Library building method and system for increasing full-length target gene yield by coupling 10*single cell sequencing and application
By employing a two-step amplification strategy involving asymmetric and nested amplification, the problems of low target gene amplification efficiency and poor full-length coverage in 10× single-cell sequencing are solved. This strategy enables efficient and specific full-length amplification of target genes, making it suitable for the detection of genes with low expression levels and for genetic and transcriptional variation studies at the single-cell level.
Patent Information
- Application Number
- CN202511623710.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-01-09
AI Technical Summary
Existing 10× single-cell sequencing technologies suffer from low target gene amplification efficiency, poor full-length coverage, and poor reproducibility, making it difficult to achieve efficient, specific, and full-length target gene library construction.
A two-step amplification strategy combining asymmetric and nested amplification was adopted. Through extreme primer design and primer combination, the target gene template was preferentially enriched and full-length amplification with high fidelity was performed.
It improves the success rate and signal intensity of target gene amplification, achieves full-length target gene coverage, supports the accurate identification of events such as mutation, splicing, and fusion, and maintains compatibility with 10× sequencing systems.
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Figure CN121294614A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a library construction method, system, and application for increasing the yield of full-length target genes by coupling 10× single-cell sequencing. Background Technology
[0002] Single-cell sequencing, particularly high-throughput single-cell transcriptome sequencing (10× scRNA-seq) based on the 10× Genomics platform, has become an important tool for analyzing cellular heterogeneity, identifying rare cell subpopulations, and studying tumor evolution. However, the 10× platform primarily uses short-read sequencing, and its library structure is designed for 3' or 5' tag sequencing of the transcriptome, making it impossible to directly obtain the full-length sequence information of target genes. In downstream studies, such as single-cell mutation detection, allele-specific analysis, or splice variant identification, researchers often need to amplify specific target genes, especially those with low expression levels, based on the 10× library preparation products and then construct libraries accordingly.
[0003] Currently used target gene amplification strategies have the following shortcomings: 1) Low amplification efficiency: The proportion of target template in 10× products is extremely low, and the target gene primers compete severely with 10× adapters, resulting in insufficient amplification yield; 2) Poor full-length coverage: Single-step amplification is prone to bias, making it difficult to obtain complete reads of the target gene; 3) Poor reproducibility: Non-specific products and primer dimers in multiple rounds of amplification affect the quality of subsequent sequencing.
[0004] Therefore, how to achieve efficient, specific and full-length target gene library construction in the context of 10× single-cell sequencing remains a key technical bottleneck in this field. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this application proposes a library construction method coupled with 10× single-cell sequencing to increase the yield of full-length target genes. This method includes a step of enriching the target gene template through asymmetric amplification, and a step of amplifying the library through nested amplification. Specifically, the full-length target gene sequence template is enriched through asymmetric amplification, and then the full-length target gene is amplified with high fidelity through nested amplification.
[0006] The library construction method of this application, which couples 10× single-cell sequencing to increase the yield of full-length target genes, firstly utilizes an extreme ratio of primers to achieve one-end biased amplification, thereby preferentially enriching specific template sequences of the target genes and reducing non-specific amplification. Secondly, it introduces a near-equal ratio of nested target gene primers to R1 primers to further amplify the product from the first step, achieving high-fidelity full-length amplification.
[0007] As some embodiments of this application, the target gene is a gene with low expression levels, which means that the expression level of the target gene in the cell is ≥ 7% of the average expression level of the housekeeping gene Actb.
[0008] The ratio of the expression level of the target gene with low expression level in the cell to the average expression level of the housekeeping gene Actb can be any of the following: 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
[0009] As some embodiments of this application, the step of enriching the target gene template through asymmetric amplification includes: amplifying the DNA sample using a primer combination in a ratio of target gene primer to 10× platform R1 primer of 2000:1 to 100000:1.
[0010] The ratio of the target gene primer to the 10× platform R1 primer can be selected from 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000:1, 70000:1, 80000:1, 90000:1, and 100000:1.
[0011] As some embodiments of this application, the step of obtaining the library by nested amplification includes: further amplifying the product after asymmetric amplification using a primer combination in a ratio of target gene primers to 10× platform R1 primers of 1:1 to 1:10.
