Multiplex PCR amplification efficiency adjusting method, system and equipment

Abstract

The invention discloses a method, a system and equipment for adjusting the amplification efficiency of multiple PCR (Polymerase Chain Reaction). In order to solve the problem of non-uniformity of multi-PCR amplification, the invention provides a multi-PCR amplification efficiency adjustment method, and the method adjusts the primer input concentration in the multi-PCR amplification reaction process through a primer concentration adjustment coefficient formula so as to achieve the purpose of uniformity of multi-PCR amplification. The method is simple and convenient to operate and high in accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection. Specifically, the present invention relates to a method, system and device for adjusting the amplification efficiency of multiplex PCR. Background Art

[0002] Multiplex PCR amplification technology is a technology used in molecular biology to simultaneously amplify multiple DNA or RNA fragments. That is, multiple pairs of specific amplification primers are simultaneously added to an amplification reaction system, and multiple amplification products can be obtained in one reaction. The detection results can be obtained by further analyzing the amplification products. In actual nucleic acid analysis and detection, the use of multiplex PCR can greatly simplify the operation and shorten the time. At the same time, compared with conventional PCR, no additional operations and instruments are required. Since the first report in 1988, multiplex PCR has become a rapid and convenient method for detecting target nucleic acids in clinical and research laboratories. Multiplex PCR has been successfully applied in many fields, including gene deletion analysis, gene mutation and gene polymorphism analysis, mRNA quantitative analysis, RNA detection, and genomic sequence analysis. In the diagnosis of infectious diseases, multiplex PCR has also played an important role in the identification of viruses, bacteria, parasites, bacteria, and the analysis of drug-resistant genes.

[0003] Although multiplex PCR amplification technology has broad application prospects, it also faces many difficulties and challenges in actual operation. Usually, a very careful primer design and multiple rounds of screening are required to establish an effective multiplex PCR program. A common problem in multiplex PCR is the uneven amplification between different primer pairs for target fragments. Even some primers in the multiplex system do not have any effective amplification at all, and its reproducibility is poor. For a successful multiplex PCR reaction system, the following factors need to be considered, including the balance between the usage concentration of primers, the concentration of PCR buffer, the concentration of magnesium ions and dNTPs, the temperature of each step in the PCR cycle, the amount of template DNA, and the amount of Taq DNA polymerase. The optimal combination of the annealing temperature and buffer system in PCR is very necessary to ensure the specificity of multiplex PCR. A certain ratio needs to be maintained between the concentration of magnesium ions and dNTPs, and at the same time, the adjustment of primer concentration is also very necessary.

[0004] It tends to amplify one or certain sequences, that is, the proportion of the final product is different from that of the starting template, resulting in detection bias, which is a common phenomenon in multiplex PCR. This is mainly because the amounts of enzymes and single nucleotides in the multiplex PCR amplification system are limited. All primer pairs in the reaction compete for these limited enzymes and single nucleotides, but the amplification efficiencies of these primer pairs are not the same. The influencing factors for the non-uniform amplification efficiency of multiplex PCR include primer sequences, target region sequences, and primer input concentrations. Under specific detection targets, the adjustment spaces for primer sequences and target regions are relatively small. The adjustment of the uniform amplification efficiency of multiplex PCR mainly starts from the primer concentration. Therefore, determining the final concentration of each primer becomes a key factor in establishing a multiplex PCR system. Regarding the primer concentration problem, generally, after amplifying with a single primer pair, the amplified primers are subjected to gel electrophoresis, and the primer concentration is adjusted according to the color intensity of the gel electrophoresis, or new primers are replaced. However, the judgment of the color intensity of gel electrophoresis often depends on the subjective observation of the experimenter, which may lead to inaccurate and inconsistent judgment results. In addition, this method has a cumbersome operation process and is time-consuming, increasing the experimental cost and time cost; the results between batches are not comparable, and when there are many primer pairs, the results are not referenceable; there is interference between primers, and the amplification situation of a single primer pair cannot directly reflect the true performance of multiplex amplification. Therefore, it is of great significance to provide a new method for adjusting the concentration of multiplex PCR primers. Summary of the Invention

[0005] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method for adjusting the primer concentration to ensure the uniformity of multiplex PCR amplification.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for adjusting the amplification efficiency of multiplex PCR.

[0008] Further, the method includes:

[0009] 1): Detect the multiplex PCR amplification products to obtain the quantity information of each amplicon;

[0010] 2): Calculate the Gini coefficient of all amplicons according to the quantity information of each amplicon;

[0011] 3): If the Gini coefficient > a, according to Formula 1: Adjust the input concentration of the multiplex PCR primers and remix the primers; where index i is the concentration adjustment coefficient corresponding to the i-th primer pair, a is the expected Gini coefficient, b is the base number, and R i$R_i$ is the quantity information of the $i$-th pair of primers in the previous round of detection, $R$ is the expected target value of the quantity of each amplicon, $K$ is the segmented adjustment coefficient, which is $K_1$ when $R_i < R$ and $K_2$ when $R_i \geq R$;

[0012] 4): Repeat steps 1) to 3) until the Gini coefficient $\leq a$.

