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 preparation, and sequencing methods Technical Field

[0001] This 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 preparation, and sequencing method. Background Technology

[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 lysis of single cells, the amount of RNA extracted is relatively small.

[0003] Reverse transcription polymerase chain reaction (RT-PCR) is a routine detection technique that can be used to target RNA samples and amplify the abundance of specific RNA samples, making it highly valuable for the detection of viral RNA. In recent years, combining droplet high-throughput technology with RT-PCR technology for clinical testing can 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 are mainly focused on detecting specific gene information and cannot be applied to high-throughput single-cell transcriptomics technologies. Furthermore, some existing technologies suffer from inaccurate selectivity of semipermeable membranes and difficulties in purification.

[0005] Therefore, there is a need 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 discoveries and understanding of the following problems:

[0008] The inventors discovered that, on the one hand, current intradroplet RT-PCR technology has limitations. First, due to the small volume of the droplets, RT-PCR amplification within each droplet leads to incomplete amplification (only retaining nucleic acid material larger than 300 bp), resulting in incomplete transcript information. Second, this technology can only detect specific genes and cannot obtain comprehensive transcript information within the cell. 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, achieving high throughput is challenging due to the difficulty in controlling many of the tiny reactors within the microplate. Furthermore, this method is more expensive compared to droplet-based RT-PCR.

[0010] Therefore, the inventors developed a method based on 5' droplet technology to obtain cDNA through reverse transcription within a droplet, while simultaneously performing amplification within the droplet. The PCR product is then purified for library construction and sequencing. This one-step reverse transcription and amplification within the droplet not only increases throughput but also simplifies the operation, shortens the time, and reduces costs.

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

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

[0013] According to embodiments of this application, the concentration of Tris-HCl in the first buffer solution is 20–30 mM. In some examples of this application, the concentration of Tris-HCl in the first buffer solution 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 this application, when the concentration of Tris-HCl in the first buffer solution is 25 mM, the stability and specificity of the PCR reaction are better.

[0014] According to embodiments of this application, the concentration of NaCl in the first buffer solution is 25–75 mM. In some examples of this application, the concentration of NaCl in the first buffer solution may optionally be 25 mM, 30 mM, 35 mM, 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, 60 mM, 65 mM, 70 mM, or 75 mM. In some preferred examples of this application, when the concentration of NaCl in the first buffer solution is 50 mM, non-specific binding of primers can be avoided.

[0015] According to embodiments of this application, the concentration of MgCl2 in the first buffer solution is 3–8 mM. In some examples of this application, the concentration of MgCl2 in the first buffer solution is optionally 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. In some preferred examples of this application, a concentration of 5 mM of MgCl2 in the first buffer solution can increase the specificity and amplification efficiency of the PCR product.

[0016] According to embodiments of this application, the first buffer further comprises betaine, dNTPs, dCTP, trihydroxypropylphosphine, a nuclease inhibitor, S2 primers (cDNA amplification primers), and KAPA high-fidelity DNA polymerase. The inventors ensured the full execution of the reverse transcription reaction by adding dCTP to the first buffer.

[0017] According to embodiments of this application, the final concentration of betaine in the first buffer solution is 0.5–2 M. In some examples of this application, the final concentration of betaine in the first buffer solution may optionally be 0.5 M, 1 M, 1.5 M, or 2 M. In some preferred examples of this application, the final concentration of betaine in the first buffer solution is 1 M.

[0018] According to embodiments of this application, the final concentration of the dNTP in the first buffer solution is 0.2–2 mM. In some examples of this application, the final concentration of the dNTP in the first buffer solution may optionally be 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, or 2 mM. In some preferred examples of this application, the final concentration of the dNTP in the first buffer solution may optionally be 1 mM.

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

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

[0021] According to embodiments of this application, the final concentration of trihydroxypropylphosphine in the first buffer solution is 0.01–0.03 M. In some examples of this application, the final concentration of trihydroxypropylphosphine in the first buffer solution is optionally 0.01 M, 0.02 M, or 0.03 M. In some preferred examples of this application, the final concentration of trihydroxypropylphosphine in the first buffer solution is 0.01 M.

[0022] According to embodiments of this application, the final concentration of the nuclease inhibitor in the first buffer solution is 1–3 U / μL. In some examples of this application, the final concentration of the nuclease inhibitor in the first buffer solution is optionally 1 U / μL, 1 U / μL, or 3 U / μL. In some preferred examples of this application, the final concentration of the nuclease inhibitor in the first buffer solution is 1 U / μL.

