A novel high-throughput single-cell multi-omics analysis method

Abstract

This invention provides a novel high-throughput single-cell multi-omics analysis method, comprising the following steps: fixation, lysis, nuclear permeation, and enzyme digestion of a population of cells; adding an A-tail and adjacent ligation to the DNA ends; and performing a transposition reaction. The pretreated cells are loaded onto a microfluidic platform, where single or multiple cells are co-encapsulated in droplets with beads containing capture adapters and a first molecular tag. The DNA and RNA of the same cell are simultaneously labeled using the capture adapters. The cells are released from the droplets and dispensed into multi-well plates. After decrosslinking, lysis, and DNA purification, primers with a second molecular tag are added to simultaneously amplify gDNA and cDNA, forming a unique composite tag. Library construction and sequencing are then performed. This invention improves cell throughput and data quality in a single experiment through a combined indexing approach, addressing the problems of low throughput, high cost, and significant ATAC signal background noise in existing single-cell three-omics technologies.

Description

Technical Field

[0001] This invention relates to cellular three-dimensional omics analysis technology, and more particularly to a high-throughput single-cell multi-omics analysis method. Background Technology

[0002] Single-cell three-dimensional genome sequencing methods can be roughly divided into three categories: (1) Physical separation method, such as the classic single-cell Hi-C technology, which separates cell nuclei one by one through micromanipulation or flow cytometry, and then performs high-throughput chromosome conformation capture (Hi-C) and other steps. This type of method is relatively straightforward in experimental operation, but a single 96-well plate operation can only process dozens of cells, and the throughput is extremely low. (2) High-throughput platforms based on microfluidics / droplets, such as Chromium from 10x Genomics, can co-encapsulate single cells and barcode beads in droplets to achieve high-throughput capture of thousands to tens of thousands of cells in each experiment. Existing studies have used the modified 10x platform to carry out high-throughput single-cell Hi-C experiments (such as Droplet Hi-C, dscHi-C), which can obtain three-dimensional genome maps of tens of thousands of cells at a time. (3) Split-and-pool barcode method: By performing multiple rounds of splitting, labeling, and pooling operations on cells, the barcode combinations from different wells in each round are assigned to individual cells, achieving large-scale single-cell labeling without physical sorting. Ramani et al. first combined this strategy with Hi-C in 2017 to develop sci-Hi-C, achieving single-cell throughput of tens of thousands in a single experiment. However, the multi-round operation is complex, the barcode combination design requirements are high, and the generated interaction data is relatively sparse.

[0003] In the prior art, Chinese invention patent application CN 119351531A9 (ChAIR) discloses a method for simultaneously capturing single-cell chromatin accessibility (ATAC), chromatin interaction (PET), and gene expression (RNA-seq) information using a microfluidic droplet platform combined with Tn5 transposase. The technical solution disclosed in this patent document has studied the joint dynamic changes of single-cell three-dimensional epigenome and transcriptome in cell lines and mouse brain tissue, demonstrating its ability to resolve cell type-specific gene expression regulation in complex tissues. However, the throughput of a single experiment in this disclosure is only in the thousands to tens of thousands, and the quality of ATAC data and the ability to capture rare cell types remain limited.

[0004] Therefore, existing single-cell three-omics technologies have bottlenecks in terms of throughput and data quality, and urgently need improvement. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel high-throughput single-cell multi-omics analysis method that addresses the problems existing in the background technology. By using a combined indexing method, the cell throughput and data quality of a single experiment are improved, and the problems of low throughput, high cost and large background noise of ATAC signal in the existing single-cell tri-omics technology are solved.

[0006] Therefore, the present invention adopts the following technical solution:

[0007] First, this invention provides a novel high-throughput single-cell multi-omics analysis method, comprising the following steps:

[0008] (1) Sample pretreatment: After double cross-linking fixation, lysis, nuclear permeation, and enzyme digestion of the population cell samples, A tails and adjacent ligations were added to the DNA ends, and transposition reaction was performed;

[0009] (2) First round of molecular tag introduction: The pretreated cells are loaded into a microfluidic platform, and one or more cells are co-encapsulated in droplets with glue beads with capture adapters and first molecular tags. The DNA and RNA of the same cell are simultaneously labeled through the capture adapters.