[0013] Furthermore, the method for obtaining the quantity information of each amplicon includes fluorescence PCR, digital PCR, capillary electrophoresis, microarray chips, microfluidic chips, and high-throughput sequencing.

[0014] Preferably, the method for obtaining the quantity information of each amplicon is high-throughput sequencing.

[0015] Furthermore, the parameters $a$, $b$, $K_1$, and $K_2$ are all variable parameters.

[0016] Preferably, the selectable range of parameter $a$ is 0.2 - 0.4; the selectable range of parameter $b$ is 2 - 10; the selectable range of parameter $K_1$ is 0.3 - 2; the selectable range of parameter $K_2$ is 0.1 - 1.

[0017] Furthermore, calculate index i and then according to formula 2: the primer input concentration corresponding to each amplicon = original primer concentration × index i ×V 基 / V 终 calculate the re-input concentration of the primer;

[0018] where index i is the concentration adjustment coefficient corresponding to the $i$-th pair of primers; $V$ 基 is the reference volume for each primer mixing, index i ×V 基 is the final volume of each pair of primer mixing; $V$ 终 is the re-solvation volume after all primers are mixed each time.

[0019] Furthermore, when performing the $n$-th ($n \geq 2$) round of primer concentration adjustment, after calculating index i the primer input concentration should be calculated using the composite concentration coefficient, that is, according to formula 3: the primer input concentration corresponding to each amplicon = original primer concentration × composite index i ×V 基 / V 终 calculate the re-input concentration of the primer, and the composite index i is the product of the index in the $(n - 1)$-th round i and the index in the $n$-th round i .

[0020] The second aspect of the present invention provides a system for adjusting the amplification efficiency of multiplex PCR.

[0021] Further, the system includes:

[0022] Amplification product detection module: Detect the multiplex PCR amplification products to obtain the quantity information of each amplicon;

[0023] Gini coefficient calculation module: Calculate the Gini coefficient of all amplicons according to the quantity information of each amplicon;

[0024] Primer concentration adjustment module: If the Gini coefficient > a, according to formula 1: Adjust the input concentration of the multiplex PCR primers and remix the primers; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers, a is the expected Gini coefficient, b is the base number, R i is the quantity information of the i-th pair of primers in the previous round of detection, R is the expected target value of the quantity of each amplicon, K is the segmented adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R;

[0025] Repeated adjustment module: Repeat the steps in the above amplification product detection module, Gini coefficient calculation module, and primer concentration adjustment module until the Gini coefficient ≤ a.

[0026] Further, the method for obtaining the quantity information of each amplicon includes fluorescence PCR, digital PCR, capillary electrophoresis, microarray chip, microfluidic chip, and high-throughput sequencing.

[0027] Preferably, the method for obtaining the quantity information of each amplicon is high-throughput sequencing.

[0028] Further, the parameters a, b, K1, and K2 are all variable parameters.

[0029] Preferably, the selectable range of parameter a is 0.2 - 0.4; the selectable range of parameter b is 2 - 10; the selectable range of parameter K1 is 0.3 - 2; the selectable range of parameter K2 is 0.1 - 1.

[0030] Further, after calculating index i according to formula 2: The input concentration of the primer corresponding to each amplicon = the original primer concentration × index i × V 基 / V 终 calculate the adjusted input concentration of the primer;

[0031] where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers; V 基 is the reference volume for each primer mixing, indexi ×V 基 is the final volume of the mixture of each pair of primers; V 终 is the reconstitution volume after mixing all primers each time.

[0032] Further, when performing the primer concentration adjustment in the nth (n≥2) round, calculate index i After that, the input concentration of the primer should be calculated using the composite concentration coefficient, that is, according to Formula 3: the input concentration of the primer corresponding to each amplicon = the original primer concentration × composite index i ×V 基 / V 终 Calculate the re-input concentration of the primer, and the composite index i is the product of the index in the (n - 1)th round i and the index in the nth round i .

[0033] The third aspect of the present invention provides a computer device.

[0034] Further, the computer device includes:

[0035] a memory and a processor, the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method described in the first aspect of the present invention is implemented.

[0036] The fourth aspect of the present invention provides a computer-readable storage medium.

[0037] Further, a computer program is stored on the computer-readable storage medium, and when the computer program is executed by a processor, the method described in the first aspect of the present invention is implemented.

[0038] The fifth aspect of the present invention provides an application in any of the following aspects.

[0039] Further, the application includes:

[0040] 1) The application of the method described in the first aspect of the present invention in the primer concentration adjustment of multiplex PCR amplification reaction;

[0041] 2) The application of the method described in the first aspect of the present invention in the sequencing of multiplex PCR amplification products.

