Primer and kit for single cell whole genome amplification

Abstract

The invention provides a primer and a kit for single cell whole genome amplification, and belongs to the technical field of molecular biology. According to the invention, dUTP deoxyuracil nucleotide is used for replacing conventional dTTP for the last basic group of the fixed sequence at the 5'end of the primer, and the primer can be specifically cut off subsequently according to the site, so that the interference of the primer sequence on sequencing is reduced. Meanwhile, compared with commercially available products, the kit comprising the primer is designed, the CV value of the kit is reduced by 63.60%, the kit has good amplification product uniformity and low base preference, the comparison rate in an SE50 sequencing mode is as high as 97.25%, no primer residue interference exists in the amplification process, and the kit is suitable for high-precision single cell sequencing analysis.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to primers and a kit for single-cell whole-genome amplification. Background Technology

[0002] In recent years, whole genome amplification (WGA) technology has been widely used in monogenomics research. Among the many WGA methods, degenerate oligonucleotide primer PCR (degenerate oligonucleotide primer polymerase chain reaction) has shown significant application value in preimplantation genetic screening due to its standardized operation process and low equipment dependence. However, existing DOP-PCR technology has revealed significant technical shortcomings in rapid detection, the core problem stemming from the inherent limitations of primer design.

[0003] Traditional DOP-PCR primers typically employ a three-segment structure of "fixed sequence - random degenerate sequence - fixed sequence". For example, a fixed sequence of 18-22 nucleotides (nt) is ligated to a random 6nt sequence, followed by ligation of 2-6nt fixed or degenerate nucleotides, resulting in a total primer length generally exceeding 25nt. While this primer design ensures annealing stability, it presents insurmountable technical bottlenecks during amplification: 1) The amplification products inevitably carry fixed primer sequences at both ends, with an average of more than 22 base pairs (bp) remaining at each end. When performing high-throughput sequencing, about 15-20% of the effective sequencing read length needs to be used to cover non-target primer regions, and this can even reach 40%-50% for short read lengths, reducing the effective utilization rate of direct data; 2) The primer residue sequences form chimeric reads with the genome sequence, causing alignment errors during NGS data analysis, which are more severe with shorter read lengths; 3) In time-sensitive scenarios such as preimplantation genetic screening (PGT), the conventional PE150 sequencing (paired-end 150bp) mode takes one to two days for the sequencing step alone, while the traditional DOP-PCR primer design is somewhat dependent on the sequencing read length, requiring the use of longer read lengths for sequencing. Using short reads (such as SE50, 50bp at one end) for sequencing will significantly reduce the sequencing alignment results. Sequencing takes up a large amount of time in the rapid detection cycle, so conventional DOP-PCR design cannot meet the clinical needs for rapid reporting.

[0004] Therefore, there is an urgent need to provide primers and kits for single-cell whole-genome amplification. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention designs primers and a kit for single-cell whole-genome amplification. While ensuring single-cell amplification efficiency, it also eliminates residual primer sequences in the amplification products, increasing the utilization rate of effective sequencing data to over 95%, and maintaining high sensitivity even with trace amounts of DNA template.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0007] In a first aspect, the present invention provides primers for single-cell whole-genome amplification, the primers comprising pre-amplification primers and final amplification primers; the 5' to 3' ends of the pre-amplification primers are, in sequence, a universal sequence, a dUTP, and a random degenerate sequence; the 5' to 3' ends of the final amplification primers are, in sequence, a universal sequence and a dUTP.

[0008] In some embodiments, the nucleotide sequence of the pre-amplification primer is shown in SEQ ID NO:1; and the nucleotide sequence of the final amplification primer is shown in SEQ ID NO:2.

[0009] In a second aspect, the present invention provides a kit for single-cell whole-genome amplification, the kit comprising primers as described in any one of claims 1-2.

[0010] Specifically, the concentration of the pre-amplification primers is 10 μM, and the concentration of the final amplification primers is 1 μM.

[0011] In some embodiments, the kit further includes lysis buffer, stop solution, stop solution pre-amplification buffer, pre-amplification reaction enzyme, final amplification buffer, and final amplification reaction enzyme.

[0012] In some embodiments, the components of the kit satisfy at least one of the following characteristics:

[0013] (1) The lysis buffer comprises 5% (v / v) NP-40, 0.5 mg / mL proteinase K, and 10 mM Tris-HCl;

[0014] (2) The terminating solution includes 1 mM PMSF.

[0015] (3) The pre-amplification buffer comprises 10% (v / v) 10×Taq Buffer, 400 μM each dNTPs, 10 μM pre-amplification primers, and 0.2 mg / mL BSA;

[0016] (4) The final amplification buffer comprises 10% (v / v) 10×TaqBuffer, 400 μM each dNTPs, 1 μM final amplification primers, and 0.2 mg / mL BSA.

