Construction method and sequencing method of single cell DNA library

Abstract

The invention provides a construction method and a sequencing method of a single cell DNA library. The construction method comprises the following steps: a) cracking a single cell, mixing the cracked single cell with a first primer, and carrying out first amplification to obtain a first amplification product; b) mixing the first amplification product with a second primer, and performing second amplification to obtain a single-cell DNA library; wherein the first primer comprises a universal amplification fragment and a random amplification fragment; the second primer comprises an index fragment and a complementary fragment; the single cell DNA library is a library suitable for BGI platform or Illumina platform sequencing, and correspondingly, the universal amplification fragment is a fragment suitable for BGI platform or Illumina platform; the fragment suitable for a BGI platform is 8-9 nt, and the fragment suitable for an Illumina platform is 10 nt. The problem that in the prior art, a single-cell DNA library construction method is complex can be solved, and the method is suitable for the field of single-cell sequencing.

Description

Technical Field

[0001] This invention relates to the field of single-cell sequencing, and more specifically, to a method for constructing and sequencing a single-cell DNA library. Background Technology

[0002] Single cells are the basic functional units of life. The total amount of genomic DNA in a single human cell is only about 6 pg. Due to limitations in DNA library construction technology, the amount of DNA from a single cell is generally insufficient to meet the needs of downstream experiments such as whole-genome sequencing (which typically require μg of DNA). However, directly mixing a large amount of cellular nucleic acids for library construction can mask genomic heterogeneity between cells (such as differences in gene mutations among different cancer cells in a tumor), or may prevent the acquisition of large numbers of cells for specific applications (such as chromosomal abnormality detection in embryonic cells or single-cell sequencing of microorganisms).

[0003] Therefore, single-cell samples require "signal amplification" using single-cell DNA amplification technology to meet downstream detection needs. Single-cell DNA amplification technology is a molecular biology technique that specifically amplifies trace amounts of genomic DNA at the single-cell level, amplifying extremely small amounts of single-cell genomic DNA to the microgram level. The amplified DNA is then processed and analyzed according to different requirements.

[0004] Currently, mainstream single-cell DNA amplification technologies are mainly based on the principle of whole genome amplification (WGA). Depending on the amplification strategy, they can be divided into three categories: PCR-based methods, isothermal amplification methods (Multiple Displacement Amplification, MDA), and multiple annealing and looping based amplification cycles (MALBAC). Subsequently, the amplified DNA is fragmented using sonication or enzyme fragmentation to construct an NGS library. Therefore, the entire process of single-cell DNA detection is relatively long, including single-cell DNA amplification and amplification product library construction. PCR-based methods use random primers to bind genomic DNA and achieve amplification through denaturation-annealing-extension cycles; a representative method is DOP-PCR (degenerate oligonucleotide primer PCR). Isothermal amplification methods... 29 DNA polymerases (high-fidelity, strong strand displacement activity) are the core technology. Random primers bind to genomic DNA, continuously displacing and extending it to form long-chain DNA, achieving whole-genome amplification. Multiple-annealing circular amplification uses special primers (random sequence at the 3' end, universal sequence at the 5' end) to bind to DNA, undergoing multiple rounds of annealing and extension to form circular DNA. This amplification process is semi-linear, and finally, universal primers are used to amplify the signal. Each of the three single-cell DNA amplification techniques has its advantages and disadvantages. DOP-PCR has a short reaction time, is simple to operate, and has good amplification uniformity, but its gene coverage is low and it has strong bias. MDA has high fidelity, long amplified fragments, and high gene coverage, but its amplification uniformity is poor, it is prone to chimeras, and its reaction time is long. MALBAC has good amplification uniformity and high gene coverage, but its operation is complex and costly. The core of single-cell DNA amplification technology is to achieve efficient, uniform, and high-fidelity whole-genome amplification with a small amount of template. Different methods (DOP-PCR, MDA, MALBAC) have their own suitable scenarios. However, each amplification method has its disadvantages. No single-cell DNA amplification technology is simple to operate, has a short reaction time, high gene coverage, good amplification uniformity, or high fidelity. Moreover, conventional single-cell DNA amplification techniques require a procedure of amplification before library construction, which is complex and time-consuming. Summary of the Invention

[0005] The main objective of this invention is to provide a method for constructing and sequencing a single-cell DNA library, thereby solving the problem of the complexity of existing methods for constructing single-cell DNA libraries.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a method for constructing a single-cell DNA library is provided, the method comprising: a) lysing a single cell and mixing it with a first primer to perform a first amplification, thereby obtaining a first amplification product; b) mixing the first amplification product with a second primer to perform a second amplification, thereby obtaining the single-cell DNA library; wherein the first primer comprises a universal amplification fragment and a random amplification fragment, the 3' end of the universal amplification fragment being ligated to the 5' segment of the random amplification fragment; the second primer comprises an index fragment and a complementary fragment, the 3' end of the index fragment being ligated to the 5' segment of the complementary fragment; the single-cell DNA library is a library suitable for sequencing on the BGI platform or a library suitable for sequencing on the Illumina platform, and correspondingly, the universal amplification fragment in the first primer is a fragment suitable for the BGI platform or a fragment suitable for the Illumina platform; the length of the fragment suitable for the BGI platform is 8-9 nt, and the length of the fragment suitable for the Illumina platform is 10 nt.