[0042] Advantages and benefits of the present invention:

[0043] The present invention provides a novel method for adjusting the primer concentration of multiplex PCR, which is applicable to primers designed for any test purpose. Compared with the method of adjusting the primer concentration by the color intensity of gel electrophoresis, this method is simple and convenient to operate, reduces the experimental cost, can simultaneously adjust up to 2000 pairs of primers, and has higher accuracy. Brief Description of the Drawings

[0044] Figure 1 It is a schematic flow chart of the method for adjusting the multiplex PCR amplification efficiency provided by the present invention;

[0045] Figure 2 It is a schematic structural diagram of the system for adjusting the multiplex PCR amplification efficiency provided by the present invention;

[0046] Figure 3 It is a schematic structural diagram of the computer device provided by the present invention. Detailed Embodiments

[0047] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0048] In some processes described in the specification and claims of the present invention and the above drawings, there are multiple operations that appear in a specific order. However, it should be clearly understood that these operations can be executed not in the order in which they appear in this article or in parallel. The operation numbers such as 101, 102, etc. are only used to distinguish each different operation, and the numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations can be executed in sequence or in parallel. It should be noted that the descriptions such as "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, nor do they limit that "first" and "second" are of different types.

[0049] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods; unless otherwise specified, the reagents, materials, etc. used in the following embodiments can all be obtained from commercial channels.

[0050] Figure 1 It is a schematic flow chart of the method for adjusting the multiplex PCR amplification efficiency provided by the present invention. Specifically, the method includes:

[0051] 101: Detect the multiplex PCR amplification products to obtain the quantity information of each amplicon;

[0052] 102: Calculate the Gini coefficient of all amplicons according to the quantity information of each amplicon;

[0053] 103: If the Gini coefficient > a, according to formula 1: Adjust the input concentration of the multiplex PCR primers and remix the primers; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers, a is the expected Gini coefficient, b is the base number, R iLet \(R_i\) be the quantity information of the \(i\)-th pair of primers in the previous round of detection, \(R\) be the expected target value of the quantity of each amplicon, and \(K\) be the segmented adjustment coefficient, which is \(K_1\) when \(R_i < R\) and \(K_2\) when \(R_i\geq R\).

[0054] 104: Repeat steps 101 to 103 until the Gini coefficient \(\leq a\).

[0055] In some embodiments of the present invention, before detecting the multiplex PCR amplification products, it further includes the steps of designing multiplex PCR primers for the test purpose and mixing the primers to run the multiplex PCR library construction program. Running the multiplex PCR library construction program includes the steps of purifying the multiplex amplification products, amplifying the library, purifying the library amplification products, and quantifying the library.

[0056] In the present invention, a primer refers to a short nucleic acid molecule. It can anneal to a complementary target nucleic acid molecule through nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. The primer can be extended along the target nucleic acid molecule by a polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, where the sequence of the primer is specific to the target nucleic acid molecule.

[0057] A target nucleic acid molecule refers to a nucleic acid molecule for which detection, quantification, qualitative detection, or a combination thereof is intended. The nucleic acid molecule does not have to be in a purified form. Various other nucleic acid molecules can also coexist with the target nucleic acid molecule. For example, the target nucleic acid molecule can be a specific nucleic acid molecule intended to be amplified. If necessary, purification or separation of the target nucleic acid molecule can be carried out by methods known to those skilled in the art, such as using commercially available purification kits, etc. In the present invention, the target nucleic acid molecule is not restricted, and the method provided by the present invention is effective for adjusting the primer concentration of any target nucleic acid molecule.

[0058] In some embodiments, the method for obtaining the quantity information of each amplicon includes fluorescence PCR, digital PCR, capillary electrophoresis, microarray chips, microfluidic chips, high-throughput sequencing; in the present invention, we do not limit the method for obtaining the quantity information of each amplicon, and any method capable of obtaining the quantity information of each amplicon can be applied to the method described in the present invention. In a specific embodiment of the present invention, we use high-throughput sequencing to obtain the quantity information of each amplicon.

[0059] In some embodiments, the high-throughput sequencing can be carried out by various high-throughput sequencing platform devices including those based on reversible terminator sequencing, semiconductor sequencing, combinatorial probe anchor ligation sequencing, single molecule real-time sequencing, single molecule nanopore sequencing, solid-state nanopore sequencing, etc.

[0060] In the present invention, the Gini coefficient is used to evaluate the uniformity of primer amplification. The Gini coefficient was originally an indicator to measure the degree of inequality in the distribution of wealth in a country or region, ranging from 0 to 1. The smaller the Gini coefficient, the more evenly the wealth is distributed; the larger the Gini coefficient, the less evenly the wealth is distributed. The number of amplicons corresponding to each pair of primers is equivalent to the individual wealth level. When using the Gini coefficient to evaluate the uniformity of primer amplification, the smaller the Gini coefficient, the more evenly the number of each amplicon is distributed, that is, the better the uniformity of primer amplification; the larger the Gini coefficient, the less evenly the number of each amplicon is distributed, that is, the worse the uniformity of primer amplification.