[0017] In some embodiments, the pre-amplification enzyme is a hot-start Taq polymerase, and the final amplification enzyme is a hot-start high-fidelity Taq polymerase.

[0018] Thirdly, the present invention provides a method for amplifying a single-cell whole genome, comprising using pre-amplification primers as shown in SEQ ID No. 1 to perform a pre-amplification reaction on single-cell genomic DNA, and using final amplification primers as shown in SEQ ID No. 2 to perform an amplification reaction on the final amplification product, so as to obtain an amplified product of single-cell genomic DNA.

[0019] In some embodiments, the single cell is a lymphocyte.

[0020] In some embodiments, the pre-amplification reaction is programmed as follows: pre-denaturation at 95°C for 15 minutes, followed by 12 cycles of denaturation at 95°C for 15 seconds, followed by gradient heating from 15°C to 75°C, and holding at 4°C.

[0021] In some embodiments, the final amplification reaction is programmed as follows: pre-denaturation at 95°C for 15 minutes, followed by 14 cycles: denaturation at 95°C for 15 seconds, followed by annealing at 65°C for 1 minute, extension at 75°C for 1 minute, and holding at 4°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention replaces the conventional dTTP with dUTP deoxyuridine nucleotides at the last base of the 5' end of the primer. The primer can then be specifically excised at this site, resulting in a primer residual rate of less than 4% in the sequencing data and a sequencing data alignment error rate of 40% to 4%. This reduces the interference of primer sequences on sequencing, lowers sequencing costs, and has no impact on CNV analysis.

[0024] This invention also includes a kit containing this primer. Using a lysis buffer (containing 5% NP-40 and proteinase K) and a two-stage amplification system, this invention can obtain 5±0.5 μg of high-purity DNA with only 1-2 cells, meeting the high-precision amplification requirements for small samples. Compared with existing kits, the kit of this invention has a whole-genome amplification coefficient of variation (CV) of 9.74±3.5, exhibiting better uniformity of amplification products and low base bias. Attached Figure Description

[0025] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a flowchart of primer design according to an embodiment of the present invention.

[0027] Figure 2 These are capillary electrophoresis and agarose gel electrophoresis images of single-cell amplification products according to an embodiment of the present invention.

[0028] Figure 3 This is a distribution map of failed sequence bases for different kits in embodiments of the present invention.

[0029] Figure 4 This is a graph showing the copy number detection results of different reagent kits in embodiments of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0032] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0033] In this invention, the term "homogeneity (CV value)" refers to the statistical coefficient of variation of genome-wide coverage depth (CV%).

[0034] In this invention, the term "alignment rate" refers to the proportion of statistical sequencing data that uniquely aligns to the reference genome (hg38).

[0035] In this invention, the term "base preference" refers to the calculated A / T / C / G base ratio across the entire genome (theoretically approximately 25%).

[0036] In this invention, the single-cell amplification primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their design principle is as follows:

[0037] 1. For pre-amplification primers: their universal and random degenerate sequences should be able to amplify the entire genome evenly and uniformly, covering a wider range of genomic regions. Essentially, this involves using short, medium-GC-content, low-secondary-structure interference random degenerate sequences to randomly bind to genomic DNA under less stringent PCR conditions, achieving multi-fragment amplification across the entire genome. Primers can be verified using the CNV-Kit software to check for amplification bias.

[0038] 2. The universal sequences of the final amplification primers and the pre-amplification primers can be complementary, enabling specific amplification of the pre-amplification product.

[0039] 3. Primers are matched with the designed reaction conditions.

[0040] Example 1

[0041] Based on the principles of single-cell amplification primer design, the sequences of the pre-amplification primers and the final amplification primers are shown below:

[0042] The pre-amplification primer comprises three parts, from 5' to 3': an upstream universal sequence primer region with the same 5' end sequence, a midstream dUTP, and a downstream random degenerate sequence primer region. The specific nucleotide sequence of the pre-amplification primer is shown in SEQ ID NO:1:

[0043] Pre-amplification primers: GATTGAGAGAGATGTTGGTGGTTAGA-dUTP-NN NNNKK (SEQ ID NO:1).

[0044] The universal sequence is of non-biological origin and does not match the sequence of any organism. Because the 5' end sequences are identical, the two ends of the amplified fragment are complementary and form a circular structure during PCR, thus avoiding biased amplification. Pre-amplification with complementary beginnings and ends is visible. Figure 1 ; where NNNNN represents a 5-base random sequence (A, G, C or T), and K represents a G or T nucleotide sequence.