[0010] (3) Second round of molecular tag introduction: The cells in the droplets are released and dispensed into multi-well plates. After decrosslinking, lysis and DNA purification, primers with a second molecular tag are added to simultaneously amplify gDNA and cDNA to form a unique composite tag.

[0011] (4) Library construction and sequencing: gDNA and cDNA were purified separately, and sequencing libraries were constructed for next-generation high-throughput sequencing.

[0012] Furthermore, in step (2), the number of cells loaded on the microfluidic platform is at least 120,000, and the present invention can increase the cell processing capacity from the 10k level in the prior art to the 100k level.

[0013] Furthermore, in step (3), the decrosslinking reaction is carried out using a pre-prepared decrosslinking mixed buffer containing proteinase K, and the mixture is shaken and incubated to decrosslink the protein-DNA.

[0014] Further, the synchronous amplification reaction system of gDNA and cDNA in step (3) includes: buffer, dNTP mixture, polymerase and primer set, wherein the primer set includes Partial TSO primer, Primer_P5 primer, ADP-i5-TS-N primer and N-I7-indexN primer, N=1~96, the latter two are pore-specific primers, which are configured to introduce the second molecular tag.

[0015] Furthermore, the synchronous amplification reaction conditions for gDNA and cDNA in step (3) are: 72℃ for 5 min; 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 7 cycles; 72℃ for 3 min; and 4℃ for maintenance.

[0016] Furthermore, the PCR amplification system for preparing the gDNA library in step (4) includes: the co-amplification product in step (3), the high-fidelity PCR premix, the Primer_P5 primer, and the Primer_P7 primer.

[0017] Further, the PCR amplification conditions for preparing the gDNA library in step (4) are: 98℃ for 3 min; [98℃ for 20 s, 63℃ for 20 s, 72℃ for 30 s] cycles 7 times; 72℃ for 3 min; 4℃ for maintenance.

[0018] Further, the PCR amplification system for preparing cDNA in step (4) includes: the co-amplification product from step (3), dNTP mixture, polymerase, Partial TSO primer and P5-ADP primer; the amplification conditions are: 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 7 cycles; 72℃ for 3 min; and 4℃ for maintenance. The P5-ADP primer consists of an ADP portion and a P5 portion. The ADP portion is part of the Illumina platform adapter, and the P5-ADP is the modified Illumina platform adapter with specificity in this invention. Its gene sequence is shown in Table 2.

[0019] Further, the PCR amplification system for preparing the cDNA library in step (4) includes: cDNA ligated with adapter, buffer, dNTP mixture, KAPA HiFi polymerase, TruSeq-7N primers (N=1~8) and P5-ADP primers; the amplification conditions are: 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 6 cycles; 72℃ for 3 min; and 4℃ for maintenance.

[0020] Based on this, the present invention can provide the application of the above-mentioned novel high-throughput single-cell multi-omics analysis method in the joint analysis of cellular three-dimensional genomics, chromatin accessibility and gene expression.

[0021] The beneficial effects of this invention are:

[0022] 1. This invention combines a traditional microfluidic platform with a multi-round combined barcode strategy (introducing two rounds of molecular tags). It innovatively first encapsulates cells in microfluidic droplets, then uses a traditional split-mix method for combined labeling, employing a pair of specific barcodes to identify single cells, achieving a more than tenfold increase in the number of cells processed per microfluidic chip. While retaining the experimental principle of CN119351531A9 (ChAIR), this invention designs a two-round barcode labeling process, enabling each cell to carry a composite tag composed of multiple rounds of different tags, thereby expanding the order of magnitude of specific cell tags and increasing the throughput of a single experiment from approximately 12,000 cells to over 120,000 cells.

[0023] 2. This invention develops RNA library expansion primers that differ from the front-end structure of ATAC library amplification primers, which can minimize sequence skipping (index hopping) and library cross-contamination in PCR amplification and high-throughput sequencing.

[0024] 3. This invention achieves optimal reaction conditions by testing and optimizing the relative ratio of Tn5 and the amount of cells introduced during the transposition reaction. This optimization significantly enhances ATAC signal quality and detection sensitivity, improves enrichment of functional DNA elements, and reduces nonspecific background noise.

