Single-cell exon sequencing method and use thereof

Abstract

Disclosed is a single-cell exon sequencing method and a use thereof, the method comprising: S1, tumor single cell sorting; S2, performing MDA amplification of sorted tumor single cells to obtain a genome library; S3, measuring the uniformity of MDA amplification products, selecting multiple sites located on different chromosomes and designing corresponding amplification primers, and testing the amplification effect of the multiple sites of the MDA product of each single cell by means of qPCR, thereby determining whether the genome has been uniformly amplified; S4, gDNA library preparation: breaking the amplified DNA into 150-200 bp fragments, and obtaining a gDNA library by means of end repair, tailing, adding adapters, and amplification; S5, exon capture and library construction: hybridizing the prepared gDNA library with a specific probe, capturing a specific region, and then adding an index tag via PCR amplification to obtain an exon library; and S6, sequencing the exon library.

Description

Single-cell exome sequencing methods and their applications Technical Field

[0001] The present invention relates to the field of gene sequencing, and particularly to a single-cell exon sequencing method and applications thereof. Background Art

[0002] Single-cell sequencing refers to a new technology that performs high-throughput sequencing analysis of multiple omics, including the genome, transcriptome, and epigenome, at the single-cell level. The application of this technology makes it possible to interpret the genetic structure and gene expression status of individual cells and to decipher intercellular heterogeneity. It has been widely used in research in developmental biology, immunity, oncology, and other fields, and has produced a large number of high-quality research and scientific research results. Single-cell exome sequencing has better sensitivity and resolution for detecting gene mutations and copy number variations. Compared with tissue block exome sequencing results, single-cell exome sequencing can not only detect genomic changes such as higher frequency mutations in some tumors, but also has important significance for studying tumor heterogeneity and clonal evolution. This provides unprecedented resolution for identifying cancer characteristics, which is of great significance in the fields of cancer detection, diagnosis, and targeted therapy. It has the potential to reveal the mechanisms of cancer occurrence and development and discover drug targets.

[0003] Due to the high heterogeneity of tumor tissue, sequencing based on tissue blocks can mask low-frequency mutations. Furthermore, whole-genome sequencing is expensive and generates large amounts of data. Mutations associated with human diseases are generally concentrated in less than 5% of exonic regions. Furthermore, single-cell DNA (approximately 6 pg) does not meet the minimum sample volume requirement for next-generation sequencing, necessitating genome amplification of individual cells. For single-cell exome sequencing, we use MDA to amplify the entire genome of a single cell. The amplified products are then used for single-cell exome library construction and sequencing to obtain exonic mutation information. Because amplification bias can lead not only to copy number variation but also to amplification bias of single nucleotide variants, selecting samples with high amplification uniformity is crucial for obtaining accurate mutation information. In our experiments, we performed amplification uniformity testing on MDA products and selected samples with high amplification uniformity for subsequent experiments, significantly improving all metrics, including amplified gene coverage and fidelity, and enhancing the accuracy of single-cell amplification and sequencing.

[0004] In summary, the single-cell exome sequencing method has the advantages of high activity, simple operation, improved success rate of single-cell exome library construction, and reduced downstream experimental costs. It has greatly accelerated the application of single-cell exome sequencing in the study of tumor evolution mechanisms, precise cancer typing, tumor resistance mechanisms, and efficacy prediction. Technical issues

[0005] Because tumor tissue is highly heterogeneous, tissue block-based sequencing can mask low-frequency mutations. Whole-genome sequencing is also expensive and generates large amounts of data, while mutations associated with human diseases are generally concentrated in less than 5% of the exon region. Furthermore, single-cell DNA (approximately 6 pg) does not meet the minimum sample volume requirement for next-generation sequencing. Technical Solutions

[0006] This invention provides a method for single-cell tumor exome sequencing and its application. This invention aims to address, to a certain extent, a technical problem in the related art: how to use a simple and convenient method to isolate and sequence the exomes of single cells from ex vivo tumor tissue. To this end, the inventors have developed a simple method for obtaining and sequencing the exomes of single cells from ex vivo tumor tissue samples, enabling the wider application of single-cell sequencing technology in studying the microenvironment of ex vivo tumor tissue and reducing costs.

[0007] First aspect:

[0008] The present invention provides a method for single-cell exon sequencing of a tumor, comprising the following steps:

[0009] S1, tumor single cell sorting;

[0010] S2. Perform MDA amplification on the sorted tumor single cells to obtain a genomic library;

[0011] 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 in each single-cell MDA product to determine whether the genome is amplified uniformly.