[0061] The following are the criteria for the Gini coefficient to characterize amplification uniformity: A Gini coefficient value of 0 represents complete average, that is, the number of each amplicon is the same; a Gini coefficient between 0.2 and 0.3 indicates that the distribution of the number of amplicons is relatively average; a Gini coefficient between 0.3 and 0.4 indicates that the distribution of the number of amplicons is relatively reasonable, but there are certain gaps; a Gini coefficient between 0.4 and 0.5 indicates that the distribution gap of the number of amplicons is relatively large; a Gini coefficient higher than 0.5 indicates that the equality degree of the distribution of the number of amplicons is very low. Those skilled in the art are familiar with the method of calculating the Gini coefficient according to the number of amplicons. In a specific embodiment of the present invention, the calculation method of the Gini coefficient includes inputting the number of sequencing reads of each amplicon into the numpy module of Python for calculation.

[0062] In the present invention, the parameters a, b, K1, and K2 are all variable parameters. a is the expected Gini coefficient. In the present invention, the value of a is not limited. Those skilled in the art can select a suitable value of a as the expected Gini coefficient standard according to the specific situation of the project. Preferably, the selectable range of the value of a is 0.2 to 0.4. Generally, we hope to control the concentration coefficient range between 0.2 and 5 to facilitate primer mixing operation. If the concentration coefficient is too high, a larger primer input volume is required; if the concentration coefficient is too low, the primer input volume is smaller. Both too large and too small are not convenient for automated primer mixing operation. Therefore, the selectable range of the parameter b is 2 to 10; when the amplification efficiency of the amplicon is less than the average value, the expected concentration coefficient range is between 1 and 5, then the selectable range of the parameter K1 is 0.3 to 2; when the amplification efficiency of the amplicon is greater than the average value, the expected concentration coefficient range is between 0.2 and 1, then the selectable range of the parameter K2 is 0.1 to 1; the parameter R is the expected target value of the number of each amplicon, generally optional as the sum of the number information of all amplicons divided by the number of amplicons, or the ideal target amplicon number value considered by the researcher. In a specific embodiment of the present invention, multiplex PCR primers are designed for high-incidence tumor susceptibility SNP sites and primer concentration is adjusted to ensure the uniformity of primer amplification. Among them, the parameters a used are 0.4, b is 3, K1 is 0.5, K2 is 0.3, and R is 1200.

[0063] In the present invention, index is calculated. i After that, according to Formula 2: the input concentration of primers corresponding to each amplicon = the original primer concentration

[0064] × index i × V 基 / V 终 the re - input concentration of the primers is calculated; where index i is the concentration adjustment coefficient corresponding to the i - th pair of primers; V 基 is the reference volume for each primer mixture, and index i × V 基 is the final volume of each pair of primer mixtures; V 终 is the re - dissolution volume after all primers are mixed each time.

[0065] In an embodiment of the present invention, multiplex PCR primers are designed for high - incidence tumor - susceptible SNP sites and the primer concentration is adjusted to ensure primer amplification uniformity. The specific method is as follows:

[0066] I. Step 1: Mix the primer sets equally

[0067] 1.1 Primer design

[0068] According to the test purpose, a software for designing primer sets for ultra - multiplex amplification developed by our unit is used to design primer sets. A total of 284 pairs of primers are obtained, which amplify 284 target regions respectively to obtain 284 amplicons.

[0069] 1.2 Primer synthesis

[0070] The primer sequences are sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis. It is required to synthesize at 100 μM and purified by PAGE.

[0071] 1.3 Equal - volume mixing of primer sets

[0072] Equal - volume mixing of primers is carried out on the Beckman Coulter automated pipetting workstation Biomek NXP. The mixed primer liquid is dried by using an Eppendorf vacuum concentrator and re - dissolved with Sangon TE buffer to adjust to the primer set storage concentration.

[0073] Through the above operations, the first batch of primers is obtained for the first - round test.

[0074] II. Step 2: The first - round test experiment, calculate the Gini coefficient

[0075] 2.1 Experimental materials

[0076] Sample: Human Genomic DNA, manufacturer: Promega, catalog number: G3041. Three technical replicates were used for each batch of experiments. During data processing, after normalizing the number of sequencing reads for each amplicon of the three technical replicates (to a data volume of 0.5M), the average number of sequencing reads for each amplicon was calculated for Gini coefficient calculation and concentration coefficient calculation.