[0025] 4. This invention retains the accessibility site for Tn5 transposase insertion and the scheme for simultaneously capturing RNA and DNA sequences in droplets, ensuring that each cell retains its ability to capture open chromatin, long-range interactions, and gene expression, while avoiding sacrifice of omics coverage depth.

[0026] In summary, through the above improvements, this invention overcomes the shortcomings of low throughput in existing single-cell three-omics technologies, significantly improves experimental efficiency and data scale, and significantly reduces experimental costs. Attached Figure Description

[0027] Figure 1 This is an overall flowchart of Embodiment 1 of the present invention.

[0028] Figure 2 This is a quality control result diagram of a key step in Embodiment 2 of the present invention.

[0029] Figure 3 This is a visualization of the three-omics data quality of mouse whole brain cells in Embodiment 3 of the present invention.

[0030] Figure 4 This is a visualization of the three-omics data quality of zebrafish embryonic cells in Embodiment 4 of the present invention.

[0031] Figure 5 This is a statistical display of the three-omics data quality of mouse whole brain cells in Example 4 of the present invention.

[0032] Figure 6 This is a statistical display of the three-omics data quality of zebrafish embryonic cells in Embodiment 5 of the present invention.

[0033] Figure 7 This is a bar chart comparing the proportions of gDNA library reads in the cDNA libraries amplified by the two primers in Example 5 of this invention.

[0034] Figure 8 This is a display diagram of mouse whole brain cell types obtained through dimensionality reduction clustering annotation in Embodiment 6 of the present invention.

[0035] Figure 9 This is a diagram showing the zebrafish embryonic cell types obtained through dimensionality reduction clustering annotation in Embodiment 6 of the present invention. Detailed Implementation

[0036] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be described in detail, clearly, and completely below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Furthermore, based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of the present invention.

[0037] Example 1: A novel high-throughput single-cell multi-omics analysis method (HiChAIR).

[0038] This embodiment provides a novel high-throughput single-cell multi-omics analysis method (named HiChAIR), the specific process of which is described in the appendix. Figure 1 And described in detail below.

[0039] Step 1, Cell double cross-linking fixation:

[0040] Collect approximately 1×10 6Mouse whole brain cells were collected and washed twice with 1× PBS buffer. The cells were resuspended in 1 mL of 2% formaldehyde-PBS buffer and incubated on a shaker at room temperature for 20 min for the first cross-linking. Then, 87 μL of 2.5 M glycine solution (final concentration approximately 0.2 M) was added, and the cells were shaken at room temperature for 10 min to terminate the cross-linking. The cells were then centrifuged (2,500×g, room temperature, 10 min), the supernatant was discarded, and the cells were washed once with 1 mL of 1× PBS. The cells were then resuspended in 1 mL of 2 mM EGS-PBS buffer and incubated on a shaker at room temperature for 45 min for the second cross-linking. Then, 87 μL of 2.5 M glycine solution was added, and the cells were shaken at room temperature for 10 min to terminate the cross-linking. The cells were then centrifuged again (2,500×g, room temperature, 10 min), the supernatant was discarded, and the cells were washed once with 1 mL of 1× PBS. The treated cross-linked cells were resuspended in PBS and aliquoted into 0.5 mL centrifuge tubes containing approximately 100,000 cells each. The cells were then placed on ice for later use or stored at -80°C for long-term preservation in subsequent steps.

[0041] Step 2: Cell lysis and nuclear membrane permeation, in situ restriction enzyme digestion:

[0042] Take six tubes of double-crosslinked cell suspension (totaling 600,000 cells, thawed on ice for 20 min after being removed from -80℃), centrifuge (2,500×g, 4℃, 5 min), and discard the supernatant. Add 200 μL of 0.1% SDS lysis buffer (containing Triton X-100, sodium deoxycholate, and SDS as the main components) to each tube of cells, mix thoroughly, and lyse on ice for 1 h; then centrifuge (2,500×g, 4℃, 5 min), and discard the supernatant. Resuspend the cell pellet in 20 μL of 0.1% SDS solution and incubate at room temperature for 30 min to achieve nuclear membrane permeabilization. Then add 5 μL of 20% Triton X-100 solution to each tube and incubate at 37℃ with shaking for 15 min to neutralize residual SDS. The in situ restriction enzyme digestion reaction system was then prepared as follows: 53.5 μL of RNase-free water, 10 μL of 10×NEB rCutSmart buffer, 2.5 μL of 40 U / μL RNase inhibitor, 2 μL of 40 U / μL AluI restriction enzyme, and 2 μL of 5 U / μL HpyCH4V restriction enzyme were added to each tube. The reaction solution was incubated overnight at 37°C with a shaker. After the reaction was completed, 1 μL of the product was taken as a "digestion control" sample and stored for later use.