[0012] S4. gDNA library preparation: The amplified DNA is fragmented into 150-200 bp fragments, and the gDNA library is obtained by end repair, tailing, adding adapters, and amplification;

[0013] S5. Exon capture and library construction: The prepared gDNA library is hybridized with specific probes to capture specific regions, and then PCR amplification and index tags are added to obtain the exon library;

[0014] S6. Sequence the exon library.

[0015] Step S3 is specifically as follows:

[0016] Multiple loci located on different chromosomes were selected and corresponding amplification primers were designed. qPCR was used to detect the amplification of multiple loci in each single-cell MDA product to determine whether the genome was amplified uniformly. The positive control was gDNA extracted from tumor tissue, and the negative control was nuclease-free water.

[0017] Relative uniformity value (RUV) is calculated as follows:

[0018] Relative uniformity value (RUV) = 2-(Cti-Ct0)

[0019] Where Cti represents the Ct value of the 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 the site is relatively uniform. Only single-cell MDA products with RUV values ​​between 0.25 and 4 for at least 75% of the sites can be considered as samples that meet the conditions for the next step of exon library construction.

[0020] The amplification primers include multiple pairs of the following primer pairs:

[0021] Forward primer (F) Reverse primer (R) SEQ ID NO.1SEQ ID NO.2SEQ ID NO.3SEQ ID NO.4SEQ ID NO.5SEQ ID NO.6SEQ ID NO.7SEQ ID NO.8SEQ ID NO.9SEQ ID NO.10SEQ ID NO.11SEQ ID NO.12SEQ ID NO.13SEQ ID NO.14SEQ ID NO.15SEQ ID NO.16.

[0022] In step S5, the specific probe is SureSelect XT Human All Exon V6.

[0023] Step S1 includes the following steps:

[0024] Step 1: Sample preparation: Dissociate the tumor tissue to be sorted into a suspension, stain it with a cell viability assay reagent to detect the proportion of live cells and the total number of cells, and pass it through a cell strainer to remove double cells and debris to prepare a highly active single-cell suspension.

[0025] 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 well plate in the collection position;

[0026] Step 3: Set the single cell titration sorting parameters and adjust the titration position to the center of the bottom of the well plate;

[0027] Step 4, single cell titration sorting: Use a capillary to absorb cells, use an imaging system to observe the state of the droplets, observe the cells passing through the liquid flow, adjust the various parameters of the cells in the sample, adjust the sorted cell particle size to capture active cells, and finally form droplets with a volume of 450-600 μL, titrate them into each well of the well plate, with 1 cell per well and eliminate empty wells and multi-cell wells based on the photos of single cells retained in the nozzle area; obtain single cells.

[0028] The single-cell titration and separation system is the CellenONE X1 system.

[0029] In step 1, the tissue to be sorted is dissociated into a cell suspension using collagenase IV.

[0030] Preferably, in step 1, the tumor tissue derived from the tumor patient to be sorted is dissociated into a single cell suspension using collagenase IV, filtered using a 70 μm cell strainer, and then counted using a cell counter.

[0031] In step 2, the orifice plate is a low adsorption orifice plate, and each well is pre-cooled with calcium-free magnesium that can cover the bottom of the orifice plate.

[0032] of PBS solution.

[0033] Preferably, in step 2, the well plate is a low-skirt 96-well plate, and 1.5 μL of pre-cooled calcium- and magnesium-free PBS solution is added to each well in advance.

[0034] The application of the exon sequencing method in accurate tumor typing, tumor resistance mechanism research, clonal evolution research, and efficacy prediction research also falls within the scope of protection of the present invention.

[0035] The MDA amplification was performed using the REPLI-g® Single Cell Kit (24).

[0036] Preferably, the complete culture medium is DMEM culture medium containing 10% FBS.

[0037] Specifically, in step 1, the DNA is ultrasonically sheared using Covaris M220.

[0038] The exon library was constructed using the SureSelectXT Target Enrichment System for the Illumina Platform.

[0039] 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, and the cell particle size range is adjusted to 5-30 μm (the diameter range of the selected image batch sorting is 10-40 μm). 10 μL of cell suspension is absorbed by the PDC and then titrated and injected into the bottom of each well of the 96-well plate.