[0077] Library construction reagent: A commercial multiplex amplification library construction kit developed for the SBS high-throughput sequencing platform;

[0078] Sequencing reagent: Salus Pro sequencing reagent kit (SRM-SE75-300M), manufacturer: Shenzhen Sailotech Co., Ltd., catalog number: SRM-SE75-300M

[0079] Magnetic bead purification: Agencourt AMPure XP magnetic beads, manufacturer Beckman Coulter, catalog number A63882; absolute ethanol (analytical reagent grade), manufacturer Xilong, catalog number 1280340501600;

[0080] 2.2 Experimental methods

[0081] 2.2.1 Multiplex amplification

[0082] Prepare the reaction system on ice according to Table 1. Vortex the reaction system and centrifuge briefly (≤600g, 5s); place the reaction system in a pre-set PCR instrument and run the program according to Table 2.

[0083] Table 1 Multiplex amplification reaction system

[0084] Component Volume PanelMix-TB 5 μL mPCRMix 10 μL Nucleic acid 15 μL Total volume 30 μL

[0085] Table 2 Multiplex amplification PCR reaction program

[0086]

[0087] 2.2.2 Purification of multiplex amplification products

[0088] 1) Preparation:

[0089] i. Take out the magnetic beads from the refrigerator and equilibrate at room temperature for at least 30 min. Vortex or invert the magnetic beads thoroughly to ensure mixing.

[0090] ii. Prepare 80% ethanol, 200 μL is required for each sample.

[0091] 2) Add 30 μL of magnetic beads to the PCR product; vortex and incubate at room temperature for 5 min.

[0092] 3) Briefly centrifuge the PCR tube and place it in a magnetic stand. After the solution becomes clear (about 2 min), carefully remove the supernatant.

[0093] 4) Keep the PCR tube in the magnetic stand all the time. Add 100 μL of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 s, and carefully remove the supernatant.

[0094] 5) Repeat step 4).

[0095] 6) Keep the PCR tube in the magnetic stand all the time. Open the lid to dry the magnetic beads (2 - 5 min, judged according to the actual situation).

[0096] 7) Add 15 μL of Nuclease-Free Water to the PCR tube, vortex to mix well, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube and place it in the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 2 min), carefully pipette 13 μL of the supernatant for the next round of PCR.

[0097] 2.2.3 Library amplification

[0098] Prepare the reaction system on ice according to Table 3. Vortex the reaction system to mix well and centrifuge briefly (≤600 g, 5 s); put the reaction system into the set PCR instrument and run the program according to Table 4.

[0099] Table 3 Library amplification reaction system

[0100] Component Volume (μL) / reaction 2nd-PCRMix 15 Multiplex amplification purified product 13 Index-i5 1 Index-i7 1 Total volume 30

[0101] Table 4 Library amplification PCR reaction program

[0102]

[0103] 2.2.4 Purification of library amplification products

[0104] 1) Preparation:

[0105] a) Take out the magnetic beads from the refrigerator and equilibrate at room temperature for at least 30 min. Vortex or invert the magnetic beads thoroughly to ensure mixing.

[0106] b) Prepare 80% ethanol, 200 μL is needed for 1 sample.

[0107] 2) Add 24 μL of magnetic beads to the PCR reaction solution; vortex to mix well and incubate at room temperature for 5 min.

[0108] 3) Briefly centrifuge the PCR tube and place it in the magnetic stand. After the solution becomes clear (about 2 min), carefully remove the supernatant.

[0109] 4) Keep the PCR tube in the magnetic stand at all times, add 100 μL of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 s, and carefully remove the supernatant.

[0110] 5) Repeat step 4.

[0111] 6) Keep the PCR tube in the magnetic stand at all times, open the lid to dry the magnetic beads (2 - 5 min, judged according to the actual situation). Note: Excessive drying of the magnetic beads will affect the purification effect.

[0112] 7) Take out the PCR tube from the magnetic stand, add 25 μL of Nuclease-Free Water, vortex to mix well, and incubate at room temperature for 5 min.

[0113] 8) Briefly centrifuge the PCR tube and place it in the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 2 min), carefully aspirate the supernatant into a new centrifuge tube, and the library construction is completed.

[0114] 2.2.5 Library Quantification

[0115] 1) It is recommended to use the dsDNA HS Assay Kit for library quantification. For specific operations, please refer to the dsDNA HS Assay Kit instruction manual.

[0116] 2) Aspirate 1 μL of the library and mix it with 199 μL of the pre-prepared Qubit quantification working solution for Qubit detection, and record the library concentration.

[0117] 2.2.6 Sequencing on the Machine

[0118] Sequencing data quality requirements:

[0119] 1) Data volume: 500 k reads / library;

[0120] 2) Sequencing mode: paired-end index; single-end sequencing: 75 bp.

[0121] 2.2.7 Data Analysis

[0122] Use the in-house developed high-throughput sequencing data analysis pipeline for targeted sequencing to process and align the data from the sequencer. First, perform sequencing data quality control to remove sequencing sequences that do not meet Q20; then filter primer dimer sequences to remove sequencing sequences with too short lengths; finally, use the Bowtie2 alignment tool to perform sequencing sequence alignment analysis and output the number of detected sequences for each amplicon. The sequencing data is shown in Table 5.