[0043] In this step, optimization is performed according to different cell types. In this example, mouse whole brain cells were treated with 0.1% SDS for 30 minutes to achieve good results. If other cell types are used, the treatment should be adjusted accordingly. For example, for human K562 cell line, 0.0125% SDS was used for 1 hour, and for 24-hour zebrafish embryo cells, 0.01% SDS was used for 20 minutes.

[0044] Step 3: Add A and adjacent DNA ends:

[0045] Prepare the A-tailing reaction system: Add 2 μL of 2 mg / mL recombinant protein, 2 μL of 5 U / μL KlenowFragment (3'→5' exo–) DNA polymerase, and 1.2 μL of 10 mM dATP to each tube. Mix well and incubate at 37°C with shaking for 1 h. Then, stop the A-tailing reaction by reacting at 65°C for 10 min. After cooling to room temperature, prepare the adjacent ligation reaction system: Add 61.8 μL of RNase-free water, 20 μL of 10×T4 DNA ligation reaction buffer, 6 μL of 2 ng / μL Bridge linker, 5 μL of 40 U / μL RNase inhibitor, and 2 μL of 400 U / μL T4 DNA ligase to each tube. Mix well and incubate at 16°C with shaking overnight for in situ DNA adjacent ligation. After ligation, take 2 μL of the product as a "ligation control" sample for later use.

[0046] Step 4, in-situ transposition (Tagmentation):

[0047] Centrifuge the cells (2,500×g, 4℃, 5 min) and discard the supernatant. Then add 200 μL of ATAC wash buffer, mix well, and centrifuge again (2,500×g, 4℃, 5 min), discarding the supernatant. Prepare the transposition reaction system: add 25.5 μL of 1× PBS, 50 μL of 2× transposition buffer, 2.5 μL of 40 U / μL RNase inhibitor, and 12 μL of Tn5 transposase to each tube. Mix thoroughly and incubate at 37℃ and 1,000 rpm for 1 h for in situ transposition. After the reaction, centrifuge (2,500×g, 4℃, 5 min), discard the supernatant, and combine the cell suspensions from 6 tubes after transposition treatment.

[0048] Step 5: Generate microfluidic droplets and introduce the first round of molecular tags:

[0049] Add 300 μL of diluted nuclei buffer. Gently mix and sonicate on ice for 1 second at 43% power to break up the clustered cell nuclei. Place a 20 μm cell filter at the mouth of a 1.5 mL centrifuge tube, add the cell suspension to the filter, and allow the cells to flow naturally through the filter; then add another 300 μL of diluted nuclei buffer to rinse the filter and collect the remaining cell suspension. Centrifuge the collected cell suspension (500 × g, 4 °C, 5 min), discard the supernatant (approximately 20 μL). Determine the cell concentration using a hemocytometer or automated cell counter. Transfer approximately 120,000 cells to a 0.2 mL PCR tube and bring the volume to 15 μL with diluted nuclei buffer. Subsequently, following the instructions of the 10x Genomics Chromium NextGEM Single Cell Multiome ATAC + Gene Expression Kit, mix the cells with the reaction reagents and load them onto a Chromium Controller for GEM preparation. After completion, reverse transcription and ligation reactions were performed: 37℃ for 45 min, 25℃ for 30 min, and finally maintained at 4℃. After the reaction, the first round of molecular tags was introduced into the gDNA and cDNA of each cell. 5 μL of Quenching reagent was added to the system, gently inverted 10 times to mix, and briefly centrifuged; then 125 μL of Recovery reagent was added, gently inverted 10 times to mix, and briefly centrifuged to release the cells. 70 μL of supernatant was transferred to a new 0.2 mL PCR tube, and 150 μL of PBS was added and mixed. The cell suspension was aliquoted into 96-well PCR plates, with 2 μL of cell suspension in each well.

