A multiplex PCR amplification efficiency adjustment method, system and device

Abstract

The application discloses a multiplex PCR amplification efficiency adjusting method, system and device. In order to solve the problem of unevenness of multiplex PCR amplification, the application provides a multiplex PCR amplification efficiency adjusting method. The method adjusts the input concentration of primers in the process of multiplex PCR amplification reaction through a primer concentration adjusting coefficient formula, and then achieves the purpose of uniform multiplex PCR amplification. The method is simple and convenient to operate, and is high in accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology. Specifically, this invention relates to a method, system, and device for adjusting the amplification efficiency of multiplex PCR. Background Technology

[0002] Multiplex PCR is a technique in molecular biology used to simultaneously amplify multiple DNA or RNA fragments. It involves adding multiple pairs of specific primers to a single amplification reaction system, yielding multiple amplification products in a single reaction. Further analysis of these products allows for the determination of detection results. In practical nucleic acid analysis and detection, multiplex PCR significantly simplifies procedures and reduces time, while requiring no additional procedures or instruments compared to conventional PCR. Since its first report in 1988, multiplex PCR has become a rapid and convenient method for targeted nucleic acid detection in clinical and research laboratories. Multiplex PCR has been successfully applied in many fields, including gene deletion analysis, gene mutation and polymorphism analysis, mRNA quantification, RNA detection, and genome sequencing. In the diagnosis of infectious diseases, multiplex PCR also plays an important role in the identification of viruses, bacteria, parasites, and the analysis of antibiotic resistance genes.

[0003] While multiplex PCR amplification technology has broad application prospects, it also faces many difficulties and challenges in practical operation. Establishing an effective multiplex PCR program typically requires very careful primer design and multiple rounds of screening. A common problem in multiplex PCR is the uneven amplification of the target fragment between different primer pairs, with some primers in the multiplex system failing to amplify effectively at all, while also exhibiting poor reproducibility. For a successful multiplex PCR reaction system, the following factors need to be considered: primer concentration, PCR buffer concentration, the balance between magnesium ion concentration and dNTP concentration, the temperature of each step in the PCR cycle, and the amounts of template DNA and Taq DNA polymerase. The optimal combination of annealing temperature and buffer system in PCR is essential to ensure the specificity of multiplex PCR; a certain ratio between magnesium ion and dNTP concentrations needs to be maintained; and adjustments to primer concentration are also crucial.

[0004] A common phenomenon in multiplex PCR is the bias towards amplifying one or more sequences, meaning the final product ratio differs from the initial template ratio, leading to detection bias. This is mainly because the amount of enzymes and single nucleotides in the multiplex PCR amplification system is limited. All primer pairs compete for these limited enzymes and single nucleotides, but their amplification efficiencies are not uniform. Factors affecting the non-uniformity of multiplex PCR amplification efficiency include primer sequences, target region sequences, and primer concentrations. For specific detection targets, the adjustment space for primer sequences and target regions is limited; therefore, adjusting the uniformity of multiplex PCR amplification efficiency primarily involves adjusting primer concentrations. Thus, determining the final concentration of each primer becomes a crucial factor in establishing a multiplex PCR system. Regarding primer concentration, a common practice is to amplify with a single primer pair, then perform gel electrophoresis on the amplified primers. The primer concentration is adjusted based on the gel electrophoresis color intensity, or new primers are used. However, judging the color intensity of gel electrophoresis often relies on the experimenter's subjective observation, which can lead to inaccurate and inconsistent results. In addition, this method is cumbersome and time-consuming, increasing experimental and time costs; batch-to-batch results are not comparable, and when the number of primer pairs is large, the results are not reliable; primers interfere with each other, and the amplification of a single primer pair cannot directly reflect the true performance of multiplex amplification. Therefore, providing a novel method for adjusting the concentration of multiplex PCR primers is of great significance. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method for adjusting primer concentration to ensure the uniformity of multiplex PCR amplification.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0008] Furthermore, 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 for all amplicones based on the quantity information of each amplicon;

[0011] 3): If the Gini coefficient > a, according to formula 1: Adjust the concentration of the multiplex PCR primers and remix the primers; among which, index i Let R be the concentration adjustment factor for the i-th primer pair, a be the expected Gini coefficient, b be the base, and R be the base value. iRi 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 piecewise adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R;

[0012] 4): Repeat steps 1) to 3) until the Gini coefficient ≤ 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, K1, and K2 are all variable parameters.

