Single-cell exon sequencing methods and applications thereof

Abstract

The application discloses a single-cell exon sequencing method and application thereof, and the method comprises the following steps: S1, tumor single-cell sorting; S2, MDA amplification of the sorted tumor single cells to obtain a genomic library; S3, detection of MDA amplification product uniformity, selection of multiple sites located on different chromosomes and design of corresponding amplification primers, detection of amplification effects of the multiple sites of each single-cell MDA product by qPCR, and determination of whether the genome is uniformly amplified; S4, gDNA library preparation: the amplified DNA is broken into 150-200bp fragments, and a gDNA library is obtained through end repair, tailing, adapter addition and amplification; S5, exon capture and library construction: the prepared gDNA library is hybridized with specific probes, specific regions are captured, and an exon library is obtained by PCR amplification and addition of index tags; and S6, sequencing of the exon library.

Description

Technical Field

[0001] This invention relates to the field of gene sequencing, specifically to single-cell exon sequencing methods and their applications. Background Technology

[0002] Single-cell sequencing is a new technology that performs high-throughput sequencing analysis of multiple omics, including the genome, transcriptome, and epigenome, at the single-cell level. This technology makes it possible to interpret the gene structure and expression status of individual cells and to understand intercellular heterogeneity. It has been widely applied in research in developmental biology, immunology, and oncology, resulting in a large number of high-quality research findings. Single-cell exon sequencing offers better sensitivity and resolution for detecting gene mutations and copy number variations. Compared to tissue block exon sequencing, single-cell exon sequencing can detect genomic alterations such as mutations at higher frequencies in some tumors, and it is also significant in studying tumor heterogeneity and clonal evolution. This provides unprecedented resolution for identifying cancer characteristics, which is of great importance in cancer detection, diagnosis, and targeted therapy, and may reveal the mechanisms of cancer development and the discovery of drug targets.

[0003] Due to the high heterogeneity of tumor tissue, tissue block-based sequencing can mask low-frequency mutations. Furthermore, whole-genome sequencing is expensive and generates massive amounts of data, while mutations related to human diseases are primarily concentrated in exon regions, which comprise less than 2% of the human genome sequence. In addition, a single cell's DNA (approximately 6 pg) is insufficient for the minimum sample size required for next-generation sequencing. Therefore, single-cell genome amplification is necessary. In single-cell exon sequencing, we use MDA to amplify the entire genome of a single cell. Subsequent analysis of the amplified products involves single-cell exon library construction and sequencing to obtain exon region mutation information. Because amplification can introduce biases, leading to deviations in copy number variation and single nucleotide variant amplification, selecting samples with high amplification uniformity is crucial for obtaining accurate mutation information. In our experiments, we conducted amplification uniformity tests on MDA products, selecting samples with high amplification uniformity for subsequent experiments. This significantly improved all indicators, including amplified gene coverage and fidelity, making single-cell amplification and sequencing more accurate.

[0004] In summary, single-cell exon sequencing has advantages such as high activity, simple operation, improved success rate of single-cell exon library construction, and reduced downstream experimental costs, which greatly accelerates the application of single-cell exon sequencing in tumor evolution mechanism research, precise cancer subtyping and tumor drug resistance mechanism, and efficacy prediction research. Summary of the Invention

[0005] This invention provides a method for tumor single-cell exon sequencing and its application. The invention aims to address, to a certain extent, the technical problems in related technologies: how to isolate single cells from isolated tumor tissue and perform exon sequencing using a simple and convenient method. To this end, the inventors have developed a simple method to obtain single cells from isolated tumor tissue samples and perform exon sequencing, enabling single-cell sequencing technology to be more widely applied to the study of the microenvironment of isolated tumor tissues and saving costs.