[0007] Furthermore, the sequence of the above-mentioned fragments applicable to the BGI platform is ATCCGACTT or TCCGACTT.

[0008] Furthermore, the sequence of the fragment applicable to the Illumina platform is the sequence shown in SEQ ID NO: 2.

[0009] Furthermore, the sequence of the above-mentioned randomly amplified fragment includes: a sequence consisting of 2-4 NKK bases, or a sequence consisting of 6-8 B bases.

[0010] Furthermore, the sequence of the first primer suitable for the BGI platform is any one or more sequences shown in SEQ ID NOs: 1, 3 or 7-16; the sequence of the first primer suitable for the Illumina platform is the sequence shown in SEQ ID NO: 4.

[0011] Further, after the first amplification described above, a first purification is performed to obtain the first amplification product.

[0012] Further, after the second amplification, a second purification was performed to obtain the single-cell DNA library.

[0013] Furthermore, the system described in the first amplification also contains PEG and / or Mg. 2+ Preferably, the PEG includes PEG6000; preferably, in the first amplification system, the concentration of PEG6000 is 3-20 wt%, more preferably 5-15 wt%; preferably, in the first amplification system, the concentration of Mg... 2+ The concentration is 5-10 nM, more preferably 6-9 nM; preferably, the above-mentioned Mg 2+ Derived from MgCl2; preferably, the first amplification includes gradient temperature amplification.

[0014] To achieve the above objectives, according to a second aspect of the present invention, a single-cell sequencing method is provided, the single-cell sequencing method comprising: constructing a single-cell DNA library using the above-described single-cell DNA library construction method; and sequencing the single-cell DNA library using a high-throughput sequencing platform.

[0015] Furthermore, the aforementioned high-throughput sequencing platforms include the BGI platform or the Illumina platform.

[0016] By applying the technical solution of this invention and utilizing the above-mentioned method for constructing single-cell DNA libraries, and employing first primers designed for libraries suitable for BGI platform sequencing or libraries for Illumina platform sequencing respectively, the length of the amplified fragments can directly meet the requirements for sequencing without the need for fragmentation and repair steps, which can greatly reduce the operations required for constructing single-cell DNA libraries. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 The diagram shows the distribution of amplified product fragments according to Example 1 of the present invention.

[0019] Figure 2A The diagram shows the distribution of amplified product fragments according to Example 2 of the present invention.

[0020] Figure 2B The diagram shows the distribution of amplified product fragments according to Embodiment 2 of the present invention.

[0021] Figure 2C The diagram shows the distribution of amplified product fragments according to Example 2 of the present invention.

[0022] Figure 2D The diagram shows the distribution of amplified product fragments according to Example 2 of the present invention.

[0023] Figure 3 The diagram shows the distribution of amplified product fragments according to Example 3 of the present invention.

[0024] Figure 4 The diagram shows the results of single-cell DNA amplification according to Example 4 of the present invention.

[0025] Figure 5 The diagram shows the results of single-cell DNA amplification according to Example 5 of the present invention. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0027] Terminology Explanation:

[0028] Next-generation sequencing (NGS): refers to high-throughput sequencing technology that can sequence a large number of DNA fragments at once. The library fragments must be within a specific length range (usually 200-800 bp) to meet the sequencing requirements. The sequenced nucleic acids must contain a fixed sequence that the sequencer can recognize. Different sequencing platforms have different fixed sequences.

[0029] Whole-genome amplification (WGA): A technique that amplifies genomic DNA to the microgram level at the single-cell or trace DNA level using molecular biology techniques to meet downstream detection needs.

[0030] Degenerate oligonucleotide primer PCR (DOP-PCR): A PCR-based WGA method that uses random degenerate primers to bind to genomic DNA and amplifies it through denaturation-annealing-extension cycles. It features short reaction time and simple operation.

[0031] Multiple substitution amplification (MDA): The isothermal WGA method, which uses DNA polymerase (high fidelity, strong strand displacement activity) as its core, can obtain long fragment amplification products, but the amplification uniformity is poor and the reaction time is long.

[0032] Multiple Annealing Circular Amplification (MALBAC): A semi-linear WGA method that uses special primers to form circular DNA. It has good amplification uniformity, but is complex to operate and costly.

[0033] Coverage: In the process of single-cell amplification, the percentage of genome fragments that are effectively amplified. The higher the value, the wider the amplification range and the better the amplification effect of the kit.

[0034] Uniformity of coverage (ROC): Its value is directly related to the uniformity of genomic coverage of the amplification products. The smaller the value, the smaller the amplification bias and the better the uniformity. The larger the value, the more likely there is over-amplification or under-amplification in local regions.