[0016] 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.

[0017] Furthermore, calculate index i and 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;

[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 mixture, index i ×V 基 is the final volume of each pair of primer mixtures; V 终 is the reconstitution volume after all primers are mixed each time.

[0019] Furthermore, when performing the n-th (n ≥ 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 = 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 i in the (n - 1)-th round and the index i in the n-th round.

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

[0021] Furthermore, the system includes:

[0022] Amplified product detection module: detecting the multiplex PCR amplified product to obtain the quantity information of each amplicon;

[0023] Gini coefficient calculation module: calculating 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 piecewise adjustment coefficient, which is K1 when Ri < R and K2 when Ri ≥ R;

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

[0026] Furthermore, 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] Furthermore, 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] 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 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 V represents the final volume of each primer pair mixture; 终 This represents the reconstitution volume after mixing all primers each time.

[0032] Furthermore, when performing the nth (n≥2) round of primer concentration adjustment, the index is calculated. i The primer concentration should then be calculated using the complexation concentration coefficient, i.e., according to formula 3: Primer concentration for each amplicon = Original primer concentration × Complexation index i ×V 基 / V 终 Calculate the primer re-injection concentration and the complex index. i For n-1 rounds, index i With n rounds index i The product of.

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

[0034] Furthermore, the computer device includes:

[0035] A memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the method described in the first aspect of the present invention.

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

[0037] Furthermore, the computer-readable storage medium stores a computer program that, when executed by a processor, implements the method described in the first aspect of the present invention.

[0038] The fifth aspect of the present invention provides for any of the following applications.

[0039] Furthermore, the applications include:

[0040] 1) The application of the method described in the first aspect of this invention in adjusting the primer concentration for multiplex PCR amplification reactions;

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

[0042] Advantages and benefits of the present invention:

[0043] This invention provides a novel method for adjusting primer concentration in multiplex PCR, applicable to primers designed for any testing purpose. Compared with the method of adjusting primer concentration by gel electrophoresis color development intensity, this method is simpler and more convenient to operate, reduces experimental costs, can adjust up to 2000 pairs of primers simultaneously, and has higher accuracy. Attached Figure Description

[0044] Figure 1 This is a schematic flowchart of the multiplex PCR amplification efficiency adjustment method provided by the present invention;

[0045] Figure 2 This is a schematic diagram of the structure of the multiplex PCR amplification efficiency adjustment system provided by the present invention;

[0046] Figure 3 A schematic diagram of the structure of the computer device provided by the present invention. Detailed Implementation

[0047] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0048] The processes described in the specification, claims, and accompanying drawings of this invention include multiple operations appearing in a specific order. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the terms "first," "second," etc., used herein are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Figure 1 This is a flowchart illustrating the multiplex PCR amplification efficiency adjustment method 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 for all amplicones based on the quantity information of each amplicon;

[0053] 103: If the Gini coefficient > a, according to formula 1: Adjust the concentration of the multiplex PCR primers and remix the primers; among which, index i Let R be the concentration adjustment factor for the i-th primer pair, a be the expected Gini coefficient, b be the base, and R be the base value. 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 piecewise 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 this invention, the Gini coefficient is used to assess the uniformity of primer amplification. The Gini coefficient, originally an indicator of wealth inequality in a country or region, ranges between 0 and 1. A smaller Gini coefficient indicates a more even distribution of wealth; a larger Gini coefficient indicates a more unequal distribution of wealth. The number of amplicons corresponding to each primer pair corresponds to an individual's wealth level. When using the Gini coefficient to assess the uniformity of primer amplification, a smaller Gini coefficient indicates a more even distribution of amplicons, meaning better primer amplification uniformity; a larger Gini coefficient indicates a more unequal distribution of amplicons, meaning worse primer amplification uniformity.