[0012] The ratio of the target gene primer to the 10× platform R1 primer can be selected from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.
[0013] The method of this application solves the following problems and achieves the following effects: 1) Improves amplification success rate and signal intensity, and solves the problem of target sequence loss caused by low template amount; 2) Achieves full-length coverage of target genes, which helps to accurately identify events such as mutation, splicing, and fusion; 3) Maintains compatibility with 10× sequencing system, and the amplification products are compatible with Nanopore or PacBio long-read sequencing, realizing the coupling of single-cell transcriptome and full-length target genes.
[0014] This application also provides a single-cell sequencing method to increase the yield of full-length target genes. The single-cell sequencing method to increase the yield of target genes includes sequencing the amplified products obtained by the library construction method coupled with 10× single-cell sequencing to increase the yield of full-length target genes using Nanopore or PacBio long-read sequencing.
[0015] This application also provides a system for increasing the yield of full-length target genes when coupling 10× single-cell sequencing. The system for increasing the yield of full-length target genes when coupling 10× single-cell sequencing includes two primer combinations: an asymmetric amplification primer combination and a nested amplification primer combination. The full-length sequence template of the target gene is enriched by the asymmetric amplification primer combination, and then the full-length amplification of the target gene with high fidelity is achieved by the nested amplification primer combination.
[0016] As some embodiments of this application, the asymmetric amplification primer combination includes a target gene primer and a 10× platform R1 primer, wherein the ratio of the target gene primer to the 10× platform R1 primer is 1000:1 to 100000:1.
[0017] As some embodiments of this application, the nested amplification primer combination includes target gene primers and 10× platform R1 primers, wherein the ratio of target gene primers to 10× platform R1 primers is 1:10 to 10:1.
[0018] This application also provides the application of the system described above for increasing the yield of full-length target genes in coupling 10× single-cell sequencing in terms of increasing target gene amplification efficiency and / or increasing full-length target gene coverage and / or maintaining compatibility with 10× sequencing systems.
[0019] This application also provides the application of the system described above for increasing the yield of full-length target genes in coupling 10× single-cell sequencing in single-cell sequencing to increase the yield of target genes.
[0020] This application also provides the application of a system for increasing the yield of full-length target genes by coupling 10× single-cell sequencing in the precise identification of any of the following: gene mutations, splice variants, fusion transcripts, and single-cell variant tracking.
[0021] In summary, the method and system of this application have significant improvements over existing technologies in terms of amplification efficiency, specificity, full-length coverage, and system compatibility. They can effectively solve the problems of low efficiency, poor specificity, and incomplete coverage of target gene full-length library construction in existing 10× single-cell sequencing systems, and have broad research and application prospects.
[0022] As described above, the library preparation method, system, and application of this application for increasing the yield of full-length target genes by coupling single-cell transcriptome sequencing have the following beneficial effects: 1. This application introduces a two-step amplification strategy (asymmetric amplification and nested amplification) based on 10× single-cell library preparation products, achieving efficient, specific, and full-length amplification of target genes without damaging the 10× library structure. The success rate of target gene amplification is several times higher than that of conventional one-step amplification methods, and it is especially suitable for the detection of low-abundance genes in the 10× system. The introduction of nested amplification significantly reduces the risk of bias and fragment deletion. Through reasonable primer design and a proportional amplification strategy (1:1), complete amplification of the target gene from the 5' end to the 3' end is achieved. In summary, it improves the amplification success rate and signal intensity, and solves the problem of target sequence loss caused by low template amount.
[0023] 2. The method of this application improves amplification specificity and reproducibility: the structured two-step amplification process effectively reduces non-specific amplification and primer dimer formation; high reproducibility between batches, and the resulting library has good signal-to-noise ratio and sequence consistency; the specific amplification rate and sequencing alignment accuracy are significantly improved, enabling high-quality reconstruction of the full-length target gene sequences at the single-cell level, which were originally difficult to obtain.
[0024] 3. The method of this application is highly compatible with the 10× single-cell sequencing system: The method of this application directly uses the 10× library preparation product as a template without additional adapter modification or system change; it retains the 10× cell barcode and molecular identifier (UMI), enabling full-length source tracing and quantitative analysis of target genes at the single-cell level; it can be seamlessly integrated with the 10× cDNA library system, and the amplified products can be directly sequenced for long reads using Nanopore or PacBio, achieving coupling between single-cell transcriptome and full-length target genes.