[0040] The application of the exon sequencing method in accurate tumor typing, tumor resistance mechanism research, clonal evolution research, and efficacy prediction research also falls within the scope of protection of the present invention. Beneficial effects

[0041] 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 studies, improve the resolution of tumor heterogeneity, and can be used to identify and study structural variations within disease-associated coding regions, helping to analyze clonal evolution (corresponding to the comparison of mutations and classification detected by tissue tumor exome sequencing and mutations and classification detected by exome sequencing of single tumor cells in the following text);

[0042] 2. Exploration of intratumor heterogeneity and clonal evolution is often limited to the level of tumor populations. Currently, increasing research focuses on studying drug resistance and recurrence in cancer patients. Therefore, exome analysis at the level of tumor populations is insufficient to meet this need. The high-resolution single-cell exome analysis technology provided by this invention provides a powerful tool for answering these questions. (This corresponds to the comparison of tissue tumor exome sequencing and single-cell exome sequencing described later.)

[0043] Due to the redundancy of whole-genome sequencing data, a large amount of data is present that is not currently needed for research. This massive amount of information often obscures low-frequency mutations carried by a small number of cells. However, single-cell exome sequencing, due to its smaller detection region (exome sequences account for approximately 1% of the human genome), allows us to achieve a depth more than ten times that of whole-genome sequencing (typically measuring 90G), even if we only sequence 15G of exome data. This significantly improves research efficiency, as shown in Table 1.

[0044] Table 1 Comparison of whole-genome sequencing and whole-exome sequencing

[0045] Sequencing technology can cover the entire genome. Sequencing cost. Required genome coverage. Whole genome sequencing >95%. High: typically >30X. Whole exome sequencing ~5% (typically coding regions). Low: typically >30X~100X.

[0046] 3. It can more efficiently discover low-frequency mutations and rare variants, increasing the average number of mutations that can be discovered from 13 in the whole exome to 533.5 in the single-cell exome.

[0047] 4. Whole genome sequencing is relatively expensive for a single sample, while single-cell exome sequencing significantly reduces sequencing costs and significantly reduces overall costs;

[0048] 5. Because amplification can produce bias, which not only leads to deviations in copy number variation but also in single nucleotide variation amplification, selecting samples with high amplification uniformity is the only way to guarantee true mutation information. During our experiment, we used the isothermal amplification technology MDA to amplify the whole genome of a single cell and verified the amplification uniformity of the MDA product through experiments. The eight selected points were also designed (to be representative of the genome). Only samples that passed the amplification uniformity experiment and had high genomic amplification uniformity were allowed to proceed with subsequent experiments. The above-mentioned unique experimental invention design has significantly improved all indicators such as amplified gene coverage and fidelity, making single-cell amplification and sequencing more accurate and guaranteeing high-quality results.

[0049] 6. Using the Agilent SureSelectXT Human Exon V6 Capture Probe, the target size is 58Mb, with excellent coverage and consistency across the target region, 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%, OMIM - cds 99%, has been unanimously recognized by the industry in the fields of genetic diseases and tumors, becoming a classic product in the field of second-generation sequencing capture, and has also been recognized by ICGC.

[0050] 7. Compared with flow cytometry, the Cellen ONE X1 single cell titration system requires a starting amount of 10 5 cells / ml, requiring a small starting cell quantity, which facilitates the sorting and enrichment of rare cells. Furthermore, flexible sorting minimizes cell damage, resulting in a high single cell rate and high activity after sorting. Visualization with image retention facilitates traceability. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0052] FIG1 is a flowchart of single-cell exon sequencing of the present invention;

[0053] FIG2 is a schematic diagram of the quality inspection results of the single cell exon library construction according to Example 1 of the present invention;

[0054] FIG3 shows the mutations detected after single-cell exome sequencing in the embodiment;

[0055] Figure 4 is a schematic diagram of the quality inspection results of the exon library constructed from tissues of Comparative Example 1;

[0056] Figure 5 is a diagram of the exon sequencing of hepatoblastoma sample tissue in comparative example 1 for mutation detection and classification;

[0057] FIG6 shows the CNV detection results of tissue and single-cell exon sequencing of the same patient in Example and Comparative Example 1. Best Mode for Carrying Out the Invention

[0058] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0059] The present invention proposes a simple and efficient method for isolating single cells from ex vivo tumor tissue and performing single-cell exome sequencing. This method is applied to studies of tumor heterogeneity, tumor clonal evolution, and drug screening at the single-cell level.