[0050] Step 6, Decrosslinking, Cleavage, and DNA Purification:

[0051] Add 18 μL of pre-prepared decrosslinking buffer (containing proteinase K, DTT, NaCl, Tris-HCl pH 8.0, Triton X-100, and EDTA) to each well of a 96-well plate and incubate at 65°C and 1,000 rpm for 2 h with shaking to decrosslink the protein-DNA. Then add 20 μL of binding / lysis buffer (Lysis / Binding Buffer), gently mix, and incubate at 55°C and 1,000 rpm for 10 min to lyse the cells. Afterward, add 4 μL of DynabeadsMyOne SILANE magnetic beads and 22 μL of isopropanol (isopropanol volume is 1 / 3 of the total volume), gently mix, and incubate at room temperature for 10 min to allow the magnetic beads to adsorb DNA. Place the reaction tube on a magnetic rack, allow it to stand magnetically, and then discard the supernatant. Add 100 μL of Wash Buffer 1, allow it to stand at room temperature for approximately 30 seconds, and then discard the supernatant. Wash again with 100 μL of Wash Buffer 2, allow it to stand at room temperature for 30 seconds, and then discard the supernatant. Finally, add 17.4 μL of Elution Buffer and incubate at room temperature for 10 minutes to release the DNA bound to the magnetic beads. Collect the supernatant and transfer it to a new 96-well plate to obtain the purified DNA product.

[0052] Step 7, Simultaneous amplification of gDNA and cDNA:

[0053] Configure a simultaneous amplification reaction system for gDNA and cDNA. Add the following to each well of a 96-well plate: 6 μL 5×KAPAHiFi PCR buffer, 1.2 μL 10 mM dNTP mixture, 0.6 μL 1 U / μL KAPA HiFi polymerase, and primers: 1.2 μL 10 µM Partial TSO primer (primer sequences are shown in Table 1), 1.2 μL 10 µM Primer_P5 primer (primer sequences are shown in Table 1), 1.2 μL 10 µM ADP-i5-TS-N primer (primer sequences are shown in Table 2), and 1.2 μL 10 µM N-I7-indexN primer (primer sequences are shown in Table 3). The latter two primers are well-specific primers used to introduce the second round of molecular tags. N in indexN is the well number, ranging from 1 to 96. The gene sequences of the primers are shown in Tables 1 and 2. PCR amplification was performed using a thermal cycler with the following program: 72℃ for 5 min; 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 7 cycles; 72℃ for 3 min; and hold at 4℃. After amplification, 15 μL of PCR product from each of 24 wells was collected and combined into a 1.5 mL centrifuge tube. 360 μL (1.0 × volume) of AMPure XP magnetic beads was added to recover the DNA, and the final elution was carried out in 40 μL of Elution Buffalo. The remaining PCR product can be stored at -80℃ for long-term use.

[0054] Step 8, gDNA library preparation:

[0055] The PCR amplification system for preparing the gDNA library consisted of: 20 μL of the co-amplification product from step seven, 25 μL of NEBNext® High-Fidelity 2×PCR Master Mix, 2.5 μL of 10 µM Primer_P5 primer, and 2.5 μL of 10 µM Primer_P7 primer. PCR amplification was performed using a thermal cycler with the following program: 98 °C for 3 min; [98 °C for 20 s, 63 °C for 20 s, 72 °C for 30 s] for 7 cycles; 72 °C for 3 min; and hold at 4 °C. After amplification, the PCR product was purified using 50 μL of 1.0× volume AMPure XP magnetic beads. Then, paired-end selection was performed (adding 0.45× volume magnetic beads first, collecting the supernatant, and then adding another 0.45× volume magnetic beads) to remove excessively short or long fragments. The library concentration was determined using the Qubit dsDNA high-sensitivity kit, and fragment distribution was detected using an Agilent 2100 bioanalyzer high-sensitivity chip. Finally, Illumina NovaSeq 6000 PE150 sequencing was performed.