[0023] According to embodiments of this application, the final concentration of the S2 primer in the first buffer solution is 0.6–1.2 μM. In some examples of this application, the final concentration of the S2 primer in the first buffer solution may optionally be 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 this application, the final concentration of the S2 primer in the first buffer solution is 0.9 μM.

[0024] According to embodiments of this application, the first buffer further comprises SSIV reverse transcriptase; the final concentration of the SSIV reverse transcriptase in the first buffer is 3–5 U / μL. In some examples of this application, the final concentration of the SSIV reverse transcriptase in the first buffer may 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 this application, the final concentration of the SSIV reverse transcriptase in the first buffer is 4.5 U / μL. According to embodiments of this application, adding SSIV reverse transcriptase to the first buffer allows for one-step RT-PCR reaction within a droplet, reducing operational steps and saving time and cost.

[0025] According to embodiments of this application, the first buffer further comprises dithiothreitol (DTT); the final concentration of dithiothreitol in the first buffer is 0.01–0.03 M. In some examples of this application, the final concentration of dithiothreitol in the first buffer may optionally be 0.01 M, 0.02 M, or 0.03 M. In some preferred examples of this application, the final concentration of dithiothreitol in the first buffer is 0.01 M. According to embodiments of this application, in RT-PCR, DTT is mainly used to reduce disulfide bonds within RNA molecules, converting them into monosulfide bonds, thereby making it easier for the RNA molecule to unravel and facilitating the action of reverse transcriptase. DTT can also maintain the activity of thermostable reverse transcriptase at high temperatures and prevent RNA template degradation or the formation of complex secondary structures, thereby increasing the efficiency and accuracy of cDNA synthesis.

[0026] According to embodiments of this application, the first buffer further comprises: Poly dT; the final concentration of Poly dT in the first buffer is 1–4 μM. In some examples of this application, the final concentration of Poly dT in the first buffer is optionally 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, or 4 μM. In some preferred examples of this application, the final concentration of Poly dT in the first buffer is 2.5 μM. According to embodiments of this application, in RT-PCR, Poly dT is mainly used to capture mRNA molecules, which are then used as templates for reverse transcription, thereby transcribed into cDNA.

[0027] According to an embodiment of this application, the oil droplet encapsulation process is carried out by mixing the single-cell suspension of the sample to be tested, the microbead suspension, and the droplet-forming oil.

[0028] For example, 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 this application, the microbead suspension includes microbeads and a second buffer solution, the second buffer solution including FS, the FS including Tris-HCl, NaCl and MgCl2.

[0030] According to embodiments of this application, the concentration of Tris-HCl in the first buffer solution is 20–30 mM. In some examples of this application, the concentration of Tris-HCl in the first buffer solution 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 this application, when the concentration of Tris-HCl in the first buffer solution is 25 mM, the stability and specificity of the PCR reaction are better.

[0031] According to embodiments of this application, the concentration of NaCl in the first buffer solution is 40–60 mM. In some examples of this application, the concentration of NaCl in the first buffer solution may optionally be 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 this application, when the concentration of NaCl in the first buffer solution is 50 mM, non-specific binding of primers can be avoided.

[0032] According to embodiments of this application, the concentration of MgCl2 in the first buffer solution is 3–8 mM. In some examples of this application, the concentration of MgCl2 in the first buffer solution is optionally 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. In some preferred examples of this application, a concentration of 5 mM of MgCl2 in the first buffer solution can increase the specificity and amplification efficiency of the PCR product.

[0033] According to embodiments of this application, the second buffer solution further comprises: Triton X-100, digitalis saponins, and ficoll.

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

[0035] According to embodiments of this application, the concentration of digitalis saponin in the second buffer solution is 0.001–0.003% (w / v). In some examples of this application, the concentration of digitalis saponin in the second buffer solution 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 this application, the concentration of digitalis saponin in the second buffer solution is 0.025% (w / v).

[0036] According to embodiments of this application, the final concentration of the Ficoll aqueous solution in the second buffer solution is 2-10% (w / v). In some examples of this application, the final concentration of the Ficoll aqueous solution in the second buffer solution may optionally be 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 this application, the final concentration of the Ficoll aqueous solution in the second buffer solution is 4% (w / v). According to embodiments of this application, adding Ficoll can prevent cells and magnetic beads from sinking during droplet running, thus avoiding blockage of microfluidic channels.

[0037] In a second aspect, this application proposes a method for constructing a sequencing library. According to an embodiment of this application, the method includes: performing a PCR amplification reaction on a sample to be tested based on the method described in the first aspect of the invention to obtain amplification products, wherein the amplification products constitute the sequencing library. According to an embodiment of this application, using the above-described intradroplet RT-PCR method for sequencing library construction can simplify the operation steps, shorten the library construction time, and save costs.