[0006] First aspect:

[0007] This invention provides a method for tumor single-cell exon sequencing, comprising the following steps:

[0008] S1, Tumor single-cell sorting;

[0009] S2. The sorted tumor cells are subjected to MDA amplification to obtain a genomic library;

[0010] S3. Detect the uniformity of MDA amplification products. Select multiple sites located on different chromosomes and design corresponding amplification primers. Use qPCR to detect the amplification effect of multiple sites of MDA products in each single cell, and then determine whether the genome is amplified uniformly.

[0011] S4. gDNA library preparation: The amplified DNA is broken into 150-200bp fragments, and gDNA libraries are obtained through end repair, tailing, adapter addition, and amplification.

[0012] S5. Exon capture and library construction: The prepared gDNA library is hybridized with a specific probe to capture a specific region. After PCR amplification and addition of index tags, an exon library is obtained.

[0013] S6. Sequencing of the exon library.

[0014] Step S3 is as follows:

[0015] Multiple loci located on different chromosomes were selected and corresponding amplification primers were designed. The amplification effect of multiple loci of MDA product in each single cell was detected by qPCR to determine whether the genome was amplified uniformly. The positive control was gDNA extracted from tumor tissue and the negative control was enzyme-free water.

[0016] The relative uniformity value (RUV) is calculated using the following formula:

[0017] Relative uniformity value(RUV)=2 -(Cti-Ct0)

[0018] Wherein, Cti represents the Ct value of that site in sample i, and Ct0 represents the Ct value of the same site in the unamplified genomic DNA; the RUV of the unamplified genomic DNA should be 1; an RUV close to 1 indicates that the amplification of that site is relatively uniform; among the multiple sites located on different chromosomes, at least 75% of the sites have RUV values ​​between 0.25 and 4. Only single-cell MDA products can be considered as samples that meet the conditions for the next step of exon library construction.

[0019] The amplification primers include multiple pairs of the following primer pairs:

[0020]

[0021] In step S5, the specific probe is Agilant SureSelect XT HumanAll ExonV6.

[0022] Step S1 includes the following steps:

[0023] Step 1: Sample preparation. The tumor cells to be sorted are dissociated into a suspension, and the proportion of live cells and the total number of cells are detected by staining with cell viability detection reagent. The suspension is then passed through a cell filter to remove double cells and debris, and a highly active single-cell suspension is prepared.

[0024] Step 2, Sample loading: Start the single-cell titration separation system, add the tumor cell suspension from Step 1 to the starting sample well, and then place the plate in the collection position;

[0025] Step 3: Set the single-cell titration sorting parameters and adjust the titration position to the center of the bottom of the well plate;

[0026] Step 4, Single-cell titration and sorting: Cells are aspirated using a capillary tube, and the state of the droplets is observed using an imaging system. Cells passing through the liquid flow are observed, and various parameters of the cells in the sample are obtained by adjusting the sorting of cell particle size to capture viable cells. Finally, a droplet with a volume of 450-600 pL is formed and titrated into each well of the well plate, one cell per well. Empty wells and multi-cell wells are discarded based on the photograph of the single cell in the nozzle area; thus, a single cell is obtained.

[0027] The single-cell titration separation system was the CellenONE X1 system.

[0028] In step 1, the cells to be sorted are dissociated into a cell suspension using collagenase IV.

[0029] Preferably, in step 1, the tumor cells from the tumor patients to be sorted are dissociated into a single-cell suspension using collagenase IV, filtered through a 70μm cell filter, and then counted using a cell counter.

[0030] In step 2, the well plate is a low-adsorption well plate, and a pre-cooled calcium- and magnesium-free PBS solution that can cover the bottom of the well plate is added to each well in advance.

[0031] Preferably, in step 2, the well plate is a low-skirted 96-well plate, and 1.5 μL of pre-cooled calcium- and magnesium-free PBS solution is added to each well beforehand.

[0032] The application of the exon sequencing method described herein in precise tumor subtyping, tumor drug resistance mechanism research, clonal evolution research, and efficacy prediction research also falls within the scope of protection of this invention.

[0033] The MDA amplification used is Single Cell Kit.

[0034] Preferably, the complete culture medium is DMEM culture medium containing 10% FBS.