[0035] Allele loss (ADO): During single-cell amplification, a certain allele is not amplified, resulting in its undetectability. The smaller the value, the smaller the proportion of lost alleles during amplification, and the better the amplification effect.

[0036] Non-invasive embryo testing (niPGT): Preimplantation genetic testing is performed on embryos by detecting cell-free DNA (cfDNA) in spent embryo culture medium (SCM) and blastocoel fluid (BF) to determine the source of genetic material.

[0037] As mentioned in the background section, existing methods for constructing single-cell DNA libraries are complex and struggle to balance efficiency and quality. In this application, the inventors attempt to develop a novel method for constructing single-cell DNA libraries, and based on this, propose a series of protection schemes.

[0038] In a first typical embodiment of this application, a method for constructing a single-cell DNA library is provided. The method includes: a) lysing a single cell and mixing it with a first primer to perform a first amplification and obtain a first amplification product; b) mixing the first amplification product with a second primer to perform a second amplification and obtain the single-cell DNA library; wherein the first primer includes a universal amplification fragment and a random amplification fragment, and the 3' end of the universal amplification fragment is ligated to the 5' segment of the random amplification fragment; the second primer includes an index fragment and a complementary fragment, and the 3' end of the index fragment is ligated to the 5' segment of the complementary fragment.

[0039] In this application, the inventors have developed a simple method for constructing a single-cell DNA library. This method requires only two amplification steps without additional operations such as fragmentation or repair to obtain a single-cell DNA library suitable for sequencing. The first primer used in the first amplification (also referred to as pre-amplification in this application) includes a universal amplification fragment at the 5' end and a random amplification fragment at the 3' end. The random amplification fragment binds to the DNA released after single-cell lysis and serves as a primer to guide the first amplification. Those skilled in the art can flexibly select or design the random amplification fragment based on the type of target single cell and its DNA, using existing technologies. The first amplification product and the second amplification product obtained using the above method have lengths sufficient for NGS sequencing, allowing for direct sequencing without additional operations such as fragmentation, repair, or adapter ligation.

[0040] Similar to MALBAC in existing technologies, during the first amplification process, the ends of the first amplification product obtained by PCR amplification contain a universal amplification fragment and a complementary fragment of the universal amplification fragment, respectively. The newly synthesized DNA can form a hairpin structure on its own and will not participate in PCR amplification as a template strand in subsequent first amplifications. This suppresses the exponential amplification effect, allowing the first amplification to proceed in an approximately linear amplification manner, improving the uniformity of amplification, and reducing the generation and accumulation of amplification errors.

[0041] After obtaining the first amplification product using the first amplification, a second amplification (also referred to as Index PCR amplification in this application) is performed on the first amplification product using a second primer. The purpose of the second amplification is to label the product with the index fragment, thereby facilitating data separation after subsequent sequencing of the mixed samples. The second primer includes an index fragment at the 5' end and a complementary fragment at the 3' end. The complementary fragment can pair complementaryly with the complementary fragment of the universal amplification fragment, thus serving as a primer to guide the second amplification. The product obtained after the second amplification is a single-cell DNA library suitable for sequencing.

[0042] In a preferred embodiment, the single-cell DNA library is a library suitable for sequencing on the BGI platform or a library suitable for sequencing on the Illumina platform, and correspondingly, the universal amplification fragment in the first primer is a fragment suitable for the BGI platform or a fragment suitable for the Illumina platform.

[0043] In a preferred embodiment, the length of the fragment suitable for the BGI platform is 8-9 nt, and the length of the fragment suitable for the Illumina platform is 10 nt; preferably, the sequence of the fragment suitable for the BGI platform is ATCCGACTT or TCCGACTT; preferably, the sequence of the fragment suitable for the Illumina platform is the sequence shown in SEQ ID NO: 2.

[0044] SEQ ID NO: 2: GATCCGACTT.

[0045] The core of the aforementioned single-cell DNA library construction method lies in its seamless compatibility with the two major sequencing platforms—BGI and Illumina. This significantly broadens the application scope of single-cell sequencing technology while simplifying experimental procedures and improving experimental efficiency and reliability. Specifically, when the target sequencing platform is BGI, the constructed library will be specifically designed and optimized for the sequencing characteristics and requirements of the BGI platform, ensuring that the library can be directly used in the BGI sequencing system. Similarly, for the Illumina platform, the constructed library will be precisely adapted to its technical parameters, allowing for unimpeded operation within the Illumina sequencing environment.

[0046] The above construction method, through the unique design of fragments suitable for BGI or Illumina platforms within the universal amplification fragment, ensures that the length of the first amplification product meets the requirements of different sequencing platforms, especially next-generation sequencing platforms, preventing the first amplification product from exceeding the readable length of next-generation sequencing. Therefore, the single-cell DNA library obtained by the above amplification method can be sequenced without additional fragmentation or repair operations. Because the sequencing-related adapters and sequences used by BGI and Illumina platforms differ, the fragments suitable for BGI and Illumina platforms are also different.