[0025] 4. The method of this application significantly improves the resolution and application value of single-cell research: it can obtain complete target gene sequences at the single-cell scale, supporting the accurate identification of complex events such as gene mutations, splice variants, fusion transcripts, and single-cell variation tracking; it is particularly suitable for high-precision research scenarios such as tumor heterogeneity, immune receptor rearrangement, and gene editing verification; and it provides new experimental tools and data acquisition methods for the analysis of genetic variations and functions at the single-cell level.
[0026] 5. This application has significant advantages in coupling single-cell sequencing and obtaining the full-length sequence of specific target genes, especially for genes with low expression levels, significantly increasing amplification yield and improving the success rate and specificity of amplifying low-abundance target genes. Achieving full-length sequence resolution of specific genes at the single-cell level provides an efficient and reliable technical approach for genetic and transcriptional variation research at the single-cell level, and provides key technical support for precision oncology and single-cell genetics research. Attached Figure Description
[0027] Figure 1A schematic diagram illustrating the working principle of one embodiment of this application is shown; Figure 2 The comparison between the target gene amplification efficiency and the one-step amplification efficiency of the implementation scheme of Example 1 is shown; Figure 3 The comparison of target gene amplification specificity and one-step amplification specificity of the implementation scheme of Example 1 is shown; Figure 4 The expression level of the target gene in single-cell transcriptomes is shown. In cells where the target gene was successfully amplified and sequenced using the method of this application, the average expression level of the target gene was 7% of the average expression level of the housekeeping gene Actb. Figure 5 The primer ratio ranges for asymmetric amplification and nested amplification are shown. The primer ratio for Round 1 can be selected from 2000:1 to 100000:1, and the primer ratio for Round 2 can be selected from 1:1 to 1:10. Detailed Implementation
[0028] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0030] Example 1 This embodiment illustrates the working principle of this application using an implementation scheme, see details below. Figure 1 The library construction method for increasing the yield of full-length target genes in this application includes a step of enriching the target gene template through asymmetric amplification and a step of amplifying the library through nested amplification. The full-length target gene sequence template is enriched through asymmetric amplification, and then the full-length target gene is amplified with high fidelity through nested amplification. For genes with low expression levels, a primer combination of target gene primers and 10× platform R1 primers at a ratio of 10000:1 is used to amplify the DNA sample. Then, a primer combination of target gene primers and 10× platform R1 primers at a ratio of 1:1 is used to further amplify the product after asymmetric amplification. The amplification system and conditions for the two steps are as follows: 1.1 Asymmetric Amplification The reaction system is as follows:
[0031] Table 1
[0032] The reaction conditions are as follows: Table 2
[0033] After amplification, the amplification system was purified and recovered using 1×Agencourt AMPure XP magnetic beads. The specific steps are as follows: 1. Remove the Agencourt AMPure XP reagent bottle and shake to mix, to prevent the magnetic beads from settling.
[0034] 2. Add 90 μL of AMPure to each well containing 50 μL of PCR product, and mix by pipetting 10 times.
[0035] 3. Incubate at room temperature for 10 min (to improve recovery efficiency).
[0036] 4. Place the reaction vessel on the magnetic base and let it stand for 2 minutes until the solution becomes clear.
[0037] 5. Keep the reaction vessel on the magnetic base, retain 5 μL of supernatant, and carefully discard the rest.
[0038] 6. Add 200 μL of 70% ethanol to each well, incubate at room temperature for 30 s, and carefully discard the supernatant.
[0039] 7. Immediately repeat step 6.
[0040] 8. Remove the reaction vessel from the magnetic base, add 25 μL of ultrapure water (elution buffer) to each well, blow and aspirate and repeatedly wash the vessel wall to ensure that the magnetic beads enter the water, and incubate in a water bath for 10 min.
[0041] 9. Place the reaction vessel on the magnetic base for 1 minute to allow the magnetic beads to separate from the solution.
[0042] 10. Transfer the eluent to a new reaction vessel.
