Dual-strand dna library quantitative detection kit and method compatible with multiple sequencing platforms

Abstract

This application discloses a kit and method for quantitative detection of double-stranded DNA libraries compatible with multiple sequencing platforms, relating to the field of sequencing technology. The kit includes standards and a mixed primer combination. The standards include a set of serially diluted double-stranded DNA fragments of known concentration, each fragment including a PCR primer binding site sequence of a sequencing adapter from a first sequencing platform or a PCR primer binding site sequence of a paired-end barcode sequencing adapter from a second sequencing platform. The mixed primer combination includes a first primer pair and a second primer pair. The first primer pair specifically binds to the PCR primer binding region in the sequencing adapter from the first sequencing platform, and the second primer pair specifically binds to the PCR primer binding region in the paired-end barcode sequencing adapter from the second sequencing platform. The molar ratio of the first primer pair to the second primer pair in the mixed primer combination is 1:1. This kit can perform quantitative detection of libraries from different sequencing platforms. All samples are calculated using the same standard curve run, reducing systematic errors.

Description

Technical Field

[0001] This application relates to the field of sequencing technology, and in particular to a double-stranded DNA library quantification kit and method compatible with multiple sequencing platforms. Background Technology

[0002] In high-throughput sequencing (NGS) workflows, accurate quantification of constructed sequencing libraries is a crucial step in ensuring accurate loading of sequencing instruments and guaranteeing the yield and quality uniformity of sequencing data. Quantitative real-time PCR (qPCR) has become the gold standard for library quantification due to its high sensitivity, high accuracy, and wide dynamic range. Currently, mainstream qPCR library quantification kits on the market are typically designed for library structures specific to a single sequencing platform. Their standards and primer systems are platform-specific. When laboratories use or switch between different sequencing platforms simultaneously, different quantification kits need to be purchased and operated separately, which not only increases costs and complicates the experimental process, but also reduces work efficiency and may introduce systematic errors due to the use of different standard curves.

[0003] Therefore, there is an urgent need to develop a universal library quantification kit that is compatible with multiple sequencing platforms to achieve unified, efficient and accurate quantification of libraries from different platforms. Summary of the Invention

[0004] The main objective of this application is to propose a double-stranded DNA library quantification kit and method compatible with multiple sequencing platforms, aiming to solve the problem that existing double-stranded DNA library quantification kits cannot be used across platforms.

[0005] To achieve the above objectives, in a first aspect, this application proposes a double-stranded DNA library quantification kit compatible with multiple sequencing platforms, the kit comprising standards and a mixed primer combination; The standard comprises a set of serially diluted double-stranded DNA fragments at known concentrations, wherein the double-stranded DNA fragments include PCR primer binding site sequences of sequencing adapters of a first sequencing platform or PCR primer binding site sequences of paired-end barcode sequencing adapters of a second sequencing platform. The hybrid primer combination includes a first primer pair and a second primer pair. The first primer pair can specifically bind to the PCR primer binding region in the sequencing adapter of the first sequencing platform, and the second primer pair can specifically bind to the PCR primer binding region in the paired-end barcode sequencing adapter of the second sequencing platform. The molar ratio of the first primer pair to the second primer pair in the hybrid primer combination is 1:1. In one embodiment, the concentration range of the standard is 0.0002~20 pM; The concentration range of the standards covers at least six orders of magnitude. In one embodiment, the concentration of the standard includes 0.0002 pM, 0.002 pM, 0.02 pM, 0.2 pM, 2 pM, and 20 pM.

[0006] In one embodiment, the kit includes a premix for providing an amplification reaction system for the DNA library to be tested.

[0007] In one embodiment, the premix comprises DNA polymerase, dNTPs, stabilizers, fluorescent dyes, and magnesium ions.

[0008] In one embodiment, the kit includes a buffer solution for diluting the DNA library to be tested.

[0009] In one embodiment, the first sequencing platform is the Illumina platform, and the second platform is the MGI platform.

[0010] In one embodiment, the double-stranded DNA fragment includes the PCR primer binding site sequence of a sequencing adapter for a first sequencing platform.