[0038] According to embodiments of this application, the method further includes demulsifying and fragmenting the amplification product; and ligating the fragmented product to a sequencing adapter. According to embodiments of this application, demulsification is performed to release the amplified PCR product. The PCR product is then fragmented and ligated to a sequencing adapter to form a sequencing library.

[0039] According to embodiments of this application, the demulsification process is performed by mixing the amplification product with a surfactant (e.g., PFO or sodium octyl sulfonate) to obtain a demulsified product. In one example of this application, the amplification product is mixed with PFO (Sigma-Aldrich, catalog number: 370533-25G), and the mixture is centrifuged at 1000g for 2 minutes to obtain a demulsified product. In some examples of this 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 no specific restrictions are imposed in this application.

[0041] According to embodiments of this application, the fragmentation process is performed in the presence of a fragmentation enzyme.

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

[0043] In a fourth aspect, this application proposes a method for sequencing a target nucleic acid molecule. According to an embodiment of this application, the method includes: constructing a sequencing library for a target sequence according to the method described in the third aspect of the invention; 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 this application, constructing a library suitable for high-throughput sequencing based on the method described in this application reduces manual operation steps, shortens time, and lowers costs.

[0044] In a fifth aspect of this application, a droplet PCR amplification kit is provided. According to an embodiment of this application, the kit includes: a first buffer and a second buffer as defined in the method of the first aspect of this invention; and droplet-generating oil. According to an embodiment of this application, the kit enables rapid PCR amplification, is easy to operate, and has a low cost. Both reverse transcription and PCR amplification reactions can be completed within the droplet, making it more efficient than previous amplification methods.

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

[0046] According to embodiments of this application, the kit further includes a digestive enzyme and at least one of the ingredients specified in the instructions.

[0047] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[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 taken in conjunction with the following drawings, in which:

[0049] Figure 1 is a schematic diagram of the sequencing quality control report results of experimental group 1-1 according to an embodiment of the present invention;

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

[0051] Figure 3 is a schematic diagram of the sequencing quality control report results of experimental group 2-1 according to an embodiment of the present invention;

[0052] Figure 4 is a schematic diagram of the sequencing quality control report results of experimental group 2-2 according to an embodiment of the present invention;

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

[0054] Figure 6 is a schematic diagram of the distribution of the PCR amplification product 2100 fragment in the 5xFS buffer of experimental group 2-1 according to the embodiment of the present invention when potassium ions are present; wherein, the 5XFS components are: 125mM Tris-HCl, 250mM NaCl, 25mM MgCl2;

[0055] Figure 7 is a schematic diagram of the distribution of PCR amplification product 2100 fragments under different salt ion concentrations according to an embodiment of the present invention; RP1-4 represent the distribution of PCR amplification product 2100 fragments in experimental groups 1-4; RP2-4 represent the distribution of PCR amplification product 2100 fragments in experimental groups 2-4; RP3-4 represent the distribution of PCR amplification product 2100 fragments in experimental groups 3-4; RP4-4 represent the distribution of PCR amplification product 2100 fragments in experimental groups 4-4.

[0056] Figure 8 is a schematic diagram of droplet generation according to an embodiment of the present invention. Detailed Implementation

[0057] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0058] Prior art related to this application:

[0059] Currently, the application of droplet RT-PCR to detect specific genes in single cells involves using a specific material to form a droplet resembling a semi-permeable membrane. This semi-permeable membrane is selectively permeable, allowing cells to be encapsulated within the droplet, lysed, and releasing specific nucleic acid substances. The solution is then replaced, the lysis buffer is removed, and 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). Its advantage lies in removing the inhibitory effect of the lysis buffer on the enzyme, thus not affecting subsequent reactions. However, it also has significant drawbacks. On the one hand, semipermeable membranes can only retain nucleic acid materials larger than 300 bp, while primers used for high-throughput labeling are all smaller than 300 bp, thus allowing droplets to pass freely and preventing labeling. On the other hand, the droplets forming the semipermeable membrane are not easily destroyed, making it impossible to obtain information about the entire cell transcript through purification. Its current main application is in detecting specific gene information, and it cannot be applied to high-throughput single-cell transcriptomics technology.

[0060] Currently, RT-PCR applications for single-cell transcriptome sequencing are primarily based on single-tube technology. Because single-tube sequencing allows for high-temperature lysis—the cells in the well plate are lysed by heating before the RT-PCR reaction reagents are added—the issue of high incompatibility between lysis and RT-PCR is resolved. Examples include Smart-seq3 and Flash-seq, both of which are plate-based (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-seq 3. 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.). While these technologies simplify the single-cell library preparation process to some extent, FLASH-seq, for example, shortens the entire library preparation process to 4.5 hours. Although there has been a significant improvement in time cost, orifice plate-based technologies have difficulty achieving high throughput.

