μL of DNA Clean Beads into the PCR tube prepared in step 9.9 and mix thoroughly by gently pipetting up and down at least 10 times. Make sure all the liquid and beads in the pipette tip are pipetted into the PCR tube.

[0167] 9.11 Incubate at room temperature for 5 minutes;

[0168] 9.12 After a brief centrifugation, place the PCR tube on a magnetic rack and let it sit for 2-5 minutes until the liquid becomes clear. Carefully aspirate the supernatant with a pipette and transfer it to a new PCR tube.

[0169] 9.13 Pipette 15 μL of DNA Clean Beads into the supernatant of the PCR tube from the previous step and pipette gently up and down at least 10 times to mix thoroughly.

[0170] 9.14 Incubate at room temperature for 5 minutes;

[0171] 9.15 After a brief centrifugation, place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Discard the supernatant.

[0172] 9.16 Keep the PCR tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds and discard the supernatant.

[0173] 9.17 Repeat the previous step and try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, centrifuge it immediately. After separation on the magnetic stand, use a small-scale pipette to remove the liquid from the bottom of the tube.

[0174] 9.18 Place the PCR tube on the magnetic rack, open the tube cap, and leave it at room temperature until the surface of the magnetic beads is free of reflections and cracks.

[0175] 9.19 Remove the PCR tube from the magnetic stand, add 48 μL of NF H2O, and mix thoroughly by pipetting gently up and down at least 10 times.

[0176] 9.20 Incubate at room temperature for 5 minutes;

[0177] 9.21 Briefly centrifuge and place the PCR tube on a magnetic rack. Let stand for 2-5 minutes until the liquid becomes clear. Transfer 46 μL of the supernatant to a new 0.2 mL PCR tube.

[0178] 10. PCR amplification

[0179] 10.1 Add 4 μL of scRNA Barcode Primer to the PCR tube in step 9.21;

[0180] 10.2 Add 50 μL of PCR Amp Enzyme to the reaction mixture in the previous step, vortex three times for 3 seconds each time, and briefly centrifuge to collect the reaction mixture at the bottom of the tube;

[0181] 10.3 Place the PCR tubes described in step 9.5.2 on a PCR instrument and perform the reaction according to the conditions in Table 11;

[0182] Table 11:

[0183] 11. Screening of PCR amplification product fragments

[0184] Remove the DNA Clean Beads in advance and equilibrate them at room temperature for at least 30 minutes. Vortex mix thoroughly before use.

[0185] 11.1 Pipette 55 μL of DNA Clean Beads into the PCR product and gently pipette up and down at least 10 times to mix thoroughly. The last time, ensure that all the liquid and beads in the pipette tip are pumped into the PCR tube.

[0186] 11.2 Incubate at room temperature for 5 minutes;

[0187] 11.3 After a brief centrifugation, place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the liquid becomes clear. Carefully aspirate the supernatant with a pipette and transfer it to a new PCR tube.

[0188] 11.4 Pipette 15 μL of DNA Clean Beads into the supernatant of the PCR tube in the previous step and pipette gently up and down at least 10 times to mix thoroughly.

[0189] 11.5 Incubate at room temperature for 5 minutes;

[0190] 11.6 After a brief centrifugation, place the PCR tube on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Discard the supernatant.

[0191] 11.7 Keep the PCR tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls. Let it stand for 30 seconds and discard the supernatant.

[0192] 11.8 Repeat the previous step and try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, centrifuge it immediately. After separation on the magnetic stand, use a small-scale pipette to remove the liquid at the bottom of the tube.

[0193] 11.9 Place the PCR tube on the magnetic rack, open the tube cap, and let it sit at room temperature until the surface of the magnetic beads is free of reflections and cracks.

[0194] 11.10 Remove the PCR tube from the magnetic stand, add 32 μL of TE Buffer, and pipette gently aspirate at least 10 times to mix thoroughly.

[0195] 11.11 Incubate at room temperature for 5 minutes;

[0196] 11.12 Briefly centrifuge and place the PCR tube on a magnetic rack. Let stand for 2-5 minutes until the liquid becomes clear. Transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0197] 11.13 Take 1 μL of the fragment screening product and use the Qubit dsDNA HS Kit to determine the concentration. Take 1 μL of the fragment screening product and use the appropriate Agilent DNA analysis kit to determine the fragment distribution.

[0198] QC (quality control) standard: concentration greater than 5ng / μL, and the main peak of fragment distribution is between 300 and 500bp.