[0011] In one embodiment, the nucleic acid sequences of the first primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid sequences of the second primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0012] Secondly, this application also proposes a method for quantitative detection of double-stranded DNA libraries, using a double-stranded DNA library quantitative detection kit compatible with multiple sequencing platforms provided in the first aspect of this application, including the following steps: S1. Use the mixed primer combination to perform qPCR amplification on the standard, and generate a standard curve based on the obtained standard Ct value and the known concentration of the standard; S2. Adjust the double-stranded DNA library to be tested to the preset concentration, perform qPCR amplification using the same qPCR amplification reaction system as in S1, and obtain the Ct value; S3. Data Analysis: The initial concentration of the double-stranded DNA library to be tested is obtained based on the Ct value and the standard curve.

[0013] The proposed multi-sequencing platform compatible double-stranded DNA library quantification kit enables the quantification of double-stranded DNA libraries on at least two sequencing platforms in a single experiment, simplifying laboratory procedures and reducing testing costs. Furthermore, all samples can be calculated using a standard curve generated from the same run, reducing batch-to-batch and platform-to-platform systematic errors and enhancing data comparability.

[0014] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 The standard qPCR amplification reaction curve provided for this application; Figure 2 qPCR amplification reaction curves of Illumina library samples of unknown concentration provided in this application; Figure 3 qPCR amplification reaction curve of MGI Barcode library samples of unknown concentration provided in this application; Figure 4 The standard qPCR amplification reaction curve provided in this application is shown at an annealing temperature of 58℃. Figure 5 The standard qPCR amplification reaction curve provided for this application at an annealing temperature of 62℃ is shown.

[0017] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0019] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0020] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0021] In next-generation sequencing (NGS) workflows, accurate quantification of the constructed sequencing library is a crucial quality control step that determines the success of subsequent sequencing and data quality. Accurate library concentration is the sole basis for calculating the loading amount on the sequencer, directly affecting the density of sequencing clusters, the total amount of data produced, the uniformity of sequencing signals, and the quality of the final read length. Insufficient loading leads to inadequate effective cluster density on the chip or flow cell, resulting in wasted sequencing capacity and substandard data output; excessive loading causes inter-cluster signal interference, increasing the sequencing error rate and severely impacting data usability. Therefore, achieving accurate library quantification is a prerequisite for ensuring efficient, economical, and reliable sequencing experiments.

[0022] Currently, commonly used nucleic acid quantification methods include broad-spectrum quantification based on fluorescent dyes (such as PicoGreen) and absolute quantification based on quantitative PCR (qPCR). Among these, qPCR quantification, with its unparalleled advantages, is widely recognized as the "gold standard" for library quantification. Its principle lies in the fact that the primers used in qPCR specifically target constant regions of sequencing adapters, selectively detecting "effective" library molecules with complete and correct adapters at both ends, and then exponentially amplifying and detecting these molecules in real time. By comparing with standards of known concentrations, the molar concentration of effective libraries in the sample can be absolutely quantified. This method boasts extremely high sensitivity (detecting femtomolar concentrations), excellent accuracy (overcoming interference from dye methods due to primer dimers, free adapters, and other invalid molecules), and a wide dynamic range (spanning multiple orders of magnitude), thus providing sequencers with the most accurate and reliable sample loading concentration data.

[0023] However, current mainstream commercial qPCR library quantification kits are typically designed with strict platform limitations. The core components of these kits—including standards for establishing standard curves and primer pairs for initiating specific amplification—are designed and optimized for specific adapter sequences and structures specific to a single sequencing platform (especially the Illumina platform, which holds a major market share). For example, their primers can only recognize and bind to the PCR primer-binding regions of Illumina platform adapters, and their standards are double-stranded DNA fragments that mimic the Illumina platform library structure.

[0024] However, with the diversification of sequencing technologies, different sequencing platforms (such as BGI DNBSEQ series from BGI Genomics and Ion Torrent series from Thermo Fisher Scientific) employ sequencing adapters with drastically different sequences and structures due to their different technical principles. This results in qPCR quantitative kits designed based on the Illumina platform being completely incompatible with libraries from other platforms. When research or clinical laboratories need to run multiple sequencing platforms simultaneously or switch between them at different times to meet the needs of different projects, they are forced to purchase dedicated quantitative kits for each platform separately. This situation leads to the following problems: (1) High cost: The laboratory needs to repeatedly invest in purchasing multiple sets of reagent kits with similar functions but incompatible with each other, which increases reagent inventory and financial pressure.