[0061] Amplification methods based on droplet PCR

[0062] Therefore, based on the deficiencies of the prior art, this application develops an amplification method based on droplet PCR. The method includes: encapsulating a single cell sample to be tested in oil droplets to obtain single-cell oil droplets; and performing PCR amplification on the single-cell oil droplets; wherein the single cell sample to be tested is provided in the form of a cell suspension, the cell suspension comprising the single cell sample to be tested and a first buffer, the first buffer comprising FS, and the FS comprising Tris-HCl, NaCl, and MgCl2.

[0063] According to the embodiments of this application, reverse transcriptase, DTT (dithiothreitol), and Poly dT can be added to the first buffer solution to achieve a one-step intradroplet reaction of reverse transcription and PCR amplification. This not only achieves high throughput but also greatly simplifies experimental steps and shortens reaction time, while also saving experimental costs. Compared with previous droplet RT-PCR (ddRT-PCR) and plate-based single-cell RT-PCR transcriptome technologies used in clinical testing, the method described in this application can obtain complete transcript information from a single cell and rapidly achieve high throughput, providing a research foundation for future automated library construction applications. Therefore, this application has significant advantages and application value for the future development of single-cell high-throughput transcriptome sequencing technology.

[0064] Specifically, for ease of understanding, the above methods and technical solutions will be explained and described in detail below.

[0065] 1. Preparation of single-cell suspension: Digest the tissue (grind or digest with enzymes), wash it in PBS (containing 0.04% BSA), filter it through a cell sieve, and resuspend it in cell buffer 1 (formulation shown in Table 1) to obtain a single-cell suspension.

[0066] 2. Preparation of microbeads: First, resuspend the index carrier (short DNA sequence used to label the sample) in the prepared bead buffer (preparation method is shown in Table 3), mix thoroughly, then resuspend the cell beads and mix thoroughly.

[0067] It should be noted that the microbeads were pre-linked with barcode1, barcode2, UMI and TSO primer (rGrGrG) sequences during preparation.

[0068] 3. Droplet generation: After mixing the droplet generation oil, cell suspension (prepared in step 1) and cell microbeads (prepared in step 2), the mixture is transferred to a PCR tube (an eight-tube tube can also be selected), covered with mineral oil, and then subjected to PCR reaction.

[0069] It should be noted that during droplet running, after cell lysis, mRNA is captured by polydT, and a reducing agent (such as DTT) releases the oligos (DNA primers) on the surface of the magnetic beads into the droplet. Under the action of RT enzymes (reverse transcriptases), reverse transcription occurs using the mRNA as a template to synthesize the first cDNA strand. A 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 template conversion. Then, the strands are melted at 95°C, and a complementary second cDNA strand is synthesized using the first cDNA strand as a template. PCR is then performed using PCR primers at both ends to amplify the target fragment.

[0070] Sequencing library construction methods

[0071] In a second aspect, this application proposes a method for constructing a sequencing library. According to an embodiment of this application, a PCR amplification reaction is performed on the test sample based on the above-described droplet PCR amplification method to obtain amplification products, which constitute the sequencing library.

[0072] It should be noted that after the above PCR amplification, the amplified cell droplets are demulsified to release the amplified cDNA. The amplified cDNA is then purified (removing impurities such as PCR primers, salts, and buffers), and the purified product is fragmented (usually using fragmentation enzymes, but other methods can also be used for fragmentation). After adapter ligation and single-strand circularization, the sequencing library is constructed.

[0073] sequencing libraries

[0074] In a third aspect, this application provides a sequencing library. According to embodiments of this application, the sequencing library is constructed using the method described above.

[0075] Methods for sequencing target nucleic acid molecules

[0076] In a fourth aspect, this application proposes a method for sequencing a target nucleic acid molecule. According to an embodiment of this application, the method involves high-throughput sequencing of the constructed sequencing library 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 this application, a droplet PCR amplification kit is provided. According to embodiments of this application, the kit comprises: a digestive enzyme; a first buffer and a second buffer (as described in the first aspect); and droplet-generating oil.