[0199] 12. Denaturation (cDNA library and Oligo library)

[0200] 12.1 For the cDNA library and oligo library, transfer 300 ng each to a PCR tube for subsequent denaturation and circularization. Any amount less than 300 ng should be added (enough for quality control). After sampling, add TE Buffer to a total volume of 45 μL.

[0201] 12.2 Add 5 μL of scRNA Splint Oligo to the PCR tube from step 12.1, vortex to mix, and briefly centrifuge to collect the reaction mixture at the bottom of the tube;

[0202] 12.3 Place the PCR tubes described in step 12.2 on a PCR instrument and perform the reaction according to the conditions in Table 12;

[0203] Table 12:

[0204] 12.4 After the 95°C reaction is completed, immediately transfer the PCR tube to ice. After standing for 5 minutes, add the single-stranded circularization reaction system.

[0205] 13. Single-chain cyclization

[0206] 13.1 Prepare the single-strand cyclization reaction system on ice according to Table 13;

[0207] Table 13:

[0208] 13.2 Use a pipette to draw 10 μL of the prepared single-stranded circularization reaction mixture into the PCR tube prepared in step 12.4. Vortex to mix thoroughly, and briefly centrifuge to collect the reaction mixture at the bottom of the tube.

[0209] 13.3 Place the PCR tubes described in step 13.3 on a PCR instrument and perform the reaction according to the conditions in Table 14;

[0210] Table 14:

[0211] 13.4 After the reaction is completed, centrifuge the PCR tube briefly and place it on ice before proceeding to the next step.

[0212] 14. Enzymatic digestion

[0213] 14.1 Prepare the enzymatic digestion reaction system on ice according to the recipe in Table 15;

[0214] Table 15:

[0215] 14.2 Use a pipette to draw 4 μL of the prepared enzyme digestion reaction system into the PCR tube prepared in step 13.4. Vortex to mix thoroughly, and briefly centrifuge to collect the reaction system at the bottom of the tube.

[0216] 14.3 Place the PCR tubes described in step 14.2 on a PCR instrument and perform the reaction according to the conditions in Table 16;

[0217] Table 16:

[0218] 14.4 After the reaction is completed, add 3 μL Frag Stop Buffer, vortex to mix, and briefly centrifuge to collect the reaction mixture at the bottom of the tube.

[0219] 15. Purification of enzyme digestion products

[0220] Note: (1) Take out the DNA Clean Beads in advance and equilibrate them at room temperature for at least 30 minutes. Vortex mix thoroughly before use.

[0221] (2) Please read carefully Appendix B regarding magnetic beads and purification before operation.

[0222] 15.1 Transfer the digested product from 14.4 to a 1.5 mL centrifuge tube, add 160.8 μL of DNA Clean Beads, and mix thoroughly by gently pipetting at least 10 times. Make sure all the liquid and beads in the pipette tip are pumped into the centrifuge tube.

[0223] 15.2 Incubate at room temperature for 10 minutes;

[0224] 15.3 Briefly centrifuge, place the centrifuge tube on a magnetic rack, let it stand for 2-5 minutes until the liquid becomes clear, and discard the supernatant.

[0225] 15.4 Keep the centrifuge tube on the magnetic stand and add 500 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds and discard the supernatant.

[0226] 15.5 Repeat the previous step and try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, centrifuge the tube briefly. After separation on a magnetic stand, use a small-scale pipette to remove the liquid from the bottom of the tube.

[0227] 15.6 Keep the centrifuge tube on the magnetic rack, open the tube cover, and dry it at room temperature until the surface of the magnetic beads is free of reflections and cracks;

[0228] 15.7 Remove the centrifuge tube from the magnetic stand, add 32 μL of TE Buffer, and pipette gently up and down at least 10 times to mix thoroughly.

[0229] 15.8 Incubate at room temperature for 10 minutes;

[0230] 15.9 Briefly centrifuge, place the tube on a magnetic stand, and let it stand for 2-5 minutes until the liquid becomes clear. Transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube (Note: Be careful not to aspirate the magnetic beads during this step, as this will significantly affect the quality of the library sequencing).

[0231] 15.10 Take 1 μL of the enzyme digestion product and use ssDNA Assay Kit was used to detect the concentration.

[0232] QC standard: concentration greater than 0.5 ng / μL.

[0233] 16. Start sequencing on the machine.

[0234] Example 2:

[0235] In this example, the FS components in the cell resuspension buffer and the bead buffer in Example 1 were adjusted, and then droplet RT-PCR high-throughput single-cell transcriptome sequencing was performed. The remaining experimental conditions and steps were the same as in Example 1.