[0025] (2) Cumbersome process: Technicians must switch between different quantitative processes, standards and experimental conditions for different platform libraries, which significantly increases the complexity of operation and labor costs.

[0026] (3) Inefficient: It is impossible to perform unified and batch quantitative detection on samples from different platforms, which reduces the overall work efficiency.

[0027] (4) Potential systematic error risk: Since different kits may use different standardized products, enzyme systems, buffer components and quantitative algorithms, using multiple independent standard curves to quantify libraries on different platforms may introduce systematic biases that are difficult to calibrate at the data level, affecting the comparability of data output between different platforms, and thus potentially causing problems for integrated analysis projects involving multi-platform data.

[0028] Therefore, there is an urgent need in this field for a solution that can overcome platform limitations and achieve universal quantification, in order to simplify experimental operations, save costs, improve efficiency, and ensure consistent and comparable quantitative results across different sequencing technologies, thereby adapting to the new trend of diversified and high-throughput development in modern sequencing laboratories.

[0029] Based on the above problems, in the first aspect, this application proposes a double-stranded DNA library quantification kit compatible with multiple sequencing platforms, including standards and mixed primer combinations.

[0030] The standard includes a set of serially diluted double-stranded DNA fragments of known concentration, wherein the double-stranded DNA fragments include PCR primer binding site sequences of sequencing adapters of a first sequencing platform or PCR primer binding site sequences of barcode sequencing adapters of a second sequencing platform.

[0031] The mixed primer combination includes a first primer pair and a second primer pair. The first primer pair can specifically bind to the PCR primer binding region in the sequencing adapter of the first sequencing platform, and the second primer pair can specifically bind to the PCR primer binding region in the paired-end barcode sequencing adapter of the second sequencing platform. The molar ratio of the first primer pair and the second primer pair in the mixed primer combination is 1:1.

[0032] The key design of the double-stranded DNA library quantification kit compatible with multiple sequencing platforms provided in this application is that the standard only carries the PCR primer binding site sequence of the sequencing adapter of the first sequencing platform or the PCR primer binding site sequence of the paired-end barcode sequencing adapter of the second sequencing platform, while the mixed primer combination includes a first primer pair that can specifically bind to the PCR primer binding region in the sequencing adapter of the first sequencing platform, and a second primer pair that can specifically bind to the PCR primer binding region in the paired-end barcode sequencing adapter of the second sequencing platform.

[0033] In qPCR reactions: For double-stranded DNA libraries from the first sequencing platform, the primer binding sites are completely consistent with the standards. The first primer pair can bind and amplify efficiently, and its amplification efficiency is highly consistent with the standards, resulting in accurate quantitative results.

[0034] For double-stranded DNA libraries from a second sequencing platform, the primer binding sites differ from those of the standard, but the second primer pair can specifically bind to them and initiate amplification. Although the primer sequences differ from the standard, under the same and optimized qPCR system and thermal cycling conditions, as long as the primer efficiencies are similar, the amplification kinetics (the linear relationship between Ct value and the number of initial template logs) still follow the same rules. Therefore, by using the same standard curve, the equivalent concentration of double-stranded DNA libraries with paired-end barcode sequencing adapters from the second sequencing platform can also be calculated.

[0035] In other words, this setup ensures that two double-stranded DNA libraries with different sequencing adapters can be simultaneously labeled using a single standard curve, achieving universal quantification.

[0036] If the standard includes two types of sequences—one containing the PCR primer binding site sequence of the sequencing adapter from the first sequencing platform, and the other containing the PCR primer binding site sequence of the paired-end barcode sequencing adapter from the second sequencing platform—unnecessary complexity will be introduced due to the slight difference in the efficiencies of the two primers, affecting the accuracy of the standard curve. Using a sequence from a single sequencing platform as the standard ensures that the establishment of the standard curve is based on a single, stable amplification efficiency.

[0037] By controlling the molar ratio of the first primer pair to the second primer pair in the mixed primer combination to 1:1, the qPCR amplification efficiency of double-stranded DNA libraries with sequencing adapters from different sequencing platforms is kept consistent, reducing errors and making the detection results more accurate.

[0038] In some implementations, the concentration range of the standard is 0.0002 to 20 pM, and the concentration range of the standard covers at least 6 orders of magnitude.