[0061] The following are the standards for characterizing amplification uniformity using the Gini coefficient: a Gini coefficient of 0 represents perfect uniformity, meaning the number of amplicones is the same; a Gini coefficient between 0.2 and 0.3 indicates a relatively even distribution of amplicon numbers; a Gini coefficient between 0.3 and 0.4 indicates a reasonably reasonable distribution of amplicon numbers, but with some variation; a Gini coefficient between 0.4 and 0.5 indicates a large variation in the distribution of amplicon numbers; and a Gini coefficient higher than 0.5 indicates a very low degree of equality in the distribution of amplicon numbers. Those skilled in the art are familiar with methods for calculating the Gini coefficient based on the number of amplicones. In a specific embodiment of this invention, the method for calculating the Gini coefficient includes inputting the sequencing read count of each amplicon into a Python numpy module for calculation.

[0062] In this invention, parameters a, b, K1, and K2 are all variable parameters. 'a' represents the expected Gini coefficient. The value of 'a' is not limited in this invention; those skilled in the art can select a suitable value of 'a' as the expected Gini coefficient standard based on the specific circumstances of the project. Preferably, the value of 'a' can be selected within the range of 0.2 to 0.4. Generally, we aim to control the concentration coefficient within the range of 0.2 to 5 to facilitate primer mixing. A high concentration coefficient requires a larger primer volume, while a low concentration coefficient requires a smaller primer volume; both excessively large and small concentration coefficients hinder automated primer mixing. Therefore, parameter b can be selected from 2 to 10; when the amplicon amplification efficiency is less than the average, the expected concentration coefficient is between 1 and 5, so parameter K1 can be selected from 0.3 to 2; when the amplicon amplification efficiency is greater than the average, the expected concentration coefficient is between 0.2 and 1, so parameter K2 can be selected from 0.1 to 1; parameter R is the expected target value for the number of each amplicon, which can generally be selected as the sum of the quantity information of all amplicons divided by the number of amplicons, or the value of the ideal target number of amplicons as considered by the researcher. In a specific 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 the uniformity of primer amplification. The parameters used are a = 0.4, b = 3, K1 = 0.5, K2 = 0.3, and R = 1200.

[0063] In this invention, the index is calculated. i Then, according to Formula 2: the primer concentration for each amplicon = the original primer concentration

[0064] ×index i ×V 基 / V 终 Calculate the primer re-injection concentration; where, index i V is the concentration adjustment coefficient corresponding to the i-th primer pair; 基 The index is the baseline volume for each primer mix. i ×V 基 V represents the final volume of each primer pair mixture; 终 This represents the reconstitution volume after mixing all primers each time.

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

[0066] Step 1: Mix equal amounts of primer sets

[0067] 1.1 Primer Design

[0068] Based on the testing objectives, primer sets were designed using our self-developed ultra-multiplex amplification primer set design software. A total of 284 primer pairs were obtained, amplifying 284 target regions and yielding 284 amplicones.

[0069] 1.2 Primer Synthesis

[0070] The primer sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis. The required synthesis concentration was 100 μM, and the primers were purified using PAGE.

[0071] 1.3 Primer sets were mixed in equal amounts

[0072] Primers were mixed in equal volumes using a Beckman Coulter automated pipetting workstation (Biomek NXP), and the mixed primer liquid was dried using an Eppendorf vacuum concentrator. The mixture was then reconstituted using Sangon Biotech TE buffer to adjust the primer set concentration for preservation.

[0073] Through the above operations, the first batch of primers was obtained for the first round of testing.

[0074] II. Step 2: First round of testing experiments, calculating the Gini coefficient.

[0075] 2.1 Experimental Materials

[0076] Sample: Human genomic DNA, manufacturer: Promega, catalog number: G3041. Each batch of experiments used three replicates. During data processing, the number of sequences from each amplicon in the three replicates was normalized (to 0.5M data volume), and the average number of sequences from each amplicon was calculated for the Gini coefficient and concentration coefficient calculations.

