Noise reduction method based on multiple PCR targeted sequencing library molecules and application thereof

Abstract

The invention discloses a noise reduction method based on multiple PCR targeted sequencing library molecules and application of the noise reduction method. The noise reduction method comprises the following steps that multiple PCR amplification products are obtained, and the multiple PCR amplification products comprise one or more of a first round of multiple PCR amplification products subjected to targeted sequence amplification and a second round of multiple PCR amplification products added with sequence connectors; the multiplex PCR amplification product is sequentially purified through magnetic beads and a purification solution, the purification solution comprises magnetic bead supernate and enzyme-free water, and the magnetic bead supernate comprises polyethylene glycol, disodium ethylene diamine tetraacetate and sodium chloride. According to the method, the purification liquid is introduced for separation, so that non-specific amplification products can be effectively reduced, the target targeting rate is improved, and the method is low in cost, convenient to operate and suitable for a multiplex PCR amplification capture library building method of a conventional laboratory.

Description

Technical Field

[0001] The present application relates to the fields of nucleic acid detection and biomedicine, and specifically to a noise reduction method based on multiplex PCR targeted sequencing library molecules and its application. Background Art

[0002] High-throughput sequencing technology, also known as next generation sequencing (NGS), has a higher throughput than Sanger sequencing and can read the sequences of hundreds of thousands to millions of DNA molecules in one run. It has unique advantages in identifying pathogens, revealing the variation and evolution of pathogenic microorganisms, and the composition of microbial communities, and has been widely used in the fields of life sciences and medicine.

[0003] NGS technology is maturing year by year, which makes the cost of whole genome sequencing lower and lower. However, the analysis of the extremely large and complex data obtained after whole genome sequencing has not become simpler. On the contrary, the development of sequencing technology has shown two extreme directions: one is large and comprehensive whole genome sequencing, and the other is small and precise targeted resequencing. Targeted sequencing enriches the genomic region of interest through a capture kit and then sequences it. It can achieve ultra-high sensitivity and accuracy with a small amount of data, and realize rapid screening of variant sites. Compared with whole genome sequencing and whole exome sequencing, targeted sequencing can focus on the area of ​​concern, remove the interference of redundant data, and maximize the use of sequencing reads. The sequencing cost is low and the sequencing depth is deep, which is especially suitable for clinical applications when the sample size is small.

[0004] The targeted sequencing methods are mainly divided into two technical routes: multiplex amplicon sequencing and hybrid capture sequencing. Multiplex PCR targeted sequencing technology, also known as amplicon targeted sequencing technology, is a targeted sequencing technology that combines multiplex PCR technology with second-generation sequencing technology. This technology first uses multiplex PCR reactions to simultaneously amplify multiple target region sequences to obtain amplicon products, and then introduces the adapter sequence (adapter) required for second-generation sequencing to both sides of the amplicon product through PCR reaction or enzyme ligation reaction to obtain an amplicon library, and then performs second-generation sequencing and bioinformatics process analysis to obtain the sequence information of the target region and achieve the purpose of target region sequence detection.

[0005] Compared with the amplicon capture method, the hybridization capture experiment process is too complicated and cumbersome, and the manual operation time is too long. Too much manual intervention may cause uncontrollable impact on the experimental results. The simplification of the experimental process of amplicon capture technology greatly reduces the professional threshold of operators, allowing more people to complete the experiment and completely eliminating the risk of insufficient manpower.

[0006] Multiplex amplicon library construction has high sequencing depth, simple library construction process, fast speed, high flexibility and low cost, so it has received widespread attention in scientific research and clinical applications. However, due to the inherent error rate of high-throughput sequencing and errors in PCR amplification, some background noise is caused, which affects the authenticity of the test results. There are currently several methods for high-throughput sequencing noise reduction. First, the use of single molecule tags (Unique Molecular Identifier, UMI) to correct or correct residual noise; second, the use of special tools in bioinformatics analysis algorithms, such as GATK false positive removal tool (GATK VariantFiltration), VarScan false positive removal tool (fpfilter), etc.; third, the optimization and improvement of experimental processes. The first two noise reduction methods are more often introduced in multiplex capture targeted sequencing, but there are fewer noise reduction examples in the third experimental process optimization. At present, there is a technology that can simultaneously complete thousands of primer amplifications through special primer design (a hairpin primer structure), but it has high requirements for primer design and detection area; there is also a technology that uses extremely low cycle numbers and two rounds of magnetic bead sorting with increased Carry DNAs to improve amplification uniformity and specific product efficiency, but it has limited application for low starting amount or rare samples; there is also a technology that completes library construction through primer-specific Mocode barcodes, enzyme digestion and enzyme ligation, but the design and operation are cumbersome and have certain limitations. At present, there is no public universal experimental process in the multiplex library reconstruction method that can directly reduce nonspecific amplification and achieve noise reduction at the experimental level. Summary of the invention

[0007] Based on this, it is necessary to provide a noise reduction method based on multiplex PCR targeted sequencing library molecules and its application.