[0047] Especially for the BGI platform, due to the special nature of the sequencing adapter sequences of this platform, it is difficult to simply design and obtain the relevant primers. When developing methods suitable for the BGI platform, it was found that the length of the first amplification product is relatively long, making it difficult to use directly in subsequent next-generation sequencing.

[0048] After repeated experiments, we determined the optimal length to be 9 nt. This length not only meets the read length requirements of the BGI sequencing system, but also effectively controls the length of the product fragment during the pre-amplification process, avoiding the reduction of sequencing efficiency and data analysis quality due to excessively long fragments. It also ensures the PCR efficiency in the subsequent second amplification, preventing the universal amplification fragment from being too short and thus failing to bind to the second primer, thereby affecting the amplification efficiency.

[0049] In a preferred embodiment, the sequence of the randomly amplified fragment includes: a sequence consisting of 2-4 NKK bases, or a sequence consisting of 6-8 B bases.

[0050] To further enhance the coverage and uniformity of the library construction method, we innovatively designed the sequences of the randomly amplified fragments. The sequences of the randomly amplified fragments can consist of 2-4 NKK bases, where K is a degenerate base and is G / T degenerate. This design increases the diversity of primer-template binding, contributing to improved amplification breadth and efficiency.

[0051] Another design approach uses sequences consisting of 6-8 B bases, where B is a degenerate base, specifically G / T / C degenerate. The higher degeneracy of B bases provides a wider range of template binding sites, which is particularly important for ensuring uniform amplification of the entire genome. This design strategy further enhances the library construction method's ability to handle various types of single-cell samples, improving genome coverage and the accuracy of allele detection.

[0052] In a preferred embodiment, the sequence of the first primer suitable for the BGI platform is any one or more sequences shown in SEQ ID NOs: 1, 3 or 7-16.

[0053] SEQ ID NO: 1: ATCCGACTTNKKNKKNKKNKK.

[0054] SEQ ID NO: 3: ATCCGACTTBBBBBB.

[0055] SEQ ID NO: 7: TCCGACTTNKKNKKNKKNKK.

[0056] SEQ ID NO: 8: ATCCGACTTNKKNKNKNKK.

[0057] SEQ ID NO: 9: ATCCGACTTNKKNKK.

[0058] SEQ ID NO: 10: ATCCGACTTBBBBB.

[0059] SEQ ID NO: 11: ATCCGACTTBBBB.

[0060] SEQ ID NO: 12: TCCGACTTNKKNKNKNKK.

[0061] SEQ ID NO: 13: TCCGACTTNKKNKK.

[0062] SEQ ID NO: 14: TCCGACTTBBBBBB.

[0063] SEQ ID NO: 15: TCCGACTTBBBBB.

[0064] SEQ ID NO: 16: TCCGACTTBBBB.

[0065] In the above construction method, for the selection of the first primer, any one of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 7-16 can be used as the first primer alone, or two primers can be used at the same time in the first amplification, such as selecting a primer containing NKK bases and a primer containing 6-8 B bases, including but not limited to using SEQ ID NO: 1 and SEQ ID NO: 3 as primers and using two primers to perform the first amplification at the same time.

[0066] In a preferred embodiment, the sequence of the first primer suitable for the Illumina platform is the sequence shown in SEQ ID NO:4.

[0067] SEQ ID NO: 4: CTTCCGATCTNKKNKKNKKNKK.

[0068] In a preferred embodiment, after the first amplification described above, a first purification is performed to obtain the first amplification product.

[0069] After the first amplification, the product is purified using methods including but not limited to magnetic bead purification to obtain the first amplification product. This step removes non-specific products, residual primers, enzymes, and other components from the reaction buffer, thereby improving the purity of the amplification product and reducing interference with subsequent second amplification and sequencing processes.

[0070] In a preferred embodiment, after the second amplification described above, a second purification is performed to obtain the single-cell DNA library described above.

[0071] Following the second amplification, a second purification process is performed using methods including, but not limited to, magnetic bead purification to effectively remove non-specific products and residual primers and enzymes, thereby obtaining a high-quality single-cell DNA library. This purification step ensures the purity and stability of the library, providing a reliable sample basis for subsequent sequencing analysis.

[0072] In a preferred embodiment, the system of the first amplification described above also contains PEG and / or Mg. 2+ .

[0073] Preferably, the PEG includes PEG6000; preferably, in the first amplification system, the concentration of PEG6000 is 3-20 wt% (including but not limited to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 wt%), more preferably 5-15 wt%.

[0074] Preferably, in the above-mentioned first amplification system, the above-mentioned Mg 2+ The concentration is 5-10 nM (including but not limited to 5, 6, 7, 8, 9 or 10 nM), more preferably 6-9 nM; preferably, the above-mentioned Mg 2+ It originates from MgCl2.