[0039] In absolute quantitative qPCR, a standard curve covering the expected concentration range of the sample (double-stranded DNA library) can be constructed using standards. Ensuring that the concentration range of the standards covers at least six orders of magnitude is the cornerstone of obtaining reliable data. Covering less than six concentration ranges significantly increases the uncertainty and risk of the quantitative results.

[0040] Preferably, in some embodiments, the concentration of the standard includes 0.0002 pM, 0.002 pM, 0.02 pM, 0.2 pM, 2 pM, and 20 pM.

[0041] In some implementations, the kit includes a premixed solution to provide an amplification reaction system for the DNA library to be tested.

[0042] In some embodiments, the premix includes DNA polymerase, dNTPs, stabilizers, fluorescent dyes, and magnesium ions.

[0043] The premixed solution used to provide the amplification reaction system for the DNA library to be tested can provide a consistent amplification kinetic environment for the first primer pair and the second primer pair to their respective templates (double-stranded DNA libraries with adapters from different sequencing platforms). This eliminates the possible degradation in amplification efficiency caused by adapter sequence differences due to sequencing platform differences, thereby ensuring the accuracy and reliability of quantifying multi-platform libraries using a single standard curve.

[0044] Specifically, the DNA polymerase includes a DNA polymerase that prevents nonspecific amplification, preferably a hot-start DNA polymerase, and more preferably a hot-start Taq DNA polymerase. The fluorescent dye includes SYBR Green I fluorescent dye, which has a sensitive fluorescence signal and can stably reflect the amount of DNA amplification. Preferably, the magnesium ions are MgCl2.

[0045] In some implementations, the kit also includes a buffer solution for diluting the DNA library to be tested.

[0046] In some implementations, the first sequencing platform is the Illumina platform, and the second platform is the MGI platform. The double-stranded fragment includes the PCR primer binding site sequence of the sequencing adapter of the first sequencing platform (Illumina platform) or the PCR primer binding site sequence of the paired-end barcode sequencing adapter of the second sequencing platform (MGI platform).

[0047] Preferably, the double-stranded DNA fragment includes the PCR primer binding site sequence of the sequencing adapter of the first sequencing platform (Illumina platform). Further, the nucleic acid sequences of the first primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid sequences of the second primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0048] This application also proposes a method for quantitative detection of double-stranded DNA libraries, using a double-stranded DNA library quantitative detection kit compatible with multiple sequencing platforms as described above, including the following steps: S1. Use the mixed primer combination to perform qPCR amplification on the standard, and generate a standard curve based on the obtained standard Ct value and the known concentration of the standard; S2. Adjust the double-stranded DNA library to be tested to the preset concentration, perform qPCR amplification using the same qPCR amplification reaction system as in S1, and obtain the Ct value; S3. Data Analysis: The initial concentration of the double-stranded DNA library to be tested is obtained based on the Ct value and the standard curve.

[0049] In some implementations, the premixed solution described above is used in qPCR amplification, as shown in Table 1. Its function is the same as previously stated and will not be repeated here.

[0050] Table 1. Composition of the premixed solution

[0051] In some implementations, the qPCR reaction program is: 95°C, 5 min; 95°C, 3 s; 58–62°C, 45 s; for a total of 35 cycles. For example, the annealing temperature can be 58°C, 59°C, 60°C, 61°C, or 62°C, etc.

[0052] The following specific examples provide further details.

[0053] Example 1 This embodiment provides a double-stranded DNA library quantification detection kit compatible with multiple sequencing platforms, specifically including: (1) Standards: a series of double-stranded DNA fragments containing Illumina primer binding sites, with concentrations of 20 pM, 2 pM, 0.2 pM, 0.02 pM, 0.002 pM and 0.0002 pM, respectively.

[0054] (2) 2×qPCR premix: hot-start Taq DNA polymerase, dNTPs, MgCl2, SYBR Green I dye and enhancer.

[0055] (3) 10× mixed primer combination: The first primer pair contains universal primer pairs for the sequencing adapters of the Illumina sequencing platform, specifically primer pairs for the Illumina P5 and P7 adapters, as shown in SEQ ID NO.1 and SEQ ID NO.2; The second primer pair contains universal primer pairs for the sequencing structure of the MGI sequencing platform, specifically primers for double-stranded sequencing adapters for the MGI paired-end barcode, as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0056] The concentrations of the first and second primer pairs were both 10 μM. After mixing, the final working concentration of each primer was 0.2 μM. The specific primer sequences are shown in Table 2.