[0056] Step 9, cDNA library preparation:

[0057] The PCR amplification system for cDNA was prepared as follows: 20 μL of the co-amplification product from step seven, 10 μL of 5×KAPA HiFi PCR buffer, 2 μL of 10 mM dNTP mixture, 1 μL of 1 U / μL KAPA HiFi polymerase, 2.5 μL of 10 µM Partial TSO primers, and 2.5 μL of 10 µM P5-ADP primers (gene sequences are shown in Table 1). The PCR amplification program was: 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 7 cycles; 72℃ for 3 min; and held at 4℃. After amplification, the PCR product was purified with 0.8 × volume (40 μL) of AMPureXP magnetic beads, and the cDNA was eluted in 40 μL of Elution Buffer.

[0058] The fragmentation reaction system was then prepared as follows: 10 μL of purified cDNA, 5 μL of fragmentation buffer, 10 μL of fragmentation enzyme, and 25 μL of Elution Buffer. The reaction was carried out at 32 °C for 5 min, followed by heat inactivation at 65 °C for 30 min. The fragmented cDNA was purified using 0.8 × 40 μL of AMPure XP magnetic beads and eluted with 50 μL of Elution Buffer.

[0059] The adapter ligation system was then prepared as follows: 50 μL fragmented cDNA, 20 μL adapter DNA, 20 μL ligation buffer, and 10 μL DNA ligase; after mixing, the mixture was incubated at 20 °C for 15 min to complete adapter ligation. The adapter-ligated cDNA was purified with 0.8 × volume (80 μL) of AMPure XP magnetic beads and eluted with 32 μL of Elution Buffer.

[0060] Finally, the PCR amplification system for the cDNA library was prepared as follows: 32 μL of adapter-ligated cDNA, 10 μL of 5×KAPA HiFi PCR buffer, 2 μL of 10 mM dNTP mixture, 1 μL of 1 U / μL KAPA HiFi polymerase, 2.5 μL of 10 µM TruSeq-7N primers (gene sequence shown in Table 1), and 2.5 μL of 10 µM P5-ADP primers. The PCR amplification program was: 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 30 s] for 6 cycles; 72℃ for 2 min; and hold at 4℃. After amplification, the cDNA library was purified using 0.8 × volume (40 μL) of AMPure XP magnetic beads.

[0061] Library concentration was determined using Qubit, and fragment size distribution was detected using an Agilent 2100 analysis chip. Finally, Illumina NovaSeq 6000 PE150 sequencing was performed.

[0062] Table 1

[0063]

[0064] Table 2

[0065]

[0066] Table 3

[0067]

[0068] Example 2: Quality control inspection of key steps.

[0069] This embodiment performs quality control testing on the DNA used in each step of Example 1. The DNA tested in this embodiment includes an enzyme digestion control (Step 2), a ligation control (Step 3), a gDNA library before paired-end selection (Step 8), a gDNA library after paired-end selection (Step 8), cDNA (Step 9), and a cDNA library (Step 9). Approximately 1 ng of each of the above DNAs was loaded onto a Bioanalyzer 2100 HS DNA chip, and a preset injection and analysis program was run using an Agilent 2100 Bioanalyzer to detect the size distribution of DNA fragments. The results are as follows: Figure 2 As shown, the 35bp and 10380bp peaks are DNA markers at both ends. The main peak of the restriction enzyme control DNA fragment is at 5500bp, indicating sufficient enzyme digestion. The main peak of the ligation control is at 6500bp, a significant increase compared to the restriction enzyme control, indicating sufficient ligation. The final gDNA and cDNA library fragment sizes are mainly distributed within 200-1000bp, with no primer dimer contamination, meeting the library preparation requirements for next-generation sequencing, and the library peak shapes are as expected.

[0070] Example 3: Visualization and comparison of the three-omics data quality of HiChAIR and ChAIR.