[0035] Specifically, in step 1, the DNA ultrasonication is performed using a Covaris M220.

[0036] The exon library was constructed using the Agilent SureSelectXT Target Enrichment System for the Illumina Platform.

[0037] Specifically, in step 2, when the CellenONE X1 single-cell titration separation system is started, the well plate tray is pre-cooled and moved to the starting point, the air pump is turned on, the cell particle size range is adjusted to 5-30μm (the diameter range of the selected image batch is 10-40μm), 10μL of cell suspension is aspirated by the PDC and then titrated and injected into the bottom of each well of the 96-well plate.

[0038] The application of the exon sequencing method described herein in precise tumor subtyping, tumor drug resistance mechanism research, clonal evolution research, and efficacy prediction research also falls within the scope of protection of this invention.

[0039] Beneficial effects:

[0040] 1. This invention uniquely combines single-cell processing with exome sequencing to create single-cell exome sequencing. Compared to whole-exome sequencing of tissues, our single-cell exome sequencing can provide single-cell-level research, improve the resolution of tumor heterogeneity, and can be used to identify and study structural variations in disease-related coding regions, which helps to analyze clonal evolution (corresponding to the comparison of mutations and classifications detected by tissue tumor exome sequencing and those detected by single tumor cell exome sequencing in the following text).

[0041] 2. Exploration of intratumoral heterogeneity and clonal evolution is largely limited to the population-level study. Current research increasingly focuses on investigating drug resistance and recurrence progression in cancer patients. Therefore, population-level exome analysis is insufficient to meet the needs. The high-resolution single-cell exome analysis technology provided by this invention offers a powerful tool for answering these questions. (This corresponds to the comparison between tissue tumor exome sequencing and single-cell exome sequencing discussed later.)

[0042] Due to the redundancy of whole-genome sequencing data, there is a large amount of data that is not needed for current research. This massive amount of information often masks low-frequency mutations carried by a few cells. However, the single-cell exon sequencing of this invention, because it detects a smaller region (exon sequences account for less than 2% of the human genome sequence), can achieve a depth of more than ten times that of whole-genome sequencing (usually 90G) even if we only sequence 15G of exome data, thus greatly improving research efficiency, as shown in Table 1.

[0043] Table 1 Comparison of whole-genome sequencing and whole-exome sequencing

[0044]

[0045] 3. It can more efficiently detect low-frequency mutations and rare variants, increasing the average number of detectable mutations from 13 in the whole exome to 533.5 in the single-cell exome.

[0046] 4. Whole genome sequencing is expensive per sample, while single-cell exome sequencing significantly reduces the cost and overall cost.

[0047] 5. Because amplification can introduce bias, leading to deviations not only in copy number variation but also in single nucleotide variant amplification, selecting samples with high amplification uniformity is crucial for obtaining accurate mutation information. In our experiments, we used MDA to amplify the entire genome of single cells and verified the amplification uniformity of the MDA products through a uniformity test. The eight selected sites were carefully designed (representative across the genome), and only samples with high genome amplification uniformity that passed the uniformity test were used for subsequent experiments. This unique experimental design significantly improves all indicators, including amplified gene coverage and fidelity, making single-cell amplification and sequencing more accurate and ensuring high-quality results.

[0048] 6. The Agilent SureSelectXT Human Whole Exon V6 capture probe has a target size of 58Mb, offering superior coverage and consistency, which helps improve capture quality. It contains the core content of relevant databases and can target more exons, including difficult-to-capture regions. Database coverage is RefSeq 99%, CCDS 99%, GENCODe 99%, HGMD-CDS 99%, and OMIM-CDS 99%. It has gained industry-wide recognition in the fields of genetic diseases and oncology, becoming a classic product in the next-generation sequencing capture field, and has also received accreditation from the International Cancer Genome Consortium (ICGC).