[0123] 2.2.8 Calculation of Gini Coefficient

[0124] Perform calculations using the numpy module in Python.

[0125] After calculation, the Gini coefficient of the number of 284 amplicon sequences in Run1 experiment before adjustment is 0.73, which is much higher than expected. Among them, the number of 59 amplicon sequences is less than 50, not meeting the experimental quality control requirements. Therefore, it is necessary to adjust the input concentration of each pair of primers based on this protocol.

[0126] III. Step 3 According to the sequencing results of the first-round experiment, calculate the concentration input coefficient (Index1) of each pair of primers in the second-round experiment

[0127] 3.1 Convert the sequencing reads into concentration input coefficients

[0128] Use the formula: where: index i is the concentration coefficient of primer pair i, b is the base, Ri is the number of reads of primer pair i in the previous round of sequencing, R is the expected reads target value, K is the segmented adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R.

[0129] a, b, K1, K2, and R are all variable parameters. Generally, we hope to control the concentration coefficient range between 0.2 and 5 to facilitate primer mixing operations. If the concentration coefficient is too high, a larger primer input volume is required; if the concentration coefficient is too low, the primer input volume is smaller. Both too large and too small are not convenient for automated primer mixing operations. Therefore, the selectable range of parameter b is 2 to 10; when the amplification efficiency of the amplicon is less than the average value, the expected concentration coefficient range is 1 to 5, and the selectable range of parameter K1 is 0.3 to 2; when the amplification efficiency of the amplicon is greater than the average value, the expected concentration coefficient range is 0.2 to 1, and the selectable range of parameter K2 is 0.1 to 1; parameter R is the expected sequencing reads target value, which can generally be selected as the total effective reads of sequencing divided by the number of amplicons, or the ideal target reads number considered by the researcher.

[0130] In the specific embodiment of the present invention, the following parameter combination is used:

[0131] a = 0.4; b = 3; K1 = 0.5 (when Ri ≤ R); K2 = 0.3 (when Ri > R); R = 1200

[0132] After calculation, the input concentration adjustment coefficients of the primer pairs corresponding to each amplicon are shown in Table 5 below.

[0133] As mentioned above, the primer pairs have been dissolved to a solution with a concentration of 100 uM. After all primers are mixed, they will be vacuum-dried and then re-dissolved to a certain volume for subsequent experimental tests.

[0134] The input concentration of primers corresponding to each amplicon = the original primer concentration × index i ×V 基 / V 终 Calculate the re - input concentration of the primers; where index i is the concentration adjustment coefficient corresponding to the i - th pair of primers; V 基 is the reference volume for each primer mixing, index i ×V 基 is the final volume of each pair of primer mixing; V 终 is the re - dissolution volume after all primers are mixed each time. In this example, the original primer concentration is 100 μM, V 基 is 10 μL, V 终 is 8000 μL, then the input concentration of primers corresponding to each amplicon = 100 μM × V i / V 终 = 100 μM × index i × 10 / 8000.

[0135] 3.2 Re - mix the primers according to the concentration input coefficient

[0136] Use the calculated input volume of primers (index i ×V 基 ) to write an automated process script to perform primer mixing on the Beckman Coulter automated pipetting workstation Biomek NXP. The automated pipetting workstation Biomek NXP is configured with a flexible eight - channel pipetting module, which can aspirate the corresponding input volume of primer pairs according to the pipetting file and pipetting process script for primer mixing, ensuring the pipetting accuracy when mixing primers of different volumes.

[0137] 3.3 Concentrate the primers to the storage concentration

[0138] Use an Eppendorf vacuum concentrator to dry the mixed primer liquid, and re - dissolve it with Shengong TE buffer to adjust to the storage concentration of the primer set.

[0139] IV. Step 4 Repeat the experimental procedure described in Step 2 using the second batch of primers for the second - round experiment. Except for using primers from different batches, other parameters remain unchanged.

[0140] After one - round adjustment, it is calculated that the Gini coefficient of the sequence numbers of 284 amplicons in the Run2 experiment is 0.40, which basically meets the expectations. Among them, the sequence numbers of 12 amplicons are less than 50, not meeting the experimental quality control requirements. Therefore, it is necessary to adjust the input concentration of the primers again.

[0141] V. Step 5 According to the sequencing results of the second - round experiment, calculate the concentration input coefficient (Index2) of the primers

[0142] Calculate the primer input concentration coefficient according to the process described in Step 3. Other operations remain unchanged.

[0143] The original Index2 is shown in Table 5 below.

[0144] Since one round of primer concentration adjustment has been carried out, that is, the basis of this concentration adjustment is the adjustment coefficient of the previous round, the composite concentration coefficient should be the product of the first-round adjustment coefficient and the second-round adjustment coefficient. When multiple rounds of adjustment are required, and so on.