[0057] Table 2. Nucleic acid sequence listing of mixed primer combinations

[0058] (4) Buffer: 10 mM Tris-HCl, 0.05% Tween-20, pH 8.0.

[0059] Example 2 This embodiment uses the kit from Example 1 to perform quantitative detection on samples from a mixed-platform library. The specific steps are as follows: (1) Standard curve reaction setup: Take 4 μL of each of the 6 gradient standards and add them to 16 μL of qPCR reaction mixture (composition: 10 μL 2× premix, 2 μL 10× mixed primer combination, 4 μL nuclease-free water). Set up 3 replicates for each concentration.

[0060] (2) Sample detection: Illumina library samples of unknown concentration and MGI two-end barcode library samples were diluted 1:10000 times with buffer solution. 4 μL of the diluted sample was added to 16 μL of the same reaction mixture as in step 1. Three replicates were set for each sample.

[0061] (3) qPCR amplification: The prepared amplification systems of a series of standards, the amplification systems of Illumina library samples of unknown concentration and the amplification systems of MGI two-end barcode library samples of unknown concentration were placed in a qPCR instrument for amplification. The amplification program was: 95℃, 5 min; 95℃, 3 s; 60℃, 45 s; a total of 35 cycles.

[0062] (4) Data analysis: The instrument software automatically generates a linear regression standard curve (R²) based on the Ct value of the standard and the known concentration. 2 >0.99). Substitute the Ct value of the sample to be tested into the equation to calculate its diluted concentration, and then multiply by the dilution factor to obtain the accurate concentration of the original library. The qPCR amplification curve is shown below. Figure 1 , 2 As shown in Figure 3, where Figure 1 qPCR amplification curves for a series of standards, Figure 2 The qPCR amplification curves are for Illumina library samples of unknown concentration. Figure 3 The image shows the qPCR amplification curves for MGIBarcode library samples of unknown concentration.

[0063] Combination Figures 1-3 As can be seen, both the Illumina library and the MGI paired-end barcode library exhibited good amplification curves, with the Ct value showing an excellent linear relationship with dilution. The concentrations of both libraries calculated using the same standard curve, verified against known concentrations of control standards, showed an error rate of less than 15%, fully meeting the accuracy requirements for sequencing loading.

[0064] Comparative Example 1 This comparative example uses the kit from Example 1 to quantify samples from a mixed-platform library. The specific steps are as follows: (1) Standard curve reaction setup: Take 4 μL of each of the 6 gradient standards and add them to 16 μL of qPCR reaction mixture (composition: 10 μL 2× premix, 2 μL 10× mixed primer combination, 4 μL nuclease-free water). Set up 3 replicates for each concentration.

[0065] (2) Sample detection: Illumina library samples of unknown concentration and MGI two-end barcode library samples were diluted 1:10000 times with buffer solution. 4 μL of the diluted sample was added to 16 μL of the same reaction mixture as in step 1. Three replicates were set for each sample.

[0066] (3) qPCR amplification: The prepared amplification systems of a series of standards, the amplification systems of Illumina library samples of unknown concentration and the amplification systems of MGI two-end barcode library samples of unknown concentration were placed in a qPCR instrument for amplification. The amplification program was: 95℃, 5 min; 95℃, 3 s; 58℃, 45 s; a total of 35 cycles.

[0067] The qPCR amplification curves of a series of standards are as follows: Figure 4 As shown. From Figure 4 It can be seen that when the annealing temperature is set at 58℃, the primer binding efficiency is poor for low-concentration standards, resulting in abnormal gradients in the standard curve and the standard curve not satisfying R. 2 ≥0.99.

[0068] Comparative Example 2 This comparative example uses the kit from Example 1 to quantify samples from a mixed-platform library. The specific steps are as follows: (1) Standard curve reaction setup: Take 4 μL of each of the 6 gradient standards and add them to 16 μL of qPCR reaction mixture (composition: 10 μL 2× premix, 2 μL 10× mixed primer combination, 4 μL nuclease-free water). Set up 3 replicates for each concentration.

[0069] (2) Sample detection: Illumina library samples of unknown concentration and MGI two-end barcode library samples were diluted 1:10000 times with buffer solution. 4 μL of the diluted sample was added to 16 μL of the same reaction mixture as in step 1. Three replicates were set for each sample.