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

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

[0079] Magnetic bead purification: AgencourtAMPure XP magnetic beads, manufacturer Beckman Coulter, catalog number A63882; anhydrous ethanol (superior 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 to mix well, then briefly aliquot (≤600g, 5s); place the reaction system into the pre-configured PCR instrument and run the program according to Table 2.

[0083] Table 1 Multiplex Amplification Reaction System

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

[0085] Table 2. Multiplex PCR reaction procedure

[0086]

[0087] 2.2.2 Purification of Multiplex Amplification Products

[0088] 1) Preparation:

[0089] i. Remove the magnetic beads from the refrigerator and allow them to equilibrate to room temperature for at least 30 minutes. Vortex or thoroughly invert the magnetic beads to ensure they are mixed.

[0090] ii. Prepare 80% ethanol, 200 μL per sample.

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

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

[0093] 4) Keep the PCR tube in the magnetic rack at all times, add 100 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0094] 5) Repeat step 4).

[0095] 6) Keep the PCR tube in the magnetic rack at all times, and open the lid to dry the magnetic beads (2-5 minutes, depending on the actual situation).

[0096] 7) Add 15 μL of Nuclease-Free Water to the PCR tube, vortex to mix, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube and place it in a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 2 min), carefully aspirate 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, then briefly aliquot (≤600g, 5s); place the reaction system into the pre-configured PCR instrument and run the program according to Table 4.

[0099] Table 3 Library amplification reaction system

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

[0101] Table 4. PCR reaction procedure for library amplification

[0102]

[0103] 2.2.4 Purification of Library Amplification Products

[0104] 1) Preparation:

[0105] a) Remove the magnetic beads from the refrigerator and allow them to equilibrate to room temperature for at least 30 minutes. Vortex or thoroughly invert the magnetic beads to ensure they are mixed.

[0106] b) Prepare 80% ethanol; 200 μL is required for one sample.

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

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

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

[0110] 5) Repeat step 4.

[0111] 6) Keep the PCR tube in the magnetic rack at all times, and open the cap to dry the magnetic beads (2-5 minutes, depending on the actual situation). Note: Over-drying of the magnetic beads will affect the purification effect.

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

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

[0114] 2.2.5 Library Quantitative Analysis

[0115] 1) Recommended use For library quantification using the dsDNAHSAssay Kit, please refer to [link to instructions]. dsDNA HS Assay Kit Instruction Manual.

[0116] 2) Mix 1 μL of the library with 199 μL of the pre-prepared Qubit quantitative working solution, perform Qubit detection, and record the library concentration.

[0117] 2.2.6 Sequencing

[0118] Sequencing data quality requirements:

[0119] 1) Data volume: 500kreads / document;

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

[0121] 2.2.7 Data Analysis

[0122] The sequencing data was processed and compared using a targeted high-throughput sequencing data analysis workflow developed in-house. First, sequencing data quality control was performed to remove sequences that did not meet Q20 requirements. Then, primer dimer sequence filtering was performed to remove sequences that were too short. Finally, the Bowtie2 alignment tool was used for sequence alignment analysis, outputting the number of sequences detected for each amplicon. See Table 5 for detailed sequencing data.

[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 sequencing reads to 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 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 for convenient primer mixing operation. If the concentration coefficient is too high, a larger primer input volume is required; if the concentration coefficient is too low, a smaller primer input volume is required. Both too large and too small are not convenient for automated primer mixing operation. 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; the parameter R is the expected sequencing reads target value, which can generally be selected as the total effective reads 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 concentration adjustment coefficients of the corresponding primer pairs for each amplicon are shown in Table 5 below.

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

[0134] Primer concentration for each amplicon = original primer concentration × index i ×V 基 / V 终 Calculate the primer re-injection concentration; where, index i V is the concentration adjustment coefficient corresponding to the i-th primer pair; 基 The index is the baseline volume for each primer mix. i ×V 基 V represents the final volume of each primer pair mixture; 终 This refers to the reconstitution volume after mixing all primers each time. In this example, the original primer concentration is 100 μM, V 基 For 10 μL, V 终 If the concentration is 8000 μL, then the primer concentration for each amplicon is 100 μM × V. i / V 终 =100μM×index i ×10 / 8000.