[0060] The present invention is applicable to single-cell exome sequencing in fresh ex vivo tumor tissues from tumor patients. The contents involved mainly include six parts: tissue dissociation, single-cell isolation and acquisition quality inspection, whole genome amplification, amplification uniformity experiment, exon capture, library construction and on-machine sequencing, as shown in Figure 1.

[0061] Single cells were isolated and obtained using a Countess II FL Automated Cell Counter, and the concentration and dissociation effect were detected under a trypan blue light microscope.

[0062] The specific probe was Agilent SureSelect XT Human All Exon V6.

[0063] MDA amplification was performed using the REPLI-g® Single Cell Kit.

[0064] DNA was sheared by ultrasonication using Covaris M220.

[0065] Exome library construction was performed using the Agilent SureSelectXT Target Enrichment System for the Illumina Platform.

[0066] 1. Single cell sample preparation

[0067] Liver cancer tumor tissue and adjacent adjacent tissue were dissociated to obtain a single-cell suspension, which was then sorted using the CellenOne X1 single-cell titration system to obtain single cells.

[0068] The specific steps are as follows:

[0069] ① Tissue dissociation: Rinse the tumor tissue 2-3 times with pre-cooled DPBS and cut about 3mm 3 Tumor tissue was placed in a 2-ml centrifuge tube, and 500 μl of 0.05% collagenase IV was added. The tissue was minced on ice and incubated in an incubator at 37°C, rotating at 60 rpm / min for 8 minutes. Cells were filtered using a 70 μm cell strainer to obtain a cell suspension. 3 ml of red blood cell lysis buffer was added to the suspension, and the suspension was incubated at room temperature for 5 minutes. The suspension was centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. 5 ml of pre-chilled calcium- and magnesium-free PBS was added, mixed by pipetting, and centrifuged using the above conditions. The supernatant was discarded. 500 μl of DMEM containing 10% FBS was added for resuspending. 10 μl of the cell suspension was stained with an equal volume of trypan blue, and cell number and viability were determined using a Countess II FL Automated Cell Counter.

[0070] 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 in advance. The cell suspension obtained above was loaded into the sample well of the CellenONE X1 single-cell titration system for sorting. The titration position was adjusted to the center of the collection well 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). With the help of an imaging system, the droplet state was observed. By adjusting the parameters such as the diameter and roundness of the cells in the sample, the cell size for sorting was adjusted (10-40 μm) to accurately capture active cells. Finally, a 0.1 μL droplet volume of (450-600 pL) was formed and titrated into each well, with one cell per well. Empty wells and wells with multiple cells were eliminated based on the photos of single cells retained in the nozzle area, and single cells were obtained by sorting.

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

[0072] Multiple displacement amplification (MDA) of single cells was performed using the REPLI-g® Single Cell Kit (157046333, Qiagen).

[0073] Cell lysis: Add 0.5ul Exo-resistant random primer and 1.5ul Lysis buffer (400 mM KOH, 100 mM DTT, 10mM EDTA) to the 3ul centrifuge tube containing the single cells sorted above. Centrifuge and place on ice for 10 minutes. Add 1.5ul Stop solution (400 mM HCl Tris-HCl and 600 mM Tris-HCl 1M, pH 7.5), centrifuge and place on ice for 2 minutes.

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

[0075] Purification: Add 37.8 μl of AMPureXP purification beads to 21 μl of amplified product for purification, and quantify the purified product using Qubit 4.

[0076] Genome amplification uniformity test.

[0077] MDA products with higher concentrations than the negative control were tested for amplification uniformity to verify the uniformity of amplification of each product. The principle is to select eight loci located on different chromosomes and design corresponding amplification primers (see Table 2 for loci and primer information). qPCR is then used to detect the amplification effect of these eight loci in each single-cell MDA product to determine whether the genome is amplified uniformly (the positive control is gDNA derived from the patient's tissue, and the negative control is enzyme-free water).

[0078] The relative uniformity value (RUV) is calculated as follows:

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

[0080] Where Cti represents the Ct value of the locus in sample i, and Ct0 represents the Ct value of the same locus in unamplified genomic DNA. The RUV of unamplified genomic DNA should be 1. An RUV close to 1 indicates relatively uniform amplification of the locus. Single-cell MDA products are considered eligible for exome library construction only if at least 6 of the 8 loci have RUV values ​​between 0.25 and 4.