[0075] In the above construction method, the inventors discovered that by adding PEG to the first amplification reaction system, the length of the amplified library fragments can be controlled, avoiding the generation of excessively long amplified fragments that would affect the subsequent sequencing quality. The inventors also discovered that by adding Mg to the first amplification reaction system... 2+ It can promote amplification effect and improve the speed and quality of amplification.

[0076] In a preferred embodiment, the first amplification includes gradient temperature amplification; preferably, in one cycle of the gradient temperature amplification PCR program, the annealing temperature is gradually increased from 15°C to 75°C, and the annealing temperatures include, but are not limited to, 15°C, 25°C, 35°C, 45°C, 55°C, 65°C and 75°C, with an annealing time of 30-50 s at each temperature.

[0077] Preferably, the PCR program for gradient temperature amplification includes: 95℃ for 3 min; 95℃ for 15 s, 15℃ for 50 s, 25℃ for 40 s, 35℃ for 30 s, 45℃ for 30 s, 55℃ for 40 s, 65℃ for 40 s, 75℃ for 40 s, for a total of 16 cycles; and held at 4℃.

[0078] The aforementioned construction method improves the efficiency and quality of single-cell DNA library construction, is applicable to various trace samples, and features convenient operation, high sample compatibility, high coverage, good homogeneity, and low allele deletion. It can be used in various applications such as preimplantation genetic testing (PGT / PGD), single-cell whole-genome CNV analysis, amplification and library construction of extremely small samples, forensic medicine, and ancient DNA sample library construction. Furthermore, this construction method can amplify 100 pg of cfDNA in simulated fetal culture medium or blastocyst cavity fluid with excellent amplification results; therefore, this construction method can also be applied to non-invasive embryo testing (niPGT).

[0079] In a second typical embodiment of this application, a single-cell sequencing method is provided, comprising: constructing a single-cell DNA library using the single-cell DNA library construction method described above; and sequencing the single-cell DNA library using a high-throughput sequencing platform.

[0080] In a preferred embodiment, the high-throughput sequencing platform described above includes the BGI platform or the Illumina platform.

[0081] After obtaining a single-cell DNA library using the above construction method, the single-cell DNA library is sequenced (including but not limited to next-generation sequencing). The sequencing platform needs to correspond to the primer sequences selected in the construction method, including the BGI platform or the Illumina platform.

[0082] Optionally, before sequencing, libraries can be constructed for multiple single cells using the above method, and then the single-cell DNA libraries corresponding to different cells can be mixed and sequenced together. Due to the presence of index sequences, different samples can be distinguished during subsequent data analysis.

[0083] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0084] Example 1:

[0085] This example describes the impact of using the same random bases and universal sequences (10 nt in length) on amplification results for single-cell DNA amplification protocols across different sequencing platforms. Previously, we established a single-cell DNA library construction system for the Illumina sequencing platform. The lysis system included random amplification primers, which consisted of two parts: a universal amplification sequence and a random sequence. We aimed to complete the single-cell DNA library construction on the BGI platform, so we replaced the universal amplification sequence adapted for the Illumina platform with the one adapted for BGI. While maintaining a fixed random sequence, we also ensured consistency in the universal sequence length between the two sequencing platforms, both being 10 nt in length.

[0086] The specific experimental steps are as follows:

[0087] Step 1: Cell lysis

[0088] 1. The random amplification primers for the Illumina sequencing platform are SEQ ID NO: 4, and the random amplification primers for the BGI sequencing platform are SEQ ID NO: 5. Dilute the random amplification primers to 500µM using TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) for later use.

[0089] SEQ ID NO: 4: CTTCCGATCTNKKNKKNKKNKK;

[0090] SEQ ID NO: 5: GATCCGACTTNKKNKKNKKNKK.

[0091] 2. Remove the gDNA sample and place it on ice to thaw naturally. Mix well and centrifuge briefly for later use.

[0092] 3. If the sample volume is less than 5 μL, it can be equilibrated to a final volume of 5 μL by adding an appropriate amount of TE buffer.

[0093] 4. Prepare the reaction system in a 0.2 mL PCR tube placed on ice, according to Table 1 below:

[0094] Table 1

[0095]

[0096] 5. Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0097] 6. Place the PCR tubes into the PCR instrument and start the program shown in Table 2 below:

[0098] Table 2

[0099]

[0100] Step 2: Pre-amplification (First Amplification)

[0101] 1. Take out the Pre-Amplification Buffer, Pre-Amplification Enhancer and Pre-Amplification Master Mix and place them on ice to thaw naturally. Mix them evenly and centrifuge for later use.

[0102] 2. Prepare the reaction system in a 0.2 mL PCR tube placed on ice, according to Table 3 below:

[0103] Table 3

[0104]

[0105] 3. Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0106] 4. Place the PCR tube into the PCR instrument and start the reaction program shown in Table 4 below. Once the temperature stabilizes at 95℃, place the reaction tube into the PCR instrument.