[0071] To verify the effectiveness of this invention, second-generation sequencing data of mouse whole brain and zebrafish embryonic cells were subjected to quality control and standardization preprocessing, and then visualized using the Basic Browser platform. The results are as follows: Figure 3 and Figure 4 As shown. Compared to the ChAIR technology in the background art, the HiChAIR technology of this invention produces ATAC peaks (i.e., chromatin accessibility) with higher specificity and lower background noise, thus significantly improving data quality and accuracy. Simultaneously, by reducing the proportion of invalid data, it significantly reduces the sequencing depth requirement and experimental costs. Regarding chromatin interaction data, the HiChAIR technology can capture more effective chromatin interactions in a single experiment, significantly improving the sensitivity and coverage of interaction detection, which is beneficial for obtaining more complete and realistic three-dimensional genome structure information. Meanwhile, at the gene expression level, the method of this invention maintains consistent capture effect and data performance with the ChAIR technology, and can stably acquire transcriptome information at the cellular level. In summary, the results show that the HiChAIR technology of this invention, while ensuring the simultaneous acquisition of multi-omics information, can provide higher signal quality and detection efficiency for both open chromatin and chromatin interaction data, thus possessing comprehensive performance superior to existing technologies.

[0072] Example 4: Statistical display of the three-omics data quality of HiChAIR and ChAIR.

[0073] To verify the effectiveness of this invention, second-generation sequencing data from mouse whole brain and zebrafish embryonic cells were preprocessed, and single cells were identified using a two-round combined indexing method. After quality control screening to retain high-quality cells, single-cell matrices for RNA and ATAC were processed using Seurat and Signac software, respectively. Finally, violin plots were generated using ggplot2 in R language (as shown in the figures below). Figure 4 and Figure 5 (As shown in the figure). This figure is divided into five dimensions (PET, Fip, TSS, UMI, and Gene), examining the distribution of data volume in a single cell, with the values ​​representing the median. In a single experiment, the HiChAIR technology of this invention can obtain ten times the high-quality cells of the background ChAIR technology, and at the single-cell level, it has comparable chromatin interaction number (PET), ATAC fragment number (Fip), RNA single-molecule tag number (UMI), and detectable gene number (Gene). Furthermore, the HiChAIR technology of this invention is significantly superior to the background ChAIR technology in terms of transcription start site enrichment fraction (TSS), indicating that the ATAC signal of HiChAIR is more enriched at transcription start sites, further confirming its lower background noise and higher specificity.

[0074] Example 5: Data continuity and cleanliness verification.

[0075] The cDNA library sequencing data obtained in Example 1 (including mouse whole brain cells, human K562 cells mixed with mouse embryonic fibroblasts NIH3T3, and zebrafish embryonic cells) were distinguished according to the specific tags of gDNA and cDNA, and the proportion of each was calculated. The proportion data were then used to plot the graphs in Graphpad Prism software. The results are as follows: Figure 5 As shown in the figure, when using conventional primers, the cDNA library contains approximately 11.8% to 35.3% gDNA contamination. However, when using specially designed primers (ADP-i5-TS-N, N=1~96) containing adapters in the primer set of this invention, the contamination rate decreases to 0.3% to 1.2%, significantly reducing the contamination rate and lowering sequencing costs.

[0076] Example 6, Cell Type Annotation.

[0077] After preprocessing the cDNA library sequencing data obtained in Example 1, the dimensionality of the cDNA library sequencing data was first reduced by principal component analysis (PCA), then uniform manifold approximation and projection (UMAP) were used, and finally, Louvain clustering was performed using Seurat. In addition, Harmony was applied to integrate cDNA library sequencing data from mouse whole brain and zebrafish embryos, respectively, to mitigate batch effects.

[0078] To annotate cells, we integrated the cDNA library sequencing data described above with published scRNA-Seq data. For mouse brains, we used ChAIR mouse brain data (Nature Methods 22, 994–1007 (2025)) for annotation, and for zebrafish embryos, we used reference data (Nature Genetics 54, 328–341 (2022) and Developmental Cell 58, 3028-3047.e12 (2023)) for annotation. Then, we used the "FindTransferAnchors" and "TransferData" functions in Seurat v5 to annotate cells, and the results are as follows: Figure 8 and Figure 9 As shown.