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

[0081] The present invention will be further explained and described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

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

[0083] 1. Preparation of single-cell suspension

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

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

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

[0087] Table 1: Preparation of Cell Resuspension Buffer

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

[0089] The manufacturers and product numbers of the reagents used to prepare the above-mentioned 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 them on a magnetic rack and let them stand for 2 min, then discard the supernatant;

[0093] 2.2 Remove the PCR tube from the magnetic rack, resuspend the index carrier in the prepared bead buffer (preparation method is shown in Table 3), mix thoroughly, then resuspend the cell beads and mix thoroughly.

[0094] Table 3:

[0095] 3. Droplet formation (Figure 8)

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

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

[0098] 3.3 Place the 30ml syringe on the holder and adjust the plunger to the initial position of 17mL. Connect the syringe to end B of the connecting tube on the collection tube cap (the connecting tube that does not contact the bottom of the collection tube) using a flat-tipped needle;

[0099] 3.4 Add 100 μL of droplet-generating oil to the collection tube, tighten the collection cap, and place the collection tube vertically on the mounting bracket.

[0100] 3.5 Gently pipette to mix the cells, add 100 μL of cell suspension to the cells well of the chip, and make sure the pipette tip is in contact with the bottom of the well;

[0101] 3.6 Use a pipette to gently mix the magnetic beads, add 100 μL of magnetic beads into the well of the chip beads, and ensure that the pipette tip contacts the bottom of the well;

[0102] 3.7 Immediately add 350 μl of liquid to generate oil in the chip's Oil via;

[0103] 3.8 Quickly pull the syringe plunger to the slot position and lock the plunger in the slot;

[0104] 3.9 After the droplet is 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 let the droplet in the tube flow into the collection tube, and cut off the connecting tube on the collection tube.

[0105] 3.10 Transfer the droplet to an eight-tube strip, ensuring the droplet surface is no more than 100 μL. Then, cover the droplet surface with 50-100 μL of mineral oil. After capping the eight-tube strip, perform RT-PCR according to the following procedure (Table 4).

[0106] Table 4:

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

[0108] 4. Delaying milk production

[0109] 4.1 After RT (reverse transcription) is completed, transfer the droplet to a new 1.5 mL centrifuge tube with low adsorption. Slowly aspirate from the bottom, without aspirating the top layer of mineral oil. Insert the pipette tip to the bottom and slowly aspirate the oil generated by the droplet and discard it, leaving the middle emulsion layer.

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

[0111] 4.3 Slowly aspirate the aqueous phase 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 Remove the Novizan DNA purification beads (or Ampure XP beads) in advance and equilibrate at room temperature for at least 30 minutes. Shake well before use.

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

[0115] 5.3 Use a pipette to aspirate 0.6X Novizan DNA purified beads into the PCR product and gently pipette at least 10 times until completely mixed. On the last time, make sure that all liquid and magnetic beads in the pipette tip are pipetted into the PCR tube.

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

[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 is clear;

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

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

[0120] 5.8 Repeat the previous step to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, the centrifuge tube can be centrifuged briefly. After separation on a magnetic rack, use a small-capacity pipette to remove the liquid from the bottom of the tube.

[0121] 5.9 Keep the centrifuge tubes fixed on the magnetic rack, open the tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked;

[0122] 5.10 Remove the centrifuge tube from the magnetic rack, add 48 μl of Nuclease-Free Water to elute the cDNA, and gently pipette at least 10 times until completely mixed;

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

[0124] 5.12 Instant centrifugation: Place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Transfer 46 μl of the supernatant to a new eight-tube set.

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

[0126] 6. Purification of Oligo products

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

[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 is clear. Discard the supernatant.

[0129] 6.3 Keep the centrifuge tube on the magnetic rack, add 1 ml of freshly prepared 80% ethanol, rinse the magnetic beads and tube walls, let stand for 30 seconds, carefully aspirate and discard the supernatant;

[0130] 6.4 Repeat the previous step to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, the centrifuge tube can be centrifuged briefly. After separation on a magnetic rack, use a small-capacity pipette to remove the liquid from the bottom of the tube.

[0131] 6.5 Keep the centrifuge tubes fixed on the magnetic rack, open the tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked;

[0132] 6.6 Remove the centrifuge tube from the magnetic rack, add 23 μl of Nuclease-Free Water to elute the cDNA, and gently pipette at least 10 times until completely mixed;

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

[0134] 6.8 Perform a short-term centrifugation. Place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Transfer 21 μl of the supernatant to a new PCR tube to prepare for oligo library amplification.

[0135] 7. Oligo secondary amplification

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

[0137] Table 5:

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

[0139] Table 6: PCR Procedure

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

[0141] 7.1 Prepare the reaction interruption 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 into a new 0.2 mL PCR tube, add NF Water to bring the volume to 45 μL, and place the PCR tube on an ice box.