[0070] (3) qPCR amplification: The prepared amplification systems of a series of standards, the amplification systems of Illumina library samples of unknown concentration and the amplification systems of MGI two-end barcode library samples of unknown concentration were placed in a qPCR instrument for amplification. The amplification program was: 95℃, 5 min; 95℃, 3 s; 62℃, 45 s; a total of 35 cycles.

[0071] The qPCR amplification curves of a series of standards are as follows: Figure 5 As shown. From Figure 5 It can be seen that when the annealing temperature is set at 62℃, the primer binding efficiency is poor for low-concentration standards, resulting in abnormal gradients in the standard curve and the standard curve not satisfying R. 2 ≥0.99.

[0072] In summary, the double-stranded DNA library quantification kit compatible with multiple sequencing platforms provided in this application has the following advantages: High compatibility: A single set of reagents and a single experiment are sufficient to quantify libraries from both Illumina and MGI platforms, simplifying laboratory workflows.

[0073] Cost-effectiveness: Eliminating the need to purchase quantitative reagent kits for two different platforms reduces consumable costs.

[0074] Consistency of results: All samples are calculated using the standard curve generated in the same run, which reduces systematic errors between batches and platforms, and makes the data more comparable.

[0075] Easy to operate: Users do not need to change primers or standards for different platforms, which improves operational efficiency and reduces the probability of errors.

[0076] High precision and sensitivity: The use of the SYBR Green I dye method and standard curves of 6 orders of magnitude ensures a wide dynamic range and high sensitivity for quantitative analysis.

[0077] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A double-stranded DNA library quantitative detection kit compatible with multiple sequencing platforms, characterized in that, The kit includes standards and a mixed primer combination; The standard comprises a set of serially diluted double-stranded DNA fragments at known concentrations, wherein the double-stranded DNA fragments include PCR primer binding site sequences of sequencing adapters of a first sequencing platform or PCR primer binding site sequences of paired-end barcode sequencing adapters of a second sequencing platform. The hybrid primer combination includes a first primer pair and a second primer pair. The first primer pair can specifically bind to the PCR primer binding region in the sequencing adapter of the first sequencing platform, and the second primer pair can specifically bind to the PCR primer binding region in the paired-end barcode sequencing adapter of the second sequencing platform. The molar ratio of the first primer pair to the second primer pair in the hybrid primer combination is 1:

1.

2. The kit of claim 1, wherein The concentration range of the standard is 0.0002~20 pM; The concentration range of the standards covers at least six orders of magnitude.

3. The kit according to claim 2, characterized in that, The concentrations of the standards include 0.0002 pM, 0.002 pM, 0.02 pM, 0.2 pM, 2 pM, and 20 pM.

4. The kit according to claim 1, characterized in that, The kit includes a premixed solution for providing an amplification reaction system for the DNA library to be tested.

5. The kit according to claim 4, characterized in that, The premix includes DNA polymerase, dNTPs, stabilizers, fluorescent dyes, and magnesium ions.

6. The kit according to claim 1, characterized in that, The kit includes a buffer solution for diluting the DNA library to be tested.

7. The kit according to claim 1, characterized in that, The first sequencing platform is the Illumina platform, and the second platform is the MGI platform.

8. The kit according to claim 7, characterized in that, The double-stranded DNA fragment includes the PCR primer binding site sequence of the sequencing adapter of the first sequencing platform.

9. The kit according to claim 7, characterized in that, The nucleic acid sequences of the first primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid sequences of the second primer pair are shown in SEQ ID NO.3 and SEQ ID NO.

4.

10. A method for quantitative detection of double-stranded DNA libraries, characterized in that, The detection is performed using the double-stranded DNA library quantification kit compatible with any one of claims 1 to 9, comprising the steps of: S1. Use the mixed primer combination to perform qPCR amplification on the standard, and generate a standard curve based on the obtained standard Ct value and the known concentration of the standard; S2. Adjust the double-stranded DNA library to be tested to the preset concentration, perform qPCR amplification using the same qPCR amplification reaction system as in S1, and obtain the Ct value; S3. Data Analysis: The initial concentration of the double-stranded DNA library to be tested is obtained based on the Ct value and the standard curve.

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