[0107] Table 4

[0108]

[0109] Step 3: Purification of pre-amplification products

[0110] 1. Remove NadPrep® SP Beads in advance, vortex mix well, and use after equilibration at room temperature for 30 minutes.

[0111] 2. Add 25 μL of NadPrep® SP Beads to the pre-amplification reaction product from step two, mix well, and incubate at 25°C for 5-10 min.

[0112] 3. After briefly centrifuging the PCR tube, place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0113] 4. Slowly add 150 µL of 80% ethanol along the side wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove and discard the supernatant.

[0114] 5. Repeat step 4 once.

[0115] 6. After briefly centrifuging the PCR tube, place it on a magnetic rack and use a 10 µL pipette tip to remove a small amount of residual ethanol, being careful not to pick up the magnetic beads.

[0116] 7. Open the PCR tube cap and let it stand at room temperature for about 5 minutes until the ethanol has completely evaporated.

[0117] 8. Remove the PCR tube, add 27 µL of Nuclease Free Water to the PCR tube, and let it stand at room temperature for about 5 minutes.

[0118] 9. After briefly centrifuging the PCR tube, place it on a magnetic rack for 2 minutes until the liquid is completely clear. Use a pipette to transfer 25 µL of the supernatant to a new PCR tube, and then proceed to step four.

[0119] Step 4: Index PCR amplification (second amplification)

[0120] 1. Take out the Post-Amplification Buffer and Post-Amplification Master Mix and place them on ice to thaw naturally. Mix them evenly and then centrifuge briefly for later use.

[0121] 2. Prepare the reaction system according to Table 5 below in a 0.2 mL PCR tube placed on ice:

[0122] Table 5

[0123]

[0124] 3. Place the PCR tubes into the PCR instrument and start the program shown in Table 6 below:

[0125] Table 6

[0126]

[0127] Step 5: Amplification Library Purification

[0128] 1. Add 50 μL of NadPrep® SP Beads to the PCR tube containing the amplification reaction product from step four, mix well, and incubate at 25°C for 5-10 min.

[0129] 2. After briefly centrifuging the PCR tube, place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0130] 3. Slowly add 150 µL of 80% ethanol along the side wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove and discard the supernatant.

[0131] 4. Repeat step 3 once.

[0132] 5. After briefly centrifuging the PCR tube, place it on a magnetic rack and use a 10 µL pipette tip to remove a small amount of residual ethanol, being careful not to aspirate the magnetic beads.

[0133] 6. Open the PCR tube cap and let it stand at room temperature for about 5 minutes until the ethanol evaporates completely.

[0134] 7. Remove the PCR tube, add 20 µL of TE Solution to the PCR tube, vortex to mix, and incubate at room temperature for 5 min.

[0135] 8. After briefly centrifuging the PCR tube, place it on a magnetic rack for 2 minutes until the liquid is completely clear. Carefully transfer the supernatant to a new 0.2 mL PCR tube for storage using a pipette, being careful not to aspirate the magnetic beads.

[0136] Results analysis: such as Figure 1 As shown, the amplification product fragments using the universal sequence from the 10 nt Illumina sequencing platform have a distribution of 200-800 bp, which meets the requirements of NGS sequencing. However, the amplification product fragments using the universal sequence from the 10 nt BGI sequencing platform have a distribution of 300-2000 bp, which is too large and does not meet the requirements of NGS sequencing. Comparing SEQ ID NO: 4 and SEQ ID NO: 5 reveals significant sequence differences between the two sequencing platforms, indicating that the universal sequence base sequence of the single-cell DNA amplification protocol has a significant impact on the fragment length of the amplification product. It is suitable for the single-cell DNA amplification system and amplification conditions of the Illumina sequencing platform, but not for the BGI platform due to changes in the universal sequence of the amplification primers.

[0137] Example 2:

[0138] This example describes the impact of different lengths of the BGI universal sequence on the amplification results in single-cell DNA amplification. Based on the results of Example 1, the universal sequence of the BGI platform affects the fragment length of the single-cell amplification protocol. This example investigates whether changing the length of the universal sequence affects the amplified fragment length without altering the universal sequence of the BGI platform. We compared the effects of 13nt, 10nt, 9nt, and 8nt BGI universal sequences on the fragment length of the amplified product. The random amplification primers for the 13nt universal sequence of the BGI sequencing platform were SEQ ID NO: 6, for the 10nt universal sequence, SEQ ID NO: 5, for the 9nt universal sequence, SEQ ID NO: 1, and for the 8nt universal sequence, SEQ ID NO: 7. The random amplification primers were diluted to 500 µM with TE buffer before use. The remaining experimental procedures were performed as described in Example 1.

[0139] SEQ ID NO: 6: TACGATCCGACTTNKKNKKNKKNKK;

[0140] SEQ ID NO: 1: ATCCGACTTNKKNKKNKKNKK;

[0141] SEQ ID NO: 7: TCCGACTTNKKNKKNKKNKK.