[0043] 1.2 Nested amplification and sample tagging The product eluted from the asymmetric amplification in 1.1 was used as a template for nested amplification. The reaction system is as follows:
[0044] Table 3
[0045] The reaction conditions are as follows: Table 4
[0046] After amplification, the nested amplification system was purified using 0.8×Agencourt AMPure XP magnetic beads. The specific steps are as follows: 1. Remove the Agencourt AMPure XP reagent bottle and shake to mix, to prevent the magnetic beads from settling.
[0047] 2. Add 40 μL of AMPure to each well containing 50 μL of PCR product, and mix by pipetting 10 times.
[0048] 3. Incubate at room temperature for 10 min (to improve recovery efficiency).
[0049] 4. Place the reaction vessel on the magnetic base and let it stand for 2 minutes until the solution becomes clear.
[0050] 5. Keep the reaction vessel on the magnetic base, retain 5 μL of supernatant, and carefully discard the rest.
[0051] 6. Add 200 μL of 70% ethanol to each well, incubate at room temperature for 30 s, and carefully discard the supernatant.
[0052] 7. Immediately repeat step 6.
[0053] 8. Remove the reaction vessel from the magnetic base, add 25 μL of ultrapure water (elution buffer) to each well, blow and aspirate and repeatedly wash the vessel wall to ensure that the magnetic beads enter the water, and incubate in a water bath for 10 min.
[0054] 9. Place the reaction vessel on the magnetic base for 1 minute to allow the magnetic beads to separate from the solution.
[0055] 1.3 Coupled with 10× single-cell sequencing The purified products from nested amplification underwent quality control, and precise quantification was performed using the Qubit dsDNA HS Assay Kit. Fragment distribution was detected using an Agilent 4200 TapeStation system. After confirming the library quality was acceptable, Nanopore or PacBio sequencing was performed. The sequencing data was coupled to the single-cell transcriptome through the following steps: 1. Cell Ranger (10×Genomics) was used to identify cells and quantify gene expression in single-cell transcriptome samples.
[0056] 2. Use MiniMap2 to align target gene reads from Nanopore / PacBio sequencing data to the reference genome.
[0057] 3. Use UMI-tools for molecular tag correction to eliminate PCR duplication.
[0058] 4. Extract the full-length sequence of the target gene containing complete cell barcodes and UMIs using a custom script.
[0059] 5. The coupling of cell barcode sequences in 10X single-cell transcriptome data and Nanopore / PacBio sequencing data was achieved.
[0060] Example 2 This embodiment illustrates the method of this application using optimized amplification conditions.
[0061] 2.1 Significantly improves target gene amplification efficiency Combination Figure 2 As can be seen, the method of this application significantly improves the amplification yield of the target gene: the target fragment length is approximately 1500 bp. Compared with the traditional one-step amplification method (i.e., F2:R1=1:1 mixing, amplification for 30 cycles), the two-step amplification method used in this invention obtained a stronger target band signal under the same sample conditions, indicating a significant improvement in the amplification efficiency of the target gene. The control group (one-step method) showed only a very weak band at 1500 bp, with a target fragment yield of approximately 0.7 ng / μL; while the sample using the two-step method of this invention formed a bright band at the same position, with a yield of up to 12.6 ng / μL, providing a sufficient template basis for subsequent library construction and sequencing.
[0062] 2.2 Significantly improves amplification specificity Combination Figure 3 As can be seen, the method of this application significantly improves the amplification specificity and yield of the target gene. The expected amplified fragment length is approximately 1500 bp. It can be observed that the traditional one-step amplification method produces a weak target band at 1500 bp, accompanied by multiple non-specific bands and an unclear main band; while the two-step amplification method of this invention yields a single, clear target band with accurate positioning and no obvious non-specific bands, indicating a significant improvement in amplification specificity and purity. Quantitative results show that the target fragment yield of the one-step method in this sample was only 1.2 ng / μL, while the two-step method of this invention can achieve 8.3 ng / μL, providing a higher quality template for subsequent full-length library construction and sequencing.
[0063] 2.3 Confirm the effect of the target gene expression level on the effective amplification of the target gene in the method of this application. Different low expression levels of target genes were selected, and the effect of target gene expression levels on effective target gene amplification was detected using the same method as in Example 1. The results are shown in [Figure 1]. Figure 4 ,from Figure 4As can be seen, the average expression level of the target gene in the single-cell transcriptome, in cells where the target gene was successfully amplified and sequenced using the method of this application, is 7% of the average expression level of the housekeeping gene Actb. In other words, even if the expression level of the target gene in the cell is as low as 7% of the average expression level of the housekeeping gene Actb, it can still be effectively amplified using the method of this application.