[0236] Adjusted 5X FS composition: 125 mM Tris-HCl, 250 mM KCl, 25 mM MgCl2.

[0237] Comparative Example 1:

[0238] In this example, the FS component in the cell resuspension buffer and the bead buffer in the prior art was used to perform droplet RT-PCR high-throughput single-cell transcriptome sequencing. The remaining experimental conditions and steps were the same as in Example 1.

[0239] The components of 5X FS in the prior art are: 250 mM Tris-HCl, 375 mM NaCl, 15 mM MgCl2.

[0240] Example 3: Comparative analysis of results

[0241] Example 1 and Comparative Example 1 show that FS components with different ion concentrations are used to mainly explore the effects of different ion concentrations on the RT-PCR system and single-cell transcriptome sequencing results.

[0242] Examples 1 and 2 show that by using different FS components, K + and Na + And the effect of enzyme concentration on RT-PCR system and single-cell transcriptome sequencing results.

[0243] Among them, after experimental verification, the results of the effects of ion concentration and RT enzyme concentration on the RT-PCR system are shown in Table 17;

[0244] Table 17:

[0245] Note: R1-3 and R1-4 are a set of repeated experiments; R2-3 and R2-4 are a set of repeated experiments; R3-3 and R3-4 are a set of repeated experiments; R4-3 and R4-4 are a set of repeated experiments;

[0246] R1: 5X FS and 2.5 μL 420RT enzyme (LS-EZ-E-00027P, BGI) as described in Example 1;

[0247] R2: 5X FS and 1 μL 420RT enzyme (LS-EZ-E-00027P, BGI) as described in Example 1;

[0248] R3: 5X FS and 2.5 μL 420RT enzyme (LS-EZ-E-00027P, BGI) as described in Comparative Example 1;

[0249] R4: 5X FS described in Comparative Example 1 and 1 μL 420RT enzyme (LS-EZ-E-00027P, BGI).

[0250] The results show that the enzyme concentration has little effect on the RT-PCR system when compared to R1 and R2, and R3 and R4. However, the salt concentration significantly affects the RT-PCR system when compared to R1 and R3, and R2 and R4. Not only is the purified cDNA concentration of R1 significantly higher than that of R3, but R2 is also higher than R4. Furthermore, the cDNA peak plot (Figure 11) shows that R1 produces better results than R3, and similarly, R2 performs better than R4.

[0251] Based on the above results, the effect of ion concentration on single-cell transcriptome sequencing was verified;

[0252] After experimental verification, different ion components (K + and Na + ) on single-cell transcriptome sequencing are shown in Table 18;

[0253] Table 18:

[0254] Note: 1-1 and 1-2 represent two repeated experiments of the sample; 2-1 and 2-2 represent two repeated experiments of the sample.

[0255] Figures 1 to 4 are the sequencing quality inspection report results. It can be seen that different ion components (K + and Na +) has little effect on the results of single-cell transcriptome sequencing, among which K + The number of UMI in the group was higher than that of Na + Figures 5 to 7 are the distribution diagrams of 2100 fragments of PCR amplification products obtained with different ion components. It can be seen that K + The amplified product fragments of the group were better distributed, with longer fragments obtained and the main peak at 800-1800bp.

[0256] In summary, based on the experimental verification results above, it can be concluded that the mapping rate, exon alignment rate, UMI, and gene number all achieve relatively good results, indicating that the above scheme has broad application prospects in single-cell transcriptome high-throughput sequencing.

[0257] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0258] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0259] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A droplet-based PCR amplification method, characterized in that, Comprising: Performing an oil droplet encapsulation treatment on single cells of a sample to be tested, so as to obtain single cell oil droplets of the sample to be tested; And Performing a PCR amplification treatment on the single cells of the sample to be tested within the oil droplets; Wherein, the single cells of the sample to be tested are provided in the form of a cell suspension, the cell suspension includes the single cells of the sample to be tested and a first buffer solution, the first buffer solution includes FS, and the FS includes Tris-HCl, NaCl and MgCl2.

2. The method according to claim 1, wherein The concentration of the Tris-HCl in the first buffer solution is 20-30 mM, preferably 25 mM; The concentration of the NaCl in the first buffer solution is 25-75 mM, preferably 50 mM; The concentration of the MgCl2 in the first buffer solution is 3-8 mM, preferably 5 mM.