[0142] Results analysis: such as Figure 2A , Figure 2B , Figure 2C and Figure 2D As shown, the distribution of single-cell DNA amplification product fragments shortens with the decrease in the universal sequence length of the BGI sequencing platform. When using a 9 nt BGI universal sequence, the main peak of the amplification product is approximately 550 bp shorter than that of the 13 nt universal sequence. When using an 8 nt BGI universal sequence, the main peak of the amplification product is the smallest, but the amplification efficiency is slightly lower due to the excessively short universal sequence. This indicates that the universal sequence length has a significant impact on the library fragment length. Considering both amplification efficiency and fragment length, we selected a 9 nt BGI universal sequence as the primer for subsequent optimization tests.

[0143] Example 3:

[0144] This embodiment describes the optimization of the pre-amplification system for a single-cell DNA amplification protocol using a 9 nt BGI universal sequence. As shown in Example 2, the universal sequence length also affects the fragment length of the final library. While the 9 nt BGI universal sequence produces the smallest amplified library fragment length, shorter universal sequences negatively impact index amplification efficiency, severely affecting library yield and richness. Therefore, we aim to optimize the amplification system using 9 nt BGI universal sequence primers to control the fragment size of the single-cell amplification product to meet NGS sequencing requirements. We used cells as templates for single-cell DNA amplification and optimized the pre-amplification system, specifically step 2 (amplification system in step two). The optimized system is shown in Table 7. The remaining experimental procedures are as described in Example 1.

[0145] The constructed single-cell DNA amplification library was hybridized and captured using NadPrep® Hybrid Capture Reagents, HiSNPPanel v1.0, and NadPrep® NanoBlockers. HiSNP Panel v1.0 is a basic SNP backbone assembly that covers the entire human genome at approximately 50 kb intervals and can be used to provide information reflecting the state of genomic composition.

[0146] Table 7

[0147]

[0148] Results Analysis: As shown in Table 8, the size of the inserted fragment and... Figure 3As shown in Table 8, adding a certain proportion of PEG can control the distribution of single-cell DNA amplification product fragments. The addition of Mg... 2+ Concentration can increase the yield of single-cell DNA amplification products; increasing Mg at high concentrations of PEG can improve the yield of single-cell DNA amplification products. 2+ The concentration of PEG is increased to improve amplification coverage and uniformity, and reduce the proportion of allele deletions. Considering coverage, uniformity, and ADO performance, the optimal combination is 10% PEG and 8 mM Mg. 2+ Under these conditions, the distribution of library fragments meets the requirements of NGS and the amplification effect is optimal.

[0149] Table 8 Results of Pre-amplification System Optimization Test for Single-Cell DNA Amplification Protocol

[0150]

[0151] Example 4:

[0152] This example describes the optimization of random sequence combinations in a 9nt BGI universal sequence single-cell DNA amplification protocol. We compared the effect of the random sequence portion of the 9nt BGI random amplification primers on amplification performance in an optimized pre-amplification system. The random amplification primer SEQ ID NO: 1, which we originally used, had a K-series degenerate base (G / T degenerate). We added SEQ ID NO: 3, which has a B-series degenerate base (G / T / C). The random amplification primers were diluted to 500 µM with TE buffer. We mixed SEQ ID NO: 1 and SEQ ID NO: 3 in equal proportions, using 1 μL per reaction. The specific experimental procedure for the single-cell amplification protocol is as described in Example 1. The constructed single-cell DNA amplification library was hybridized and captured using NadPrep® Hybrid Capture Reagents, HiSNP Panel v1.0, and NadPrep® NanoBlockers.

[0153] SEQ ID NO: 3: ATCCGACTTBBBBBB.

[0154] Results analysis: such as Figure 4 As shown, the mixed random amplification primers, compared to the original single random amplification primers, exhibited higher amplification abundance in high-GC regions, further improved coverage and uniformity, and reduced the proportion of allele deletions. Therefore, supplementing random amplification primers with degenerate bases can better improve the effectiveness of single-cell DNA amplification protocols.

[0155] Example 5:

[0156] This embodiment describes the amplification effect of an optimized single-cell DNA amplification protocol on low-volume cfDNA samples. Preimplantation genetic testing (PGD) requires blastomeres or trophectoderm (TE) cell biopsies to obtain the embryo's genetic information. This invasive technique can potentially affect embryo quality and long-term fetal development. While TE cell biopsies at the blastocyst stage are relatively safe, due to embryonic mosaicism, TE cells cannot fully reflect the inner cell mass (ICM) and the genetic information of the final fetus. With the development of biotechnology, cell-free embryonic DNA has been detected in discarded embryo culture media and blastocyst fluid, providing a source of genetic material for non-invasive embryo testing. We simulated cfDNA in embryo culture media or blastocyst fluid using an optimized single-cell DNA amplification protocol to evaluate whether the existing protocol can effectively amplify cfDNA samples and whether it can be used in non-invasive embryo testing applications. We used 100 pg cfDNA samples for testing, and the specific experimental procedure for the single-cell amplification protocol was performed as described in Example 1. The constructed single-cell DNA amplification library was hybridized and captured using NadPrep® Hybrid Capture Reagents, HiSNP Panelv1.0, and NadPrep® NanoBlockers.