[0079] As can be seen, the HiChAIR technology of this invention not only boasts the throughput advantage of processing over 100,000 cells in a single experiment, but also maintains a significant advantage in data quality. For various primary samples, including mouse whole brain tissue and zebrafish embryos, the single-cell data obtained by this invention exhibit high usability and stability. After dimensionality reduction analysis, cell population distributions with good separation and clear cluster boundaries are obtained. In subsequent cell type annotation, the main cell types in the samples can be accurately identified, and the degree of confounding between different cell populations is low, demonstrating excellent cell resolution capabilities, thereby ensuring the reliability and accuracy of downstream biological analysis.

[0080] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A novel high-throughput single-cell multi-omics analysis method, characterized in that, Includes the following steps: (1) Sample pretreatment: After double cross-linking fixation, lysis, nuclear permeation, and enzyme digestion of the population cell samples, A tails and adjacent ligations were added to the DNA ends, and transposition reaction was performed; (2) First round of molecular tag introduction: The pretreated cells are loaded into a microfluidic platform, and one or more cells are co-encapsulated in droplets with glue beads with capture adapters and first molecular tags. The DNA and RNA of the same cell are simultaneously labeled through the capture adapters. (3) Second round of molecular tag introduction: The cells in the droplets are released and dispensed into multi-well plates. After decrosslinking, lysis and DNA purification, primers with a second molecular tag are added to simultaneously amplify gDNA and cDNA to form a unique composite tag. (4) Library construction and sequencing: gDNA and cDNA were purified separately, and sequencing libraries were constructed for next-generation high-throughput sequencing.

2. The novel high-throughput single-cell multi-omics analysis method according to claim 1, characterized in that, In step (2), the microfluidic platform is loaded with at least 120,000 cells.

3. The novel high-throughput single-cell multi-omics analysis method according to claim 1, characterized in that, In step (3), the decrosslinking reaction is carried out using a pre-prepared decrosslinking mixed buffer containing proteinase K, and the mixture is shaken and incubated to decrosslink the protein-DNA.

4. The novel high-throughput single-cell multi-omics analysis method according to claim 1, characterized in that, The synchronous amplification reaction system of gDNA and cDNA in step (3) includes: buffer, dNTP mixture, polymerase and primer set, wherein the primer set includes Partial TSO primer, Primer_P5 primer, ADP-i5-TS-N primer and N-I7-indexN primer, N=1~96, and the gene sequence of the primer set, such as the last two, are pore-specific primers, which are configured to introduce the second molecular tag.

5. The novel high-throughput single-cell multi-omics analysis method according to claim 4, characterized in that, The conditions for the synchronous amplification of gDNA and cDNA in step (3) are: 72℃ for 5 min; 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 7 cycles; 72℃ for 3 min; and 4℃ for maintenance.

6. The novel high-throughput single-cell multi-omics analysis method according to claim 1, characterized in that, The PCR amplification system for preparing the gDNA library in step (4) includes: the co-amplification product from step (3), high-fidelity PCR premix, Primer_P5 primer, and Primer_P7 primer.

7. The novel high-throughput single-cell multi-omics analysis method according to claim 6, characterized in that, The PCR amplification conditions for preparing the gDNA library in step (4) are as follows: 98℃ for 3 min; [98℃ for 20 s, 63℃ for 20 s, 72℃ for 30 s] for 7 cycles; 72℃ for 3 min; and 4℃ for maintenance.

8. The novel high-throughput single-cell multi-omics analysis method according to claim 1, characterized in that, The PCR amplification system for preparing cDNA in step (4) includes: the co-amplification product from step (3), dNTP mixture, polymerase, Partial TSO primer and P5-ADP primer; the amplification conditions are: 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 7 cycles; 72℃ for 3 min; and 4℃ for maintenance.

9. A novel high-throughput single-cell multi-omics analysis method according to claim 8, characterized in that, The PCR amplification system for preparing the cDNA library in step (4) includes: cDNA ligated with adapter, buffer, dNTP mixture, KAPAHiFi polymerase, TruSeq-7N primers (N=1~8) and P5-ADP primers; the amplification conditions are: 95℃ for 3 min; [98℃ for 20 s, 63℃ for 30 s, 72℃ for 1 min] for 6 cycles; 72℃ for 3 min; and 4℃ for maintenance.

10. The application of a novel high-throughput single-cell multi-omics analysis method according to any one of claims 1-9 in the combined analysis of cellular three-dimensional genomics, chromatin accessibility and gene expression.

Images