[0145] The original Index2 and the composite Index2 are shown in Table 5 below.

[0146] VI. Step 6: Repeat the experimental process described in Step 2 using the 3rd batch of primers to conduct the third-round experiment. Except for using primers of different batches, other parameters remain unchanged.

[0147] After the second-round adjustment, it is calculated that the Gini coefficient of the number of amplicons in Run3 Experiment 284 is 0.38, meeting the expectation. The number of amplicons is not less than 50, meeting the experimental quality control requirements. The concentration adjustment for this primer set is completed.

[0148] Table 5 Primer Adjustment Coefficient and Average Sequencing Sequence in the Embodiment

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] Figure 2 It is a schematic structural diagram of the multiplex PCR amplification efficiency adjustment system provided by the present invention.

[0162] The system is programmed or otherwise configured to include an amplification product detection module 201, a Gini coefficient calculation module 202, a primer concentration adjustment module 203, and a repeat adjustment module 204:

[0163] Amplification product detection module 201: Detect the multiplex PCR amplification products to obtain the quantity information of each amplicon;

[0164] Gini coefficient calculation module 202: Calculate the Gini coefficient of all amplicons according to the quantity information of each amplicon;

[0165] Primer concentration adjustment module 203: If the Gini coefficient > a, according to formula 1: Adjust the input concentration of the multiplex PCR primers and remix the primers; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers, a is the expected Gini coefficient, b is the base, and R i is the quantity information of the i-th pair of primers in the previous round of sequencing, R is the expected target value of the quantity of each amplicon, and K is the segmented adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R;

[0166] Repeat adjustment module 204: Repeat the steps in the above amplification product detection module, Gini coefficient calculation module, and primer concentration adjustment module until the Gini coefficient ≤ a.

[0167] Furthermore, the method for obtaining the quantity information of each amplicon includes fluorescence PCR, digital PCR, capillary electrophoresis, microarray chips, microfluidic chips, and high-throughput sequencing.

[0168] Preferably, the method for obtaining the quantity information of each amplicon is high-throughput sequencing.

[0169] Furthermore, the parameters a, b, K1, and K2 are all variable parameters.

[0170] Preferably, the selectable range of parameter a is 0.2 to 0.4; the selectable range of b is 2 to 10; the selectable range of parameter K1 is 0.3 to 2; the selectable range of parameter K2 is 0.1 to 1.

[0171] Furthermore, after calculating index i According to formula 2: The input concentration of the primer corresponding to each amplicon = the original primer concentration × index i × V 基 / V 终 Calculate the re-input concentration of the primer; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers; V 基 is the reference volume for each primer mixing, indexi ×V 基 is the final volume of the mixture of each pair of primers; V 终 is the reconstitution volume after mixing all primers each time.

[0172] Further, when performing the primer concentration adjustment in the nth (n≥2) round, calculate index i and then use the composite concentration coefficient to calculate the input concentration of the primers, that is, according to Formula 3: the input concentration of the primers corresponding to each amplicon = the original primer concentration × composite index i ×V 基 / V 终 to calculate the re-input concentration of the primers. The composite index i is the product of the index in the (n - 1)th round i and the index in the nth round i .

[0173] The system may be the user's electronic device or a computer system remotely located relative to the electronic device.

[0174] Figure 3 is a schematic structural diagram of the computer device provided by the present invention.

[0175] The computer device 300 includes a processor 301 and a memory 302 coupled to the processor 301. Program instructions are stored in the memory 302. When the program instructions are executed by the processor 301, the processor 301 executes the method described in the first aspect of the present invention.

[0176] Among them, the processor 301 can also be called a CPU (Central Processing Unit). The processor 301 may be an integrated circuit chip with signal processing capabilities. The processor 301 can also be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components. The general-purpose processor can be a microprocessor or the processor can also be any conventional processor, etc.

[0177] The computer device 300 can be a mobile electronic device.

[0178] It should be understood that the systems, devices, and methods described in the present invention can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the modules is only a logical function division. In actual implementation, there may be other division methods. For example, multiple modules or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the displayed or discussed coupling or direct coupling or communication connection between each other can be an indirect coupling or communication connection through some interfaces, devices, or modules, and can be in electrical, mechanical, or other forms.

[0179] The modules described as separate components may or may not be physically separated. The components shown as modules may or may not be physical units, that is, they may be located in one place or distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0180] In addition, the functional modules in each embodiment of the present invention can be integrated into one processing module, or each module can exist physically alone, or two or more modules can be integrated into one module. The above integrated modules can be implemented in the form of hardware or in the form of software functional modules.

[0181] The above is only the implementation manner of this application, and does not limit the patent scope of this application. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of this application, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of this application.