[0157] Results analysis: such as Figure 5 As shown, the optimized single-cell DNA amplification protocol amplified 100 pg cfDNA, and the amplification products showed good coverage and uniformity. It is preliminarily determined that this single-cell DNA library construction method can be applied to non-invasive embryo detection.

[0158] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: In the above method, the construction method is formed by integrating steps such as single-cell lysis and DNA release, first-round gradient temperature pre-amplification containing universal sequences, magnetic bead purification, index amplification to distinguish samples for mixed sequencing, and purification of index amplification products. Furthermore, addressing the previous lack of simple single-cell DNA amplification schemes adapted to the domestic BGI sequencing platform, the construction method of this application successfully achieves compatibility with the BGI platform by optimizing the universal sequences of random amplification primers, the pre-amplification system, and the degenerate base combination of random sequences. Specifically, a 9 nt BGI universal sequence is selected to solve the problem that 10 nt universal sequences result in amplification product fragments that are too large and do not meet the requirements of NGS sequencing, while avoiding the universal sequence being too short and affecting index amplification efficiency; the pre-amplification system is determined to contain 10% PEG and 5 mM Mg. 2+The optimal combination of primers improves amplification coverage and uniformity while reducing the proportion of allele deletions. Random sequence degenerate base combinations mix G / T degenerate primers in equal proportions with G / T / C degenerate primers to increase the amplification abundance of high-GC regions. For cell-free DNA, this method also achieves efficient amplification with good coverage and uniformity. This method can be efficiently applied to preimplantation genetic testing, single-cell whole-genome CNV analysis, and amplification and library construction of extremely small samples (such as ancient DNA and forensic micro-biological samples). It solves the problems of traditional methods, such as the need for amplification before library construction leading to long and complex procedures, easy loss of information from low starting sample volumes, poor compatibility with different sequencing platforms, and high requirements for sample integrity. This improves the efficiency, quality, and applicability of single-cell DNA library construction.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a single-cell DNA library, characterized in that, The construction method includes: a) After lysing a single cell, mix it with the first primer and perform the first amplification to obtain the first amplification product; b) Mix the first amplification product with the second primer and perform a second amplification to obtain the single-cell DNA library; The first primer includes a universal amplification fragment and a random amplification fragment, wherein the 3' end of the universal amplification fragment is connected to the 5' segment of the random amplification fragment; The second primer includes an index fragment and a complementary fragment, wherein the 3' end of the index fragment is connected to the 5' end of the complementary fragment; The single-cell DNA library is a library suitable for sequencing on the BGI platform or a library for sequencing on the Illumina platform. Correspondingly, the universal amplification fragment in the first primer is a fragment suitable for the BGI platform or a fragment suitable for the Illumina platform; The fragments suitable for the BGI platform are 8-9 nt in length, and the fragments suitable for the Illumina platform are 10 nt in length.

2. The construction method according to claim 1, characterized in that, The sequence of the fragment applicable to the BGI platform is ATCCGACTT or TCCGACTT.

3. The construction method according to claim 1, characterized in that, The sequence of the fragment applicable to the Illumina platform is the sequence shown in SEQ ID NO:

2.

4. The construction method according to claim 1, characterized in that, The sequence of the randomly amplified fragment includes: a sequence consisting of 2-4 NKK bases, or a sequence consisting of 6-8 B bases.

5. The construction method according to claim 2, characterized in that, The sequence of the first primer suitable for the BGI platform is any one or more sequences shown in SEQ ID NOs: 1, 3 or 7-16; The sequence of the first primer suitable for the Illumina platform is shown in SEQ ID NO:

4.

6. The construction method according to claim 1, characterized in that, After the first amplification, the first purification is performed to obtain the first amplification product.

7. The construction method according to claim 1, characterized in that, After the second amplification, a second purification is performed to obtain the single-cell DNA library.

8. The construction method according to any one of claims 1-7, characterized in that, The first amplification system also contains PEG and / or Mg. 2+ ; Preferably, the PEG includes PEG6000; Preferably, in the first amplification system, the concentration of PEG6000 is 3-20 wt%, more preferably 5-15 wt%. Preferably, in the first amplification system, the Mg 2+ The concentration is 5-10 nM, more preferably 6-9 nM; Preferably, the Mg 2+ Derived from MgCl2; Preferably, the first amplification includes gradient temperature amplification.

9. A single-cell sequencing method, characterized in that, The single-cell sequencing method includes: The single-cell DNA library is constructed using the method for constructing a single-cell DNA library according to any one of claims 1-8; The single-cell DNA library was sequenced using a high-throughput sequencing platform.

10. The single-cell sequencing method according to claim 9, characterized in that, The high-throughput sequencing platform includes the BGI platform or the Illumina platform.

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