[0081] Table 2: 8 sites for amplification uniformity testing and their corresponding qPCR primers

[0082] Single-cell exome library construction and sequencing

[0083] Exon library construction was performed using the Agilent SureSelectXT Target Enrichment System for the Illumina Platform. The specific steps are as follows:

[0084] Genomic DNA fragmentation: 1.5 g of amplified MDA product that passed homogeneity testing was diluted to 65 μl with low TE buffer. The fragments were fragmented into 150-200 bp fragments using a Covaris M220 ultrasonic disruptor at a peak power of 75, a duty factor of 20%, 200 cycles, a time of 250 s, and a temperature of 18-22°C. The fragments were purified using Beckman AMPure XP to obtain 24 μl of purified product.

[0085] End Repair: Add 26 µl of End Repair Mix (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 by pipetting. Centrifuge and incubate in a PCR machine at 20°C for 30 minutes. Store at 4°C. After incubation, purify using AMPure XP to obtain 15 µl of purified product.

[0086] 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 15 µl of the above product. Mix thoroughly by pipetting. Centrifuge and incubate in a PCR machine at 37°C for 30 minutes. Store at 4°C. After incubation, purify using AMPure XP to obtain 6.5 µl of purified product.

[0087] 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 6.5 µl product. Mix by pipetting, centrifuge, and incubate in a PCR machine at 20°C for 15 minutes. Store at 4°C. After incubation, purify using Beckman AMPure XP to obtain 16 µl of purified product.

[0088] Amplification: 7.5 μl of the above product was added to 17.5 μl of PCR reaction mix (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 by pipetting, centrifuge, and incubate in a PCR instrument: 98°C for 2 min, followed by 4-6 cycles of 98°C for 30 s, 65°C for 30 s, and 72°C for 1 min, followed by 72°C for 10 min, and then store at 4°C. After incubation, 15 μl of purified product was obtained using Beckman AMPure XP. The gDNA library concentration and fragment distribution were measured using a Thermo Fisher Scientific Qubit 4.0 and an Agilent 2100 Bioanalyzer, respectively.

[0089] Hybridization capture: a. Based on the above product concentration, prepare 1.7 μl of dilution solution with a concentration of 221 ng / μl for each library.

[0090] b. Prepare Hybridization Buffer: (3.315µl SureSelect Hyb 1, 0.135µl SureSelect Hyb 2, 1.325µl ​​SureSelect Hyb 3, 1.725µl SureSelect Hyb 4) and store at room temperature until use. c. Prepare SureSelect Blocking Buffer: (1.25µl SureSelect Indexing Block 1, 1.25µl SureSelect Block 2, 0.3µl SureSelect ILM Indexing Block 3). d. Add 2.8µl SureSelect Blocking Buffer to each 1.7µl gDNA library aliquot, pipette to mix thoroughly, and incubate in a PCR machine at 95°C for 5 minutes, then at 65°C for ≥5 minutes. e. Prepare Hybridization Mix: (6.5µl Hybridization Buffer (from step b), 1µl 25% RNase Block Solution, 2.5µl Probe (designed for ≥3 Mb)). f. Add 10 µL of the prepared hybridization mix to each sample from step d and incubate in a PCR instrument at 105°C with a heated lid and 65°C for 24 hours. g. Prepare streptavidin-coated magnetic beads: Resuspend Dynabeads MyOne Streptavidin T1 magnetic beads. Transfer 25 µL of beads to a new microcentrifuge tube for each sample. Wash three times with 200 µL of SureSelect Binding Buffer. Resuspend in 100 µL of the streptavidin beads prepared in step g. Transfer the sample from step f to the 100 µL streptavidin beads prepared in step g, mix by pipetting, and incubate at room temperature at 1400–1800 rpm for 30 minutes. After incubation, place on a magnetic rack, allow the solution to clear, and discard the supernatant. i. Resuspend in 100 µL of SureSelect Wash Buffer 1, incubate at room temperature for 15 minutes, place on a magnetic rack, allow the solution to clear, and discard the supernatant. j. Wash three times with 100 μl of Wash Buffer 2 preheated at 65°C, then elute with 15 μl of Nuclease-Free Water. Place on ice until ready to use.