[0049] 7. Compared to flow cytometers, the Cellen ONE X1 single-cell titration system requires a starting volume of 10 during the sorting process. 5 Cells / ml: A small initial cell count is required, which is beneficial for the sorting and enrichment of rare cells. Furthermore, the flexible sorting process causes minimal cell damage, resulting in a high single-cell rate and high viability. Visualization and image storage facilitate traceability. Attached Figure Description

[0050] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0051] Figure 1 This is a flowchart of the single-cell exon sequencing process of this invention;

[0052] Figure 2 This is a diagram showing the quality control results of single-cell exon library construction according to Example 1 of the present invention;

[0053] Figure 3 This is the mutation detection result after single-cell exon sequencing in the embodiment;

[0054] Figure 4 This is a schematic diagram of the quality control results of the construction of exon libraries from tissue source 1 in Comparison Example 1.

[0055] Figure 5 Comparative Example 1: Exon sequencing of hepatoblastoma tissue samples to detect mutations and classification;

[0056] Figure 6 This is the CNV detection result of tissue and single-cell exon sequencing of the same patient in Examples and Comparative Example 1. Detailed Implementation

[0057] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0058] This invention proposes a simple and efficient method for isolating single cells from isolated tumor tissue and performing single-cell exon sequencing. This method can be applied to studies on tumor heterogeneity, tumor clonal evolution, and drug screening at the single-cell level.

[0059] This invention is applicable to single-cell exon sequencing of fresh, ex vivo tumor tissue from cancer patients. The main components include six parts: tissue dissociation, single-cell isolation and quality control, whole-genome amplification, amplification homogeneity assay, exon capture, library construction, and sequencing. Figure 1 .

[0060] Single-cell isolation and acquisition were performed using a Countess II FL Automated Cell Counter and a trypan blue light microscope to detect concentration and dissociation efficiency.

[0061] The specific probe is the Agilent SureSelect XT HumanAll Exon V6.

[0062] MDA amplification uses Single Cell Kit.

[0063] The DNA ultrasonic fragmentation was performed using Covaris M220.

[0064] Exon library construction was performed using the Agilent SureSelectXT Target Enrichment System for the Illumina Platform.

[0065] Example 1: Single-cell exon sequencing

[0066] 1. Preparation of single cell samples

[0067] Liver cancer tumor tissue and adjacent normal tissue were dissociated to obtain single-cell suspensions. The cell suspensions were then sorted using the CellenOne X1 single-cell titration system to obtain individual cells.

[0068] The specific steps are as follows:

[0069] ① Tissue dissection: Rinse the tumor tissue 2-3 times with pre-cooled DPBS, and cut off approximately 3mm.3 Tumor tissue was placed in a 2ml centrifuge tube, and 500ul of 0.05% collagenase IV was added. The tissue was minced on ice and incubated at 37°C and 60rpm / min for 8min. The cells were then filtered through a 70µm cell filter to obtain a cell suspension. 3ml of erythrocyte lysis buffer was added to the suspension, and the mixture was incubated at room temperature for 5min. The suspension was then centrifuged at 500g and 4°C for 5min, and the supernatant was discarded. 5ml of pre-chilled calcium- and magnesium-free PBS was added, and the mixture was mixed by pipetting. The cells were then centrifuged under the same conditions, and the supernatant was discarded. The cells were resuspended in 500ul of DMEM containing 10% FBS. 10µL of the cell suspension was stained with an equal volume of trypan blue, and the cell count and viability were measured using a Countess II FL Automated Cell Counter.

[0070] ② Single-cell sorting: 1.5 μL of calcium- and magnesium-free PBS solution was added to each well of a low-adsorption 96-well plate beforehand; the cell suspension obtained above was loaded into the sample wells of the CellenONE X1 single-cell titration system for sorting; the titration position was adjusted to the center of the collection plate, and the sorting parameters were set as follows: X-axis = 19, Y-axis = 40 and Z-axis = 144, Voltage = 76 and Pulse = 49; the cell suspension was aspirated using a PDC (Piezo Dispense Capillary), and the droplet state was observed with the help of the imaging system. By adjusting the parameters such as the diameter and roundness of the cells in the sample, the cell size of the sorted cells (10-40 μm) was adjusted to accurately capture viable cells and finally form a droplet of 0.1 μL (450-600 pL) in volume, which was titrated into each well, one cell per well; empty wells and multi-cell wells were removed based on the photograph of the single cell in the nozzle area, thus obtaining single cells through sorting.