[0144] 7.3 Use a pipette to add 15 μL of the prepared interrupted reaction system to the PCR tube from step 7.2, vortex 3 times for 3 seconds each time, and briefly centrifuge to collect the reaction system to the bottom of the tube;

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

[0146] Table 8:

[0147] After the reaction is complete, briefly centrifuge 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 connectors for the reaction system on ice according to Table 9;

[0150] Table 9:

[0151] 8.2 Slowly pipette 40 μL of the prepared adapter connection reaction system into the PCR tube from step 7.4, vortex to mix, and collect the reaction system at the bottom of the tube after a short centrifugation.

[0152] 8.3 Place the PCR tube described in step 8.2 on the PCR instrument and carry out the reaction according to the conditions in Table 10;

[0153] Table 10:

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

[0155] 8.4 Remove the DNA Clean Beads in advance and allow them to equilibrate at room temperature for at least 30 minutes. Vortex them before use.

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

[0157] 9.1 Pipette 100 μL of DNA Clean Beads into the adapter ligation product from step 8.3 and gently pipette at least 10 times until completely mixed. On the last time, make sure all liquid and magnetic beads in the pipette tip are pipetted 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 is clear. Discard the supernatant.

[0160] 9.4 Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, rinse the magnetic beads and tube walls, let stand for 30 seconds, and discard the supernatant.

[0161] 9.5 Repeat the previous step, try to aspirate as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, centrifuge briefly. After separation on a magnetic rack, use a small-capacity pipette to aspirate the liquid from the bottom of the tube.

[0162] 9.6 Keep the PCR tube on the magnetic rack, open the tube cap, and let it dry at room temperature until the surface of the magnetic beads is no longer reflective and no longer cracked;

[0163] 9.7 Remove the PCR tube from the magnetic rack, add 102 μL of NF Water, and gently pipette at least 10 times until completely mixed;

[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 is clear. Transfer 100 μL of supernatant to a new 0.2 mL PCR tube.

[0166] 9.10 Pipette 55 μL of DNA Clean Beads into the PCR tube from step 9.9 and gently pipette at least 10 times until completely mixed. On the last time, make sure all liquid and magnetic 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 stand for 2–5 minutes until the liquid is clear. Carefully aspirate the supernatant with a pipette and transfer it to a new PCR tube.

[0169] 9.13 Add 15 μL of DNA Clean Beads to the supernatant of the PCR tube from the previous step, and gently pipette at least 10 times until completely mixed;

[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, add 200 μL of freshly prepared 80% ethanol, rinse the magnetic beads and tube walls, let stand for 30 seconds, and discard the supernatant.

[0173] 9.17 Repeat the previous step, try to aspirate as much liquid as possible from the tube. If a small amount remains on the tube wall, centrifuge briefly. After separation on a magnetic rack, use a small-capacity pipette to aspirate the liquid from the bottom of the tube.

[0174] 9.18 Keep the PCR tube on the magnetic rack, open the tube cap, and place at room temperature until the surface of the magnetic beads is no longer reflective and no longer cracked.

[0175] 9.19 Remove the PCR tube from the magnetic rack, add 48 μL of NF H2O, and gently pipette at least 10 times until completely mixed;

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

[0177] 9.21 Briefly centrifuge, place the PCR tube on a magnetic rack, and let it stand for 2-5 minutes until the liquid is clear. Transfer 46 μL of 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 from step 9.21;

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

[0181] 10.3 Place the PCR tube described in step 9.5.2 on the PCR instrument and carry out 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 allow them to equilibrate at room temperature for at least 30 minutes. Vortex them before use.

[0185] 11.1 Add 55 μL of DNA Clean Beads to the PCR product and gently pipette at least 10 times until completely mixed. On the last time, make sure that all liquid and magnetic beads in the pipette tip are added to 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 is clear. Carefully aspirate the supernatant with a pipette and transfer it to a new PCR tube.

[0188] 11.4 Add 15 μL of DNA Clean Beads to the supernatant of the PCR tube from the previous step, and gently pipette at least 10 times until completely mixed;

[0189] 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, add 200 μL of freshly prepared 80% ethanol, rinse the magnetic beads and tube walls, let stand for 30 seconds, and discard the supernatant.

[0192] 11.8 Repeat the previous step, try to aspirate as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, centrifuge briefly. After separation on a magnetic rack, use a small-capacity pipette to aspirate the liquid from the bottom of the tube.