Claims

1. A method for adjusting the amplification efficiency of multiplex PCR, characterized in that, The method includes: 1): Detect the multiplex PCR amplification products to obtain the quantity information of each amplicon; 2): Calculate the Gini coefficient of all amplicons according to the quantity information of each amplicon; 3): If the Gini coefficient > a, according to formula 1: Adjust the input concentration of the multiplex PCR primers and remix the primers; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers, a is the expected Gini coefficient, b is the base number, Ri is the quantity information of the i-th pair of primers in the previous round of detection, R is the expected target value of each amplicon quantity, K is the segmented adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R; 4): Repeat the above steps 1) to 3) more than once until the Gini coefficient ≤ a.

2. The method according to claim 1, wherein The method for obtaining the quantity information of each amplicon includes fluorescence PCR, digital PCR, capillary electrophoresis, microarray chip, microfluidic chip, high-throughput sequencing; Preferably, the method for obtaining the quantity information of each amplicon is high-throughput sequencing.

3. The method according to claim 1, wherein The parameters a, b, K1, and K2 are all variable parameters; Preferably, the selectable range of parameter a is 0.2 to 0.4; the selectable range of parameter b is 2 to 10; the selectable range of parameter K1 is 0.3 to 2; the selectable range of parameter K2 is 0.1 to 1.

4. The method according to claim 1, characterized in that, Calculate the index i Then, according to Formula 2: The primer input concentration corresponding to each amplicon = the original primer concentration × index i ×V 基 / V 终 Calculate the re-input concentration of the primer; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers; V 基 is the reference volume for each primer mixture, index i ×V 基 is the final volume for each pair of primer mixtures; V 终 is the reconstitution volume after mixing all primers each time; Preferably, when performing the primer concentration adjustment in the nth (n≥2) round, calculate index i After that, the primer input concentration should be calculated using the composite concentration coefficient, that is, according to Formula 3: the primer input concentration corresponding to each amplicon = the original primer concentration × composite index i ×V 基 / V 终 Calculate the re-input concentration of the primer, and the composite index i is the product of the index of the (n - 1)th round i and the index of the nth round i .

5. A multiplex PCR amplification efficiency adjustment system, characterized in that, The system includes: Amplification product detection module: Detect the multiplex PCR amplification products to obtain the quantity information of each amplicon; Gini coefficient calculation module: Calculate the Gini coefficient of all amplicons according to the quantity information of each amplicon; Primer concentration adjustment module: If the Gini coefficient > a, according to Formula 1: Adjust the input concentration of multiplex PCR primers and remix the primers; where indexi is the concentration adjustment coefficient corresponding to the i-th pair of primers, a is the expected Gini coefficient, b is the base number, Ri is the quantity information of the i-th pair of primers in the previous round of detection, R is the expected target value of each amplicon quantity, K is the segment adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R; Repeated adjustment module: Repeat the steps in the above amplification product detection module, Gini coefficient calculation module, and primer concentration adjustment module until the Gini coefficient ≤ a.

6. The system according to claim 5, wherein The method for obtaining the quantity information of each amplicon includes fluorescence PCR, digital PCR, capillary electrophoresis, microarray chip, microfluidic chip, high-throughput sequencing; Preferably, the method for obtaining the quantity information of each amplicon is high-throughput sequencing.

7. The system according to claim 5, characterized in that, The parameters a, b, K1, and K2 are all variable parameters; Preferably, the selectable range of parameter a is 0.2 to 0.4; the selectable range of b is 2 to 10; the selectable range of parameter K1 is 0.3 to 2; the selectable range of parameter K2 is 0.1 to 1; Preferably, index is calculated i After that, according to Formula 2: the input concentration of primers corresponding to each amplicon = the original primer concentration × index i ×V 基 / V 终 the re-input concentration of the primers is calculated; where index i is the concentration adjustment coefficient corresponding to the i-th pair of primers; V 基 is the reference volume for each primer mixture, index i ×V 基 is the final volume of each pair of primer mixtures; V 终 is the reconstitution volume after mixing all primers each time; Preferably, when performing the primer concentration adjustment in the nth (n≥2) round, calculate index i After that, the primer input concentration should be calculated using the composite concentration coefficient, that is, according to Formula 3: the primer input concentration corresponding to each amplicon = the original primer concentration × composite index i ×V 基 / V 终 Calculate the re-input concentration of the primer, and the composite index i is the product of the index in the (n - 1)th round i and the index in the nth round i .

8. A computer device, characterized in that, The computer device includes: A memory and a processor, the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method described in any one of claims 1-4 is implemented.

9. A computer-readable storage medium, characterized in that, A computer program is stored on the computer-readable storage medium, and when the computer program is executed by the processor, the method described in any one of claims 1-4 is implemented.

10. Use in any of the following aspects, characterized in that, The application includes: 1) The application of the method described in any one of claims 1-4 in the adjustment of the primer concentration in the multiplex PCR amplification reaction; 2) The application of the method described in any one of claims 1-4 in the sequencing of multiplex PCR amplification products.

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