[0045] The final amplification primer comprises two parts, from 5' to 3': a universal sequence primer region at the upstream 5' end that is identical to the pre-amplification primer sequence, and a dUTP. The specific nucleotide sequence of the final amplification primer is shown in SEQ ID NO:2:

[0046] Final amplification primers: GATTGAGAGAGATGTTGGTGGTTAGA-dUTP (SEQ ID NO:2).

[0047] Example 2

[0048] I. A kit for single-cell whole-genome amplification, comprising lysis buffer, stop solution, pre-amplification buffer, pre-amplification enzyme, and final amplification buffer, wherein the pre-amplification enzyme and final amplification buffer comprise the pre-amplification primers and final amplification primers of Example 1. The kit is detailed in Table 1 below:

[0049] Table 1. Reagent Kit Components

[0050]

[0051]

[0052] II. Figure 1 The following is a description of how to use a kit for single-cell whole-genome amplification:

[0053] 1. Sample preprocessing

[0054] Take a single-cell suspension of lymphocytes (containing 1-2 cells / 3μL), add it to a PCR tube, and store at -20℃ for at least 30 minutes. Set up a phosphate-buffered saline (pH=7.4) as a template-free control group (NTC).

[0055] 2. Cell lysis

[0056] Add 2 μL of lysis buffer to each well, for a total volume of 5 μL (add nuclease-free water if necessary);

[0057] Run the lysis program on the PCR instrument: 75℃ for 10 min, 95℃ for 4 min, hold at 4℃ to release genomic DNA and obtain lysis products.

[0058] 3. Pre-amplification reaction

[0059] Prepare the pre-amplification mixture: (5 μL pre-amplification buffer + 1 μL pre-amplification enzyme + 4 μL nuclease-free water);

[0060] Add 10 μL of pre-amplification mixture to the lysis product from step 2.

[0061] Run the pre-amplification program on the PCR instrument: 95℃ for 15 min, 12 cycles [95℃ for 15 s → gradient temperature increase 15-75℃ / cycle], hold at 4℃ to obtain the pre-amplification product.

[0062] 4. Whole genome amplification

[0063] Prepare the final amplification mixture: (20 μL final amplification buffer + 1 μL final amplification enzyme + 39 μL nuclease-free water);

[0064] Add 60 μL of final amplification mixture to the pre-amplification product from step 3.

[0065] Run the final amplification program on the PCR instrument: 95℃ for 15 min → 14 cycles [95℃ for 15 s → 65℃ for 1 min → 75℃ for 1 min] → hold at 4℃.

[0066] 5. Quality Inspection and Verification

[0067] Take 2 μL of amplification product and use a Qubit 4.0 fluorometer and Qubit... TM The dsDNA kit (catalog number Q32854) is used to detect DNA concentration.

[0068] The results are as follows Figure 2 As shown, the yield was 5.417±0.5μg (n=10), with fragment distribution ranging from 200-1600bp, meeting the subsequent detection standards; while the yield of the NTC group was <0.4μg, with no obvious fragments, ruling out false positive amplification from the kit.

[0069] Example 3

[0070] Analysis of the performance of the kit in Example 2

[0071] 1. Verify the differences between the kit of the present invention and commercially available single-cell amplification kits in terms of amplification uniformity (CV value), base bias, and alignment rate.

[0072] 2. Validation Sample

[0073] Standard cell line: Human fibroblasts (cell line GM00325, karyotype 47, XXY) single cell suspension.

[0074] 3. Experimental Procedure

[0075] Experimental group: The kit of the present invention was used according to the procedure in Example 2, and the sample size n = 10;

[0076] Control group 1: A commercially available single-cell whole genome amplification kit (SC301) was used, and the procedure was followed according to the instructions. The sample size was n=10.

[0077] Control group 2: The primers in the kit of this invention are replaced with non-specifically resectable primers (non-specifically resectable primers refer to the pre-amplification primers and final amplification primers in which dUTP is replaced with dTTP), and the rest is the same as the experimental group, with a sample size of n=10.

[0078] 4. Library Construction

[0079] Three sets of samples were prepared using a library construction kit (manufacturer: Fapon; reagent name: One-Step Library Prep Kit for MGI) containing UDG enzyme. The process included enzyme digestion (primer-specific excision and fragmentation), end repair, A addition, adapter ligation, purification after ligation, and amplification. The amplified samples were then purified to obtain the three sets of sample libraries.

[0080] 5. Sequencing analysis:

[0081] SE50 sequencing (single-end sequencing, 50bp read length) was used on the MGI200 sequencing platform; the operation was performed according to the instructions on the BGI website, with an average sequencing depth of 0.2×.