[0064] 2.4 To explore the effective range of the ratio of target gene primers to 10× platform R1 primers in asymmetric and nested amplification. To explore the range within which the ratio of target gene primers to 10× platform R1 primers in the two-step amplification method of this application can be expanded while still achieving effective amplification, the same method as in Example 1 was used to adjust the ratio of target gene primers to 10× platform R1 primers in the two-step amplification. The detection results are shown in [Figure 1]. Figure 5 .from Figure 5 As can be seen from A, in asymmetric amplification, a target gene primer to 10× platform R1 primer ratio of 2000:1 to 100000:1 can achieve effective amplification of the target gene. Figure 5 B shows that in nested amplification, a ratio of 1:1 to 1:10 between the target gene primer and the 10× platform R1 primer can effectively amplify the target gene.
[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A library preparation method for increasing full-length target gene yield coupled with 10x single-cell sequencing, characterized in that, The library construction method for increasing full-length target gene yield coupled with 10x single-cell sequencing comprises a step of asymmetric amplification for enriching target gene templates, and a step of nested amplification for obtaining amplified library. The asymmetric amplification enriches full-length sequence templates of the target gene, and then the step of nested amplification realizes full-length amplification of the target gene with high fidelity.
2. The library preparation method of increasing full-length target gene yield for coupled 10x single-cell sequencing of claim 1, wherein, The target gene is a low-expression gene, which refers to an expression level of the target gene in a cell being greater than or equal to 7% of an average expression level of a housekeeping gene Actb gene.
3. The library preparation method of increasing full-length target gene yield coupled with 10x single-cell sequencing of claim 1, wherein, The step of asymmetric amplification for enriching target gene templates comprises using a primer combination of a target gene primer and a 10x platform R1 primer with a ratio of 1000:1 to 100000:
1. The step of nested amplification for obtaining amplified library comprises using a primer combination of a target gene primer and a 10x platform R1 primer with a ratio of 1:10 to 10:1 to further amplify the product after asymmetric amplification.
4. A single-cell sequencing method for increasing the yield of a full-length target gene, characterized by, The single-cell sequencing method for increasing target gene yield comprises using Nanopore or PacBio long-read sequencing to sequence the amplified product obtained by the library construction method for increasing full-length target gene yield coupled with 10x single-cell sequencing according to any one of claims 1 to 3.
5. A system for coupling 10x single cell sequencing for increased full-length target gene yield, comprising, The system for increasing full-length target gene yield coupled with 10x single-cell sequencing comprises two primer combinations: an asymmetric amplification primer combination and a nested amplification primer combination; the asymmetric amplification primer combination enriches full-length sequence templates of the target gene, and then the nested amplification primer combination realizes full-length amplification of the target gene with high fidelity.
6. The system for coupling 10x single cell sequencing increasing full-length target gene yield of claim 5, wherein, The asymmetric amplification primer combination comprises a target gene primer and a 10x platform R1 primer, wherein the ratio of the target gene primer to the 10x platform R1 primer is 2000:1 to 100000:
1.
7. The system for coupling 10x single cell sequencing of claim 1, wherein, The nested amplification primer combination comprises a target gene primer and a 10x platform R1 primer, wherein the ratio of the target gene primer to the 10x platform R1 primer is 1:1 to 1:
10.
8. The system for increasing full-length target gene yield coupled with 10x single-cell sequencing according to any one of claims 5 to 7 is applied to increasing target gene amplification efficiency and / or increasing target gene full-length coverage and / or maintaining compatibility with the 10x sequencing system.
9. The system for increasing full-length target gene yield coupled with 10x single-cell sequencing according to any one of claims 5 to 7 is applied to the single-cell sequencing method for increasing target gene yield.
10. The system for increasing full-length target gene yield coupled with 10x single-cell sequencing according to any one of claims 5 to 7 is applied to accurate identification of any one of gene mutation, splice variant, fusion transcript, and single-cell variant tracking.