[0091] ⑦ Capture Library Amplification and Indexing: For each sample, prepare 15.5µL of PCR reaction mix (9.25µL Nuclease-Free Water, 5µL 5× Herculase II Reaction Buffer, 0.5µL Herculase II Fusion DNA Polymerase, 0.25µL 100mM dNTP Mix, 0.5µL SureSelect ILM Indexing Post-Capture Forward PCR Primer) in a new PCR tube. Add 2.5µL of indexing primer and 7µL of the magnetic bead-based capture library (step j in ⑥) to each tube. Mix by pipetting and place in a PCR instrument and perform amplification: 98°C for 2 minutes, followed by 12 cycles of 98°C for 30 seconds, 57°C for 30 seconds, 72°C for 1 minute, 72°C for 10 minutes, and then store at 4°C. After incubation, purify using Beckman AMPure XP to obtain 15µL of exon library product.

[0092] ⑧ The library concentration was quantified using the Thermo Fisher Scientific Qubit 4, and the prepared exon library was quality checked for abundance and fragment distribution using the Agilent 2100 Bioanalyzer (as shown in Figure 2). Exon libraries that met the standards were selected for PE150 sequencing on the Illumina NovaSeq 6000 sequencing platform, generating 10 Gb of data.

[0093] 5. Test results

[0094] Figure 3 shows the mutations detected after single-cell exome sequencing of the patient's four tumors, indicating a greater number and variety of mutations than exome sequencing of tissue blocks, with an average of 533.5 mutations detected in each tumor cell. Modes for Carrying Out the Invention

[0095] Comparative Example 1 Exon Sequencing of Tissue Sources

[0096] The difference between this comparative example and Example 1 is that this comparative example is based on sequencing of an exon library constructed from DNA extracted from tissue, while Example 1 is based on sequencing of an exon library constructed from DNA of a single cell.

[0097] (1) Genomic DNA extraction

[0098] Genomic DNA was extracted from the cancer and adjacent tissues of the same origin as in Example 1 using the QIAamp DNA Mini Kit (51306, Qiagen), and the obtained genomic DNA was quantified using a Thermo Fisher Scientific Qubit 4 fluorometer.

[0099] (2) Exome library construction

[0100] The specific steps are the same as the exon library construction steps in the above embodiment.

[0101] (3) Exome library quantification and quality control

[0102] The prepared exon library was quality-checked for abundance and fragment distribution using the Agilent 2100 Bioanalyzer, as shown in Figure 4. Only those that met the standards could be sequenced.

[0103] (4) Sequencing

[0104] PE150 sequencing was performed on the Illumina NovaSeq 6000 sequencing platform.

[0105] (5) Data Analysis

[0106] The raw sequencing data are quality controlled and screened, compared with the reference genome and germline mutations are removed, and SNP and CNV identification, filtering and annotation are performed.

[0107] The main filtering conditions are: (1) selecting sites that are filtered as PASS by the mutect2 tool; (2) the sequencing depth of the mutation site is greater than 10; (3) the site mutation frequency is greater than 0.05.

[0108] (6) Test results

[0109] Analysis revealed 13 mutations, primarily missense, detected in the exon regions of this patient (CTNNB1, LNP1, DIAPH1, NOTCH4, GABRR1, SLC25A45, PMEL, CHFR, UBN1, MYO18A, CNOT3, EIF3D, and GDI1). The mutation types and classifications are shown in Figure 5.

[0110] In this comparative example, single-cell and tissue block exome sequencing showed no significant differences in the distribution of mutations detected across different classifications. However, exome sequencing of four single tumor cells (Figure 3) detected tens of times more mutations overall than the corresponding exome sequencing (Figure 5). The discovery of these high-resolution tumor mutation profiles will facilitate prognostic analysis and provide guidance for medication use.

[0111] On the other hand, single-cell exome sequencing also demonstrates higher detection efficiency for tumor copy number variation (CNV) detection than traditional tissue block-based sequencing. As shown in Figure 6, the exome sequencing results of a patient's single tumor cell show that both copy number loss and copy number amplification detection are better than those of tissue block exome sequencing. Compared with the exome sequencing results from the same patient, it also has very significant CNV detection performance. In addition, tumor single-cell exome sequencing can also reveal CNV heterogeneity between tumor cells from the same patient, which is conducive to the study of patient tumor heterogeneity and clonal evolution.

[0112] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention. Industrial Applicability

[0113] Genomic research: Single-cell exome sequencing can be used to study the genomes of individual cells, revealing genetic variation and gene expression differences between cells. This is of great significance for understanding human development, disease mechanisms, and the functional differences between different cell types in an organism.