[0071] 2. Single-cell multiple displacement amplification technology

[0072] use The Single Cell Kit (157046333, Qiagen) is used for single-cell multiple displacement amplification (MDA).

[0073] ① Cell lysis: Add 0.5 μL of Exo-resistant Random primer and 1.5 μL of Lysis buffer (400 mM KOH, 100 mM DTT, 10 mM EDTA) to 3 μL of the centrifuge tube containing single cells as described above. After centrifugation, place on ice for 10 min. Add 1.5 μL of Stop solution (400 mM HCl Tris-HCl and 600 mM Tris-HCl 1 M, pH 7.5), centrifuge, and place on ice for 2 min.

[0074] ② Amplification: Add 16ul of amplification mixture (15ul REPLI-g sc Reaction Buffer, 1ul REPLI-g sc DNAPolymerase) to step ①, mix well by pipetting, centrifuge, and then amplify: 30℃, 90min, 65℃, 3min, and store at 4℃.

[0075] ③ Purification: Add 37.8 μL Beckman AMPureXP purification beads to 21 μL of amplification product for purification, and use Thermo Fisher Scientific Qubit 4 to quantify the purified product.

[0076] 3. Detection of uniformity of genome amplification.

[0077] The amplification uniformity of MDA products with higher concentrations than the negative control was tested to verify the amplification uniformity of each product. The principle is to select 8 sites located on different chromosomes and design corresponding amplification primers (site and primer information is shown in Table 2). The amplification effect of these 8 sites of MDA products in each single cell was detected by qPCR, thereby determining whether the genome was amplified uniformly (positive control is gDNA from the patient's tissue, negative control is enzyme-free water).

[0078] The relative uniformity value (RUV) is calculated using the following formula:

[0079] Relative uniformity value(RUV)=2 -(Cti-Ct0)

[0080] Where Cti represents the Ct value at that site in sample i, and Ct0 represents the Ct value at the same site in the unamplified genomic DNA. The RUV of the unamplified genomic DNA should be 1. A RUV close to 1 indicates that the amplification at that site is relatively uniform. Only single-cell MDA products with RUV values ​​between 0.25 and 4 at at least 6 of the 8 sites can be considered as samples that meet the criteria for the next step of exon library construction.

[0081] Table 2: Eight sites for amplification uniformity detection and their corresponding qPCR primers

[0082]

[0083] 4. Construction and sequencing of single-cell exon libraries

[0084] The exome library was constructed using the Agilant SureSelectXT Target Enrichment System for the Illumina Platform. The specific steps are as follows:

[0085] ① Genomic DNA fragmentation: Take 1.5g of MDA product that has passed the homogeneity test after amplification, dilute it to 65uL with low TE buffer, and use a Covaris M220 sonicator to fragment it into 150-200bp fragments. The fragmentation conditions are set as follows: peak power: 75, duty factor: 20%, cycles: 200, time: 250s, and temperature: 18-22℃. The fragmented product is purified using Beckman AMPure XP, and finally 24uL of purified product is obtained.

[0086] ② End Repair: Add 26 μL of end repair mixture (17.6 μL Nuclease-Free Water, 5 μL 10× End Repair Buffer, 0.8 μL dNTP Mix, 0.5 μL T4 DNA Polymerase, 1 μL Klenow DNA Polymerase, 1.1 μL T4 Polynucleotide Kinase) to 24 μL of purified product, mix thoroughly by pipetting, centrifuge, and incubate on a PCR instrument at 20 °C for 30 min, then store at 4 °C. After incubation, purify using Beckman AMPure XP to obtain 15 μL of purified product.