[0193] 11.9 Keep the PCR tube on the magnetic rack, open the tube cap, and place at room temperature until the surface of the magnetic beads is no longer reflective and no longer cracked;

[0194] 11.10 Remove the PCR tube from the magnetic rack, add 32 μL of TE Buffer, and gently pipette at least 10 times until completely mixed;

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

[0196] 11.12 Briefly centrifuge, place the PCR tube on a magnetic rack, let stand for 2-5 minutes until the liquid is clear, and transfer 30 μL of supernatant to a new 1.5 mL centrifuge tube;

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

[0198] QC (Quality Control) standard: Concentration greater than 5 ng / μL, fragment distribution main peak between 300 and 500 bp.

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

[0200] 12.1 For both cDNA and Oligo libraries, take 300 ng each into a PCR tube for subsequent denaturation and circularization. If less than 300 ng is required, add all remaining amount (reserve enough for quality control). After taking the samples, add TE buffer to bring the total volume to 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 tube 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 reaction at 95℃ is complete, immediately transfer the PCR tube to ice and let it stand for 5 minutes before adding it to the single-stranded cyclization reaction system.

[0205] 13. Single-chain cyclization

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

[0207] Table 13:

[0208] 13.2 Use a pipette to add 10 μL of the prepared single-chain cyclization reaction system to the PCR tube in step 12.4, vortex to mix, and briefly centrifuge to collect the reaction system to the bottom of the tube;

[0209] 13.3 Place the PCR tube 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 complete, briefly centrifuge the PCR tube and place it on ice before proceeding to the next step of the reaction.

[0212] 14. Enzymatic digestion

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

[0214] Table 15:

[0215] 14.2 Use a pipette to add 4 μL of the prepared enzyme digestion reaction system to the PCR tube in step 13.4, vortex to mix, and briefly centrifuge to collect the reaction system to the bottom of the tube;

[0216] 14.3 Place the PCR tube 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 complete, add 3 μL of Frag Stop Buffer, vortex to mix, and briefly centrifuge to collect the reaction system to the bottom of the tube.

[0219] 15. Purification of enzyme digestion products

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

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

[0222] 15.1 Transfer the enzyme digestion product from step 14.4 to a 1.5 mL centrifuge tube, add 160.8 μL of DNA Clean Beads, and gently pipette at least 10 times until completely mixed. On the last time, make sure that all liquid in the pipette tip and the magnetic beads are transferred into the centrifuge tube.

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

[0224] 15.3 Brief centrifugation: Place the centrifuge tube on a magnetic rack and let it stand for 2–5 minutes until the liquid is clear. Discard the supernatant.

[0225] 15.4 Keep the centrifuge tubes on the magnetic rack, add 500 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, and then discard the supernatant.

[0226] 15.5 Repeat the previous step, try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, you can briefly centrifuge the tube, separate it on a magnetic rack, and then use a small-capacity pipette to remove the liquid from the bottom of the tube.

[0227] 15.6 Keep the centrifuge tubes on the magnetic rack, open the tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked;

[0228] 15.7 Remove the centrifuge tube from the magnetic rack, add 32 μL of TE Buffer, and gently pipette at least 10 times until completely mixed;

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

[0230] 15.9 Brief centrifugation: Place the centrifuge tube on a magnetic rack and let it stand for 2–5 minutes until the liquid is clear. Transfer 30 μL of supernatant to a new 1.5 mL centrifuge tube (Note: Be careful not to pick up the magnetic beads in this step, as this will greatly affect the quality of library sequencing).

[0231] 15.10 Take 1 μL of the enzyme digestion product for use. The concentration was detected using the ssDNA Assay Kit.

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

[0233] 16. Sequencing.

[0234] Example 2:

[0235] In this embodiment, the FS component in the cell resuspension buffer and microbead buffer in Example 1 was adjusted, and 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] The adjusted 5X FS composition is: 125mM Tris-HCl, 250mM KCl, 25mM MgCl2.

[0237] Comparative Example 1:

[0238] In this embodiment, the FS component in cell resuspension buffer and microbead buffer used 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 existing 5X FS composition consists of: 250mM Tris-HCl, 375mM NaCl, and 15mM MgCl2.

[0240] Example 3: Result Comparison and Analysis

[0241] Example 1 and Comparative Example 1 illustrate the effects of different ion concentrations of FS components on the RT-PCR system and single-cell transcriptome sequencing results.

[0242] Examples 1 and 2 illustrate the investigation of K by using different FS components. + and Na + The effects of enzyme concentration on RT-PCR system and single-cell transcriptome sequencing results.

[0243] The effects of ion concentration and RT enzyme concentration on the RT-PCR system, as verified by experiments, 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 and 1 μL 420RT enzyme (LS-EZ-E-00027P, BGI) as described in Comparative Example 1.