[0114] Cancer Research: Single-cell exome sequencing can help identify genetic variations and mutations in cancer cells, revealing the heterogeneity and evolutionary processes of different cancer cell populations. This has guiding significance for early cancer diagnosis, the formulation of treatment strategies, and the development of anticancer drugs.

[0115] Immunology Research: Single-cell exome sequencing can be used to study the phenotypic and functional differences of different immune cell subsets in the immune system. This helps to reveal the developmental process of immune cells, the mechanisms of response to infection and vaccination, and the pathogenesis of autoimmune diseases.

[0116] Bioengineering: Single-cell exome sequencing can be used to screen, study, and improve microbial strains, including fermentation strains and strains that produce bioactive substances. This will help improve the yield and efficiency of microbial strains, leading to widespread applications in food, medicine, and other fields.

[0117] Agricultural Research: Single-cell exome sequencing can help study gene expression and function in crops, revealing genes related to their adaptability and yield under different environmental conditions. This has important implications for crop breeding and agricultural production improvements, potentially increasing crop stress resistance and yield. Sequence Listing Free Content

[0118] Type your sequence listing free description paragraph here.

Claims

1. A tumor single cell exome sequencing method, It is characterized in that The steps include: S1, tumor single cell sorting; S2, performing MDA amplification on the sorted tumor single cells to obtain a genomic library; S3. Detect the uniformity of MDA amplification products. Select multiple sites located on different chromosomes and design corresponding amplification primers. Detect the amplification effects of these multiple sites of each single-cell MDA product by qPCR to determine whether the genome is amplified uniformly. S4. gDNA library preparation: The amplified DNA was broken into 150-200 bp fragments, and the gDNA library was obtained by end repair, tailing, adding adapters, and amplification; S5, exon capture and library construction: hybridize the prepared gDNA library with specific probes, capture specific regions, and then amplify by PCR to add index tags to obtain the exon library; S6. Sequence the exon library.

2. The exon sequencing method according to claim 1, It is characterized in that Step S3 is specifically as follows: Select multiple sites on different chromosomes and design corresponding amplification primers. Use qPCR to detect the amplification effects of multiple sites of each single-cell MDA product to determine whether the genome is amplified uniformly. The positive control is gDNA extracted from tumor tissue, and the negative control is enzyme-free water. Relative uniformity value (RUV) is calculated as follows: Relative uniformity value (RUV) = 2 -(Cti-Ct0) Among them, Cti represents the Ct value of the 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; RUV close to 1 indicates that the amplification of the site is relatively uniform; the single-cell MDA products with RUV values ​​between 0.25-4 at least at 6 of the 8 sites can be regarded as qualified samples for the next step of exon library construction.

3. The exon sequencing method according to claim 2, It is characterized in that The amplification primers include multiple pairs of the following primer pairs: Forward primer (F) Reverse primer (R) SEQ ID NO: 1SEQ ID NO: 2SEQ ID NO: 3SEQ ID NO: 4SEQ ID NO: 5SEQ ID NO: 6SEQ ID NO: 7SEQ ID NO: 8SEQ ID NO: 9SEQ ID NO: 10SEQ ID NO: 11SEQ ID NO: 12SEQ ID NO: 13SEQ ID NO: 14SEQ ID NO: 15SEQ ID NO:

16.

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

5. The exon sequencing method according to claim 1, It is characterized in that Step S1 includes 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 in step 1 to the starting sample well, and then place the well 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 sorting: use a capillary to absorb cells, use an imaging system to observe the state of droplets, observe the cells passing through the liquid flow, adjust the various parameters of the cells in the sample, adjust the size of the sorted cells to capture active cells, and finally form droplets with a volume of 450-600 p L, titrate into each well of the well plate, with 1 cell per well, and remove empty wells and multi-cell wells based on the photos of single cells retained in the nozzle area; Obtain single cells.

6. The exon sequencing method according to claim 5, It is characterized in that In step 1, the cells to be sorted are dissociated into a cell suspension using collagenase IV.

7. The exon sequencing method according to claim 5, It is 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 is added to each well in advance to cover the bottom of the well plate.

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

9. Application of the exon sequencing method as described in any one of claims 1 to 8 in precise tumor typing, tumor resistance mechanism research, clonal evolution research, and efficacy prediction research.

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