[0135] 3.2 Remix primers according to the concentration input coefficient.

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

[0137] 3.3 Primer concentration to storage concentration

[0138] The mixed primer liquid was dried using an Eppendorf vacuum concentrator and then reconstituted with Sangon Biotech TE buffer to adjust to the primer set preservation concentration.

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

[0140] After one round of adjustments, the Gini coefficient of the 284 amplicon sequences in Run2 was calculated to be 0.40, which basically met expectations. However, 12 amplicon sequences had fewer than 50 sequences, failing to meet the experimental quality control requirements. Therefore, the primer concentration needs to be adjusted again.

[0141] Step 5: Based on the second round of sequencing results, calculate the primer concentration input factor (Index2).

[0142] Follow the procedure described in step 3 to calculate the primer concentration coefficient. All other operations remain unchanged.

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

[0144] Since one round of primer concentration adjustment has already been carried out, meaning that the basis for this concentration adjustment is the adjustment coefficient from the previous round, the composite concentration coefficient should be the product of the adjustment coefficient from the first round and the adjustment coefficient from the second round. This principle applies when multiple rounds of adjustment are required.

[0145] For details on the original Index2 and the composite Index2, please refer to Table 5 below.

[0146] VI. Step 6: Repeat the experimental procedure described in Step 2 using the third batch of primers for the third round of experiments. Except for using different batches of primers, all other parameters remain unchanged.

[0147] After the second round of adjustments, the Gini coefficient for the 284 amplicon sequences in Run3 was calculated to be 0.38, meeting expectations. The number of sequences without amplicon sequences was less than 50, meeting the experimental quality control requirements. The concentration adjustments for this primer set are now complete.

[0148] Table 5 Primer adjustment coefficients and average sequencing sequence values ​​in the examples.

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] Figure 2 This is a schematic diagram of the structure 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 based on 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 number, R i [[ID=Z16]]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, 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 - 0.4; the selectable range of b is 2 - 10; the selectable range of parameter K'1 is 0.3 - 2; the selectable range of parameter K2 is 0.1 - 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 V represents the final volume of each primer pair mixture; 终 This represents the reconstitution volume after mixing all primers each time.

[0172] Furthermore, when performing the nth (n≥2) round of primer concentration adjustment, the index is calculated. i The primer concentration should then be calculated using the complexation concentration coefficient, i.e., according to formula 3: Primer concentration for each amplicon = Original primer concentration × Complexation index i ×V 基 / V 终 Calculate the primer re-injection concentration and the complex index. i For n-1 rounds, index i With n rounds index i The product of.

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

[0174] Figure 3 A schematic diagram of the structure 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. The memory 302 stores program instructions, which, when executed by the processor 301, cause the processor 301 to perform the method described in the first aspect of the present invention.

[0176] The processor 301 can also be referred to as 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, or discrete hardware components. A general-purpose processor can be a microprocessor or any conventional processor.

[0177] Computer device 300 can be a mobile electronic device.

[0178] It should be understood that the systems, apparatuses, and methods described in this invention can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of modules is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple modules or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the couplings or direct couplings or communication connections shown or discussed may be indirect couplings or communication connections through some interfaces, apparatuses, or modules, and may be electrical, mechanical, or other forms.

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

[0180] Furthermore, the functional modules in the various embodiments of the present invention can be integrated into one processing module, or each module can exist physically separately, or two or more modules can be integrated into one module. The integrated modules described above can be implemented in hardware or as software functional modules.