[0087] ③ Add A to the 3' end: Add 10 μL of the prepared reaction mixture (5.5 μL Nuclease-Free Water, 2.5 μL 10×Klenow Polymerase Buffer, 0.5 μL dATP, 1.5 μL Exo(-)Klenow) to the 15 μL product above, mix well by pipetting, centrifuge, and incubate on a PCR instrument at 37°C for 30 min, then store at 4°C. After incubation, purify using Beckman AMPure XP to obtain 6.5 μL of purified product.

[0088] ④ Adapter ligation: Add 18.5 μL of reaction mixture (7.75 μL Nuclease-Free Water, 5 μL 5x T4 DNA Ligase Buffer, 5 μL SureSelect Adaptor Oligo Mix, 0.75 μL T4 DNA Ligase) to the above 6.5 μL product, mix well by pipetting, centrifuge, and incubate on a PCR instrument: 20 °C for 15 min, then store at 4 °C. After incubation, purify using Beckman AMPure XP to obtain 16 μL of purified product.

[0089] ⑤ Amplification: Take 7.5 μL of the above product and add 17.5 μL of PCR reaction mixture (10.5 μL Nuclease-Free Water, 0.625 μL SureSelect Primer, 0.625 μL SureSelect ILM Indexing Pre-Capture PCR Reverse Primer, 5 μL 5×Herculase II Reaction Buffer, 0.25 μL 100 mM dNTP Mix, 0.5 μL Herculase II Fusion DNA Polymerase). Mix well by pipetting, centrifuge, and incubate on a PCR instrument: 98℃ for 2 min, then 98℃ for 30 s, 65℃ for 30 s, 72℃ for 1 min, 4-6 cycles, 72℃ for 10 min, and store at 4℃. After incubation, purify using Beckman AMPure XP to obtain 15 μL of purified product. The concentration and fragment distribution of the gDNA library were detected using Thermo Fisher Scientific Qubit 4.0 and Agilent 2100 Bioanalyzer, respectively.

[0090] ⑥ Hybridization capture: a. Prepare 1.7 μL of a diluent with a concentration of 221 ng / μL for each library according to the above product concentration.

[0091] b. Prepare hybridization buffer: (3.315uL SureSelect Hyb 1, 0.135uL SureSelect Hyb 2, 1.325uL SureSelect Hyb 3, 1.725uL SureSelect Hyb 4), store at room temperature until used. c. Prepare SureSelect blocking buffer: (1.25uL SureSelect Indexing Block 1, 1.25uL SureSelect Block 2, 0.3uL SureSelect ILM Indexing Block 3). d. Add 2.8uL SureSelect blocking buffer to each 1.7uL gDNA library, mix well by pipetting, and incubate on a PCR instrument: 95℃ for 5 min, then 65℃ for storage (≥5 min). e. Prepare hybridization mixture: (6.5uL hybridization buffer (step b), 1uL 25% RNase Block solution, 2.5uL Probe (with design ≥3Mb)). f. Add 10 μL of the prepared hybridization mixture to each sample from step d, and incubate on a PCR instrument: 105°C hot-lid, 65°C, 24 h. g. Prepare streptavidin-coated magnetic beads: Resuspend Dynabeads MyOne Streptavidin T1 magnetic beads. Pipette 25 μL of the beads into a new centrifuge tube for each sample, add 200 μL of SureSelect Binding Buffer for washing, repeat 3 times, and resuspend in 100 μL of SureSelect Binding Buffer for later use. h. Transfer the samples from step f to 100 μL of the prepared streptavidin beads from step g, mix by pipetting, and incubate at room temperature, 1400–1800 rpm for 30 min. After incubation, place on a magnetic rack until the solution is clear, then discard the supernatant. i. Resuspend in 100 μL of SureSelect Wash Buffer 1, incubate at room temperature for 15 min, place on a magnetic rack, and discard the supernatant after the solution is clear. j. Wash three times with 100uL of preheated WashBuffer 2 at 65°C, followed by elution with 15uL of Nuclease-Free Water, and store on ice for later use.