3. The method according to claim 1, characterized in that, The first buffer solution further includes betaine, dNTP, dCTP, trihydroxypropylphosphine, nuclease inhibitor, S2 primer and KAPA high-fidelity DNA polymerase.

4. The method according to claim 3, characterized in that The final concentration of the betaine in the first buffer solution is 0.5-2 M, preferably 1 M.

5. The method according to claim 3, characterized in that The final concentration of the dNTP in the first buffer solution is 0.2-2 mM, preferably 1 mM.

6. The method according to claim 3, wherein The final concentration of the dCTP in the first buffer solution is 1-5 mM, preferably 5 mM.

7. The method according to claim 3, characterized in that, The final concentration of the KAPA high-fidelity DNA polymerase in the first buffer solution is 0.01-0.03 U / μL; preferably 0.02 U / μL.

8. The method according to claim 3, wherein The final concentration of the trihydroxypropylphosphine in the first buffer solution is 0.01-0.03 M, preferably 0.01 M.

9. The method according to claim 3, wherein The final concentration of the nuclease inhibitor in the first buffer solution is 1-3 U / μL, preferably 1 U / μL.

10. The method according to claim 3, characterized in that The final concentration of the S2 primer in the first buffer solution is 0.6-1.2 μM, preferably 0.9 μM.

11. The method according to claim 3, wherein The first buffer solution further includes: SSIV reverse transcriptase; the final concentration of the SSIV reverse transcriptase in the first buffer solution is 3-5 U / μL, preferably 4.5 U / μL.

12. The method according to claim 3, wherein The first buffer solution further includes: dithiothreitol; the final concentration of the dithiothreitol in the first buffer solution is 0.01-0.03 M, preferably 0.01 M.

13. The method according to claim 3, characterized in that, The first buffer solution further includes: Poly dT; the final concentration of the Poly dT in the first buffer solution is 1-4 μM, preferably 2.5 μM.

14. The method according to claim 1, wherein The oil droplet encapsulation treatment is performed in the following manner: Mixing the single cell suspension of the sample to be tested, the bead suspension and the droplet-forming oil.

15. The method according to claim 14, characterized in that The bead suspension includes beads and a second buffer solution, the second buffer solution includes FS, and the FS includes Tris-HCl, NaCl and MgCl2.

16. The method according to claim 15, wherein The concentration of the Tris-HCl in the first buffer solution is 20-30 mM, preferably 25 mM; The concentration of the NaCl in the first buffer solution is 40-60 mM, preferably 50 mM; The concentration of the MgCl2 in the first buffer solution is 3-8 mM, preferably 5 mM.

17. The method according to claim 15, wherein The second buffer solution further includes: Triton X-100, digitonin and ficoll.

18. The method according to claim 17, characterized in that, The concentration of Triton X-100 in the second buffer is 0.1-0.3% (w / v), preferably 0.1% (w / v).

19. The method according to claim 17, wherein The concentration of digitonin in the second buffer is 0.01-0.03% (w / v), preferably 0.025% (w / v).

20. The method according to claim 17, characterized in that, The final concentration of ficoll in the second buffer is 2-10% (w / v), preferably 4% (w / v).

21. A method for constructing a sequencing library, characterized in that, Comprising: Performing a PCR amplification reaction on a sample to be tested according to the method of any one of claims 1-20 to obtain an amplification product, and the amplification product constitutes the sequencing library.

22. The method according to claim 21, wherein Further comprising performing demulsification treatment and fragmentation treatment on the amplification product; and Connecting the fragmented product with a sequencing adapter.

23. The method according to claim 22, wherein The demulsification treatment is carried out through the following steps: Mixing the amplification product with a surfactant to obtain a demulsification treatment product.

24. The method according to claim 22, wherein The fragmentation treatment is carried out in the presence of a fragmentation enzyme.

25. A sequencing library, characterized in that, The sequencing library is obtained by the method of any one of claims 21-24.

26. A method for sequencing a target nucleic acid molecule, characterized in that, Comprising: Constructing a sequencing library for a target sequence according to the method of any one of claims 21-24; Sequencing the sequencing library to obtain a sequencing result; and Based on the sequencing result, determining the nucleic acid sequence of the target nucleic acid molecule.

27. A droplet PCR amplification kit, characterized in that, Comprising: The first buffer defined in the method of any one of claims 1-13; The second buffer defined in the method of claim 1 or any one of claims 15-20; and Droplet generation oil.

28. The kit according to claim 27, wherein Further comprising: Digestive enzyme.

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