[0250] The results showed that the comparison between R1 and R2, and between R3 and R4, indicated that the difference in enzyme concentration was not significant; however, the comparison between R1 and R3, and between R2 and R4, indicated that salt ion concentration had a significant impact on the RT-PCR system. Comparing R1 and R3, not only was the concentration of purified cDNA significantly higher in R1 than in R3, but R2 was also higher than R4. Furthermore, the cDNA peak diagram (Figure 11) also showed that R1 performed better than R3; similarly, R2 performed better than R4.

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

[0252] Experiments have verified that different ionic components (K) + and Na + The comparison results of the effects of single-cell transcriptome sequencing are shown in Table 18.

[0253] Table 18:

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

[0255] Figures 1-4 show the sequencing quality control report results, which indicate that different ionic components (K... + and Na +The impact on single-cell transcriptome sequencing results is relatively small, among which, K + The number of UMIs in the group is higher than that in Na. + Group. Figures 5-7 show the distribution of PCR amplification products 2100 fragments obtained with different ionic components. It can be seen that K + The amplification products of the group showed better fragment distribution, with longer fragments and a main peak in the range of 800-1800bp.

[0256] In summary, based on the experimental results above, it can be concluded that the mapping rate, exon alignment rate, UMI, and gene number can all achieve better 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 technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0258] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0259] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A droplet-based PCR amplification method, characterized by, include: The single cells of the sample to be tested are coated with oil droplets to obtain single cell oil droplets of the sample to be tested. as well as The single-cell sample within the oil droplet was subjected to PCR amplification. The single cell sample to be tested is provided in the form of a cell suspension, which includes the single cell sample to be tested and a first buffer solution. The first buffer solution includes FS, which includes Tris-HCl, NaCl and MgCl2.

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

3. The method of claim 1, wherein, The first buffer further comprises betaine, dNTP, dCTP, trihydroxypropylphosphine, nuclease inhibitor, S2 primer, and KAPA high-fidelity DNA polymerase.

4. The method of claim 3, wherein, The final concentration of betaine in the first buffer solution is 0.5–2 M, preferably 1 M.

5. The method of claim 3, wherein, The final concentration of the dNTP in the first buffer solution is 0.2–2 mM, preferably 1 mM.

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

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

8. The method of 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 of 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 of claim 3, wherein, 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 of claim 3, wherein, The first buffer further comprises SSIV reverse transcriptase; the final concentration of the SSIV reverse transcriptase in the first buffer is 3-5 U / μL, preferably 4.5 U / μL.

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

13. The method of claim 3, wherein, The first buffer solution further comprises: Poly dT; the final concentration of Poly dT in the first buffer solution is 1 to 4 μM, preferably 2.5 μM.

14. The method of claim 1, wherein, The oil droplet encapsulation process is performed in the following manner: The sample to be tested, consisting of single-cell suspension, microbead suspension, and droplet-forming oil, was mixed and processed.

15. The method of claim 14, wherein, The microbead suspension includes microbeads and a second buffer solution, the second buffer solution including FS, the FS including Tris-HCl, NaCl and MgCl2.

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

17. The method of claim 15, wherein, The second buffer solution further comprises: Triton X-100, digitalis saponins, and ficoll.

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

19. The method of claim 17, wherein, The digitalis saponins are present in the second buffer solution at 0.01–0.03% (w / v), preferably 0.025% (w / v).

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

21. A method of sequencing library construction, comprising: include: The method according to any one of claims 1-20 is used to perform a PCR amplification reaction on the test sample in order to obtain amplification products, wherein the amplification products constitute the sequencing library.

22. The method of claim 21, wherein, Further, this includes demulsification and fragmentation of the amplified product; and The fragmented products are then ligated to sequencing adapters.

23. The method of claim 22, wherein, The demulsification process is carried out through the following steps: The amplified product was mixed with a surfactant to obtain a demulsified product.

24. The method of claim 22, wherein, The fragmentation process is performed in the presence of a fragmentation enzyme.

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

26. A method of sequencing a target nucleic acid molecule, comprising: include: For the target sequence, a sequencing library is constructed according to any one of claims 21-24; Sequencing is performed on the aforementioned sequencing library to obtain sequencing results; and Based on the sequencing results, the nucleic acid sequence of the target nucleic acid molecule is determined.

27. A droplet PCR amplification kit, characterized by, include: The first buffer solution as defined in the method of any one of claims 1-13; The second buffer solution defined in the method of any one of claims 1 or 15-20; and The droplets generate oil.

28. The kit of claim 27, wherein Further includes: Digestive enzymes.

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