[0181] The above are merely embodiments of this application and do not limit the scope of this patent application. Any equivalent structural or procedural changes made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection 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 for all amplicones based on 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 the number of each amplicon, K is the segmented adjustment coefficient, when Ri < R, it is K1, when Ri ≥ R, it is K2; the selectable range of the parameter a is 0.2 - 0.4, index i the selectable range is 0.2 - 5; Calculate index i Then, according to Formula 2: Primer concentration for each amplicon = original primer concentration × index i ×V 基 / V 终 Calculate the primer re-injection concentration; where, index i V is the concentration adjustment coefficient corresponding to the i-th primer pair; 基 This is the baseline volume for each primer mix. index i ×V 基 V represents the final volume of each primer pair mixture; 终 This refers to the reconstitution volume after mixing all primers each time; When performing the nth (n≥2) round of primer concentration adjustment, the following is calculated: index i The primer concentration should then be calculated using the complexation concentration coefficient, i.e., according to formula 3: Primer concentration for each amplicon = Original primer concentration × Complexation concentration. index i ×V 基 / V 终 Calculate the primer re-injection concentration and compound. index i For n-1 rounds index i With n rounds index i The product; 4): Repeat steps 1) to 3) once more until the Gini coefficient is ≤ a.

2. The method according to claim 1, characterized in that, The methods for obtaining the quantity information of each amplicon include fluorescent PCR, digital PCR, capillary electrophoresis, microarray chips, microfluidic chips, and high-throughput sequencing.

3. The method according to claim 1, characterized in that, The method for obtaining the number of each amplicon is high-throughput sequencing.

4. The method according to claim 1, characterized in that, The parameters b, K1, and K2 are all variable parameters.

5. The method according to claim 4, characterized in that, The parameter b can be selected from 2 to 10; the parameter K1 can be selected from 0.3 to 2; and the parameter K2 can be selected from 0.1 to 1.

6. A multiplex PCR amplification efficiency adjustment system, characterized in that, The system includes: Amplification product detection module: Detects multiplex PCR amplification products to obtain the quantity information of each amplicon; Gini coefficient calculation module: Calculates the Gini coefficient of all amplicones based on 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 the number of each amplicon, K is the segmented adjustment coefficient, when Ri < R, it is K1, when Ri ≥ R, it is K2; the selectable range of parameter a is 0.2 - 0.4, index i the selectable range is 0.2 - 5; Calculate index i Then, according to Formula 2: Primer concentration for each amplicon = original primer concentration × index i ×V 基 / V 终 Calculate the primer re-injection concentration; where, index i V is the concentration adjustment coefficient corresponding to the i-th primer pair; 基 This is the baseline volume for each primer mix. index i ×V 基 V represents the final volume of each primer pair mixture; 终 This refers to the reconstitution volume after mixing all primers each time; When performing the nth (n≥2) round of primer concentration adjustment, the following is calculated: index i The primer concentration should then be calculated using the complexation concentration coefficient, i.e., according to formula 3: Primer concentration for each amplicon = Original primer concentration × Complexation concentration. index i ×V 基 / V 终 Calculate the primer re-injection concentration and compound. index i For n-1 rounds index i With n rounds index i The product; Repeat the adjustment module: Repeat the steps in the amplification product detection module, Gini coefficient calculation module, and primer concentration adjustment module until the Gini coefficient is ≤ a.

7. The system according to claim 6, characterized in that, The methods for obtaining the quantity information of each amplicon include fluorescent PCR, digital PCR, capillary electrophoresis, microarray chips, microfluidic chips, and high-throughput sequencing.

8. The system according to claim 7, characterized in that, The method for obtaining the number of each amplicon is high-throughput sequencing.

9. The system according to claim 6, characterized in that, The parameters b, K1, and K2 are all variable parameters.

10. The system according to claim 9, characterized in that, The parameter b can be selected from 2 to 10; the parameter K1 can be selected from 0.3 to 2; and the parameter K2 can be selected from 0.1 to 1.

11. A computer device, characterized in that, The computer device includes: A memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the method of any one of claims 1-5.

12. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program that, when executed by a processor, implements the method described in any one of claims 1-5.

13. The following application, characterized in that, The applications include: 1) The application of the method according to any one of claims 1-5 in adjusting the primer concentration for multiplex PCR amplification reactions; 2) The application of the method according to any one of claims 1-5 in sequencing multiplex PCR amplification products.

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