[0092] ⑦ Capture library amplification and indexing tag addition: Prepare 15.5 μL of PCR reaction mixture (9.25 μL Nuclease-Free Water, 5 μL 5×Herculase II Reaction Buffer, 0.5 μL Herculase II Fusion DNA Polymerase, 0.25 μL 100 mM dNTP Mix, 0.5 μL SureSelect ILMIndexing Post-Capture Forward PCR Primer) in a new PCR tube for each sample. Add 2.5 μL of indexing primer and 7 μL of the above magnetic bead capture library (step j in ⑥) to each tube. Mix well by pipetting and place in a PCR instrument to perform the amplification program: 98℃, 2 min, then 98℃, 30 s, 57℃, 30 s, 72℃, 1 min, 12 cycles, 72℃, 10 min, 4℃, and store. After incubation, purify using Beckman AMPure XP to obtain 15 μL of exon library product.

[0093] ⑧ The library concentration was quantified using a Thermo Fisher Scientific Qubit 4, and the abundance and fragment distribution of the successfully prepared exon library were quality checked using an Agilent 2100 Bioanalyzer (e.g., Figure 2 As shown in the figure, exon libraries that meet the standards were selected and PE150 sequencing was performed on the Illumina NovaSeq 6000 sequencing platform to obtain 10Gb of data.

[0094] 5. Test Results

[0095] Figure 3 The study presented the mutations detected after single-cell exome sequencing of the patient's four tumors, showing a greater number and variety of mutations compared to tissue exome sequencing, with an average of 533.5 mutations detected per tumor cell.

[0096] Comparative Example 1: Tissue-derived exome sequencing

[0097] The difference between this comparative example and Example 1 is that this comparative example involves sequencing an exon library constructed from DNA extracted from tissue, while Example 1 involves sequencing an exon library constructed from DNA from a single cell.

[0098] (1) Genomic DNA extraction

[0099] Genomic DNA was extracted from cancer and adjacent normal tissues of the same origin as in Example 1 using the QIAamp DNAMini Kit (51306, Qiagen), and the obtained genomic DNA was quantified using a Thermo Fisher Scientific Qubit4 fluorometer.

[0100] (2) Exon library construction

[0101] The specific steps are the same as the exon library construction steps in the above implementation list.

[0102] (3) Quantitative analysis and quality control of exome libraries

[0103] The successfully prepared exon libraries were quality checked for abundance and fragment distribution using an Agilent 2100 Bioanalyzer. Figure 4 Only those that meet the standards can be used for sequencing.

[0104] (4) Sequencing

[0105] PE150 sequencing was performed on the Illumina NovaSeq 6000 sequencing platform.

[0106] (5) Data Analysis

[0107] The raw sequencing data undergoes quality control and screening, is compared with the reference genome and germline mutations are removed, and then SNP and CNV identification, filtering and annotation are performed.

[0108] The main filtering conditions are: (1) Select sites that are filtered as PASS by the muect2 tool; (2) Sequencing depth of the mutation site is greater than 10; (3) Mutation frequency of the site is greater than 0.05.

[0109] (6) Test Results

[0110] Analysis revealed 13 mutations, predominantly missense mutations, in the exon regions of this patient (CTNNB1, LNP1, DIAPH1, NOTCH4, GABRR1, SLC25A45, PMEL, CHFR, UBN1, MYO18A, CNOT3, EIF3D, GDI1). Mutation types and classifications are as follows: Figure 5 As shown.

[0111] In this comparative study, there were no major differences in the detection distribution of exon sequencing for single cells and tissue blocks across different mutation categories, but the detection distribution of exon sequencing for four single tumor cells showed significant differences. Figure 3 ) compared to the corresponding ( Figure 5 Overall, several tens of times more mutation information was detected. The discovery of this high-resolution tumor mutation information will be beneficial for patient prognostic analysis and provide guidance on medication.