[0082] 6. Results:

[0083] After quality control, the sequencing data was aligned to a reference genome, then GC-corrected and windowed. The log2 ratio of each region was calculated using normalization, and the chromosome copy number of the sample was estimated using a statistical model. The detection results are shown in Table 2 below:

[0084] Table 2. Test results of different reagent kits

[0085]

[0086] Base preference results:

[0087] Single-cell amplification primers have a significant impact on sequencing base bias and can lead to sequence alignment failures. Therefore, sequences that failed to align are used for analysis, such as... Figure 3 As shown, due to the influence of single-cell amplification primers, the first 28 bp of sequencing in control groups 1 and 2 showed a severe base bias, while the experimental group only had a slight influence on the first 10 bp (caused by library preparation reagents, not by single-cell amplification reagents), which was better than control groups 1 and 2.

[0088] Standard chromosome copy number test results:

[0089] In SE50 sequencing mode, all three groups were able to detect 47,XXY chromosome copy number results, but one case in control group 1 reported a false positive abnormality (partial mosaic duplication of chromosome 10) that did not belong to the standard cells. Figure 4 As shown, control group 1 reported the copy number of chromosome GM00325, but one case showed a false positive result for chromosome 10: 47,XXY; +10{p15.3->p11.1(39.00Mb×3,mos[40.4%])}; +X{×2}; while the experimental group correctly reported the copy number of chromosome GM00325: 47,XXY; +X{×2}, proving that the experimental group had the best coefficient of variation (CV) and alignment rate.

[0090] In summary, this invention reduces primer sequence interference by replacing the last base of the 5' fixed sequence of the primer with dUTP (deoxyuridine triphosphate). Subsequent primer cleavage can be specifically performed at this site. Furthermore, this invention designs a kit incorporating this primer. Compared to commercially available products, this kit exhibits a 63.60% lower CV value, better amplification product uniformity, and low base bias (A / T / C / G ratio close to the theoretical value of 25%). The alignment rate in SE50 sequencing mode reaches 97.25%, indicating no primer residue interference during amplification and suitability for high-precision single-cell sequencing analysis.

[0091] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A primer for single-cell whole-genome amplification, characterized in that, The primers include pre-amplification primers and final amplification primers; the 5' to 3' ends of the pre-amplification primers are, in order, a universal sequence, a dUTP, and a random degenerate sequence; the 5' to 3' ends of the final amplification primers are, in order, a universal sequence and a dUTP.

2. The primer according to claim 1, characterized in that, The nucleotide sequence of the pre-amplification primer is shown in SEQ ID NO:1; the nucleotide sequence of the final amplification primer is shown in SEQ ID NO:

2.

3. A kit for single-cell whole-genome amplification, characterized in that, The kit includes primers as described in any one of claims 1-2.

4. The reagent kit according to claim 2, characterized in that, The kit also includes lysis buffer, stop solution, stop solution pre-amplification buffer, pre-amplification reaction enzyme, final amplification buffer and final amplification reaction enzyme.

5. The reagent kit according to claim 4, characterized in that, Each component of the kit satisfies at least one of the following characteristics: (1) The lysis buffer comprises 5% (v / v) NP-40, 0.5 mg / mL proteinase K, and 10 mM Tris-HCl; (2) The terminating solution includes 1 mM PMSF. (3) The pre-amplification buffer comprises 10% (v / v) 10×Taq Buffer, 400 μM each dNTPs, 10 μM pre-amplification primers, and 0.2 mg / mL BSA; (4) The final amplification buffer comprises 10% (v / v) 10×Taq Buffer, 400 μM each dNTPs, 1 μM final amplification primers, and 0.2 mg / mL BSA.

6. The reagent kit according to claim 4, characterized in that, The pre-amplification enzyme is a hot-start Taq polymerase, and the final amplification enzyme is a hot-start high-fidelity Taq polymerase.

7. A method for single-cell whole-genome amplification, characterized in that, The method includes using pre-amplification primers as shown in SEQ ID No. 1 to perform a pre-amplification reaction on single-cell genomic DNA, and using final amplification primers as shown in SEQ ID No. 2 to perform an amplification reaction on the final amplification product to obtain the amplified product of single-cell genomic DNA.

8. The method according to claim 7, characterized in that, The single cell mentioned is a lymphocyte.

9. The method according to claim 7, characterized in that, The procedure for the pre-amplification reaction is as follows: pre-denaturation at 95°C for 15 minutes, followed by 12 cycles: denaturation at 95°C for 15 seconds, followed by gradient heating from 15°C to 75°C, and holding at 4°C.

10. The method according to claim 7, characterized in that, The procedure for the final amplification reaction is as follows: pre-denaturation at 95°C for 15 minutes, followed by 14 cycles: denaturation at 95°C for 15 seconds, followed by annealing at 65°C for 1 minute, extension at 75°C for 1 minute, and holding at 4°C.

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