[0112] On the other hand, in the detection of tumor copy number variations (CNVs), single-cell exon sequencing has also demonstrated higher detection efficiency than traditional tissue block-based sequencing. For example... Figure 6 As shown, exon sequencing results of single tumor cells from patients exhibited superior detection rates for both copy number deletions and copy number amplifications compared to tissue block exon sequencing results. It also demonstrated significantly better CNV detection performance compared to exon sequencing results from the same patient. Furthermore, single-cell exon sequencing of tumors can reveal CNV heterogeneity among tumor cells from the same patient, which is beneficial for studying tumor heterogeneity and clonal evolution.

[0113] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for single-cell exon sequencing of tumors for non-diagnostic purposes, characterized in that, Includes the following steps: S1. Tumor single-cell sorting; including the following steps: Step 1: Sample preparation. Dissociate the tumor cells to be sorted into a cell suspension. Step 2, Sample loading: Start the single-cell titration separation system, add the tumor cell suspension from Step 1 to the starting sample well, and then place the plate in the collection position; Step 3: Set the single-cell titration sorting parameters and adjust the titration position to the center of the bottom of the well plate; Step 4, Single-cell titration and sorting: Cells are aspirated using a capillary tube, and the state of the droplets is observed using an imaging system. Cells passing through the liquid flow are observed, and various parameters of the cells in the sample are obtained by adjustment. The cell particle size is adjusted to capture viable cells and finally form a droplet with a volume of 450-600 pL. The droplet is titrated into each well of the plate, with one cell per well. Empty wells and multi-cell wells are discarded based on the photograph of a single cell in the nozzle area. Obtain single cells; S2. The sorted tumor cells are subjected to MDA amplification to obtain a genomic library; S3. To detect the uniformity of MDA amplification products, select multiple sites located on different chromosomes and design corresponding amplification primers. Use qPCR to detect the amplification effect of MDA products at these multiple sites in each single cell, thereby determining whether the genome is amplified uniformly. S4. gDNA library preparation: The amplified DNA is broken into 150-200bp fragments, and gDNA libraries are obtained through end repair, tailing, adapter addition, and amplification. S5. Exon capture and library construction: The prepared gDNA library is hybridized with a specific probe to capture a specific region. After PCR amplification and addition of index tags, an exon library is obtained. S6. Sequencing the exon library; Step S3 is as follows: Multiple loci located on different chromosomes were selected and corresponding amplification primers were designed. The amplification effect of multiple loci in the MDA product of each single cell was detected by qPCR to determine whether the genome was amplified uniformly. The positive control was gDNA extracted from tumor tissue, and the negative control was enzyme-free water. The relative uniformity value (RUV) is calculated using the following formula: Relative uniformity value RUV = 2 -(Cti-Ct0) Where Cti represents the Ct value of that site in sample i, and Ct0 represents the Ct value of the same site in unamplified genomic DNA; the RUV of unamplified genomic DNA should be 1; an RUV close to 1 indicates that the amplification of that site is relatively uniform; among multiple sites located on different chromosomes, at least 75% of the sites have RUV values ​​between 0.25 and 4. Only single-cell MDA products can be considered as samples that meet the conditions for the next step of exon library construction. In S3, the amplification primers include multiple pairs of the following primer pairs:

2. The exon sequencing method according to claim 1, characterized in that, In step S5, the specific probe is an Agilent SureSelect XT Human All Exon V6.

3. The exon sequencing method according to claim 1, characterized in that, In step 1, the cells to be sorted are dissociated into a cell suspension using collagenase IV.

4. The exon sequencing method according to claim 3, characterized in that, In step 2, the well plate is a low-adsorption well plate, and a pre-cooled calcium- and magnesium-free PBS solution that can cover the bottom of the well plate is added to each well in advance.

5. The exon sequencing method according to claim 1, characterized in that, The MDA amplification was performed using the Qiagen REPLI-g® Single Cell Kit.

6. The application of the exon sequencing method as described in any one of claims 1-5 in the study of tumor drug resistance mechanisms and clonal evolution.

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