[0008] The first aspect of the present application provides a noise reduction method based on multiplex PCR targeted sequencing library molecules, comprising the following steps:

[0009] Obtaining multiplex PCR amplification products, wherein the multiplex PCR amplification products include one or more of a first round of multiplex PCR amplification products of targeted sequence amplification and a second round of multiplex PCR amplification products with added sequence adapters;

[0010] The multiplex PCR amplification products are purified using magnetic beads and purification solution in sequence, wherein the purification solution includes magnetic bead supernatant and enzyme-free water, and the magnetic bead supernatant includes polyethylene glycol, disodium ethylenediaminetetraacetic acid and sodium chloride.

[0011] In some embodiments, the volume proportion of the magnetic bead supernatant in the purified solution is 60% to 100%.

[0012] In some embodiments, the volume proportion of the magnetic bead supernatant in the purified solution is 67% to 80%.

[0013] In some embodiments, the steps of sequentially purifying using magnetic beads and purification solution include:

[0014] adding magnetic beads to the multiplex PCR amplification products for purification;

[0015] The multiplex PCR amplification products purified by magnetic beads were directly mixed with the purification solution for purification.

[0016] In some embodiments, the volume ratio of the magnetic beads to the purification solution is 1:(1.5~2).

[0017] The second aspect of the present application provides a method for constructing a multiplex PCR targeted sequencing library, comprising purifying and reducing the noise of the multiplex PCR targeted sequencing library using the noise reduction method described in the first aspect of the present application.

[0018] In some embodiments, the construction method comprises the following steps:

[0019] The genomic DNA was extracted and the first round of multiplex PCR amplification was performed using upstream and downstream specific primers containing specific sequences complementary to the target sequence and universal sequences;

[0020] Purifying the products of the first round of multiplex PCR amplification using magnetic beads and purification solution in sequence;

[0021] Adding adapter primers to the products purified by magnetic beads and purification solution in sequence to perform a second round of PCR amplification;

[0022] The products of the second round of PCR amplification were purified using magnetic beads and purification solution in turn to obtain a multiplex PCR targeted sequencing library.

[0023] The third aspect of the present application provides a kit for constructing a multiplex PCR targeted sequencing library, the kit comprising magnetic beads and a purification solution, wherein the purification solution comprises a magnetic bead supernatant and enzyme-free water, and the magnetic bead supernatant comprises polyethylene glycol, disodium ethylenediaminetetraacetic acid and sodium chloride.

[0024] In some embodiments, the volume proportion of the magnetic bead supernatant in the purified solution is 60% to 100%. Optionally, the volume proportion of the magnetic bead supernatant in the purified solution is 67% to 80%.

[0025] In some embodiments, the kit further comprises one or more of multiple specific primers, label primers, PCR reaction solution and ethanol.

[0026] The aforementioned noise reduction method introduces purification solution without adding new raw materials or reagents, and does not rely on complex instruments and equipment. It is low-cost and easy to operate, and can effectively reduce non-specific amplification products, increase the on-target rate, and improve the quality of the library and sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0028] Figure 1 This is a flow chart of multiplex PCR amplification in one embodiment of the present application;

[0029] Figure 2 This is a purification liquid cleaning process in one embodiment of the present application;

[0030] Figure 3 A in the figure is a quality inspection graph of a library without adding purification solution in one embodiment of the present application; B is a quality inspection graph of a library with adding purification solution in one embodiment of the present application. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0033] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0034] In the present application, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0035] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0036] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0037] In this application, when referring to the unit of a data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0038] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0039] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form a new technical solution.

[0040] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0041] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0042] Currently, there are few examples of high-throughput sequencing noise reduction through experimental process optimization and improvement, and some existing optimization methods have some shortcomings, such as limited application to low starting amount or rare samples; cumbersome design and operation, etc. There is no public universal experimental process that can directly reduce nonspecific amplification and thus reduce noise at the experimental level.

[0043] Based on this, the embodiments of the present application at least provide a noise reduction method based on multiplex PCR targeted sequencing library molecules and its application. This method can effectively reduce non-specific amplification products by introducing purification liquid sorting, thereby improving the target on-target rate. This technology is a multiplex PCR amplification capture library construction method with low cost, easy operation, and suitable for conventional laboratories.

[0044] In some embodiments, the noise reduction method includes preparation of a purified solution and noise reduction experimental operation.

[0045] In some embodiments, the flow chart of multiplex PCR amplification is as follows Figure 1 shown.

[0046] In some embodiments, the purification solution cleaning process is as follows Figure 2 shown.

[0047] In a first aspect of the present application, a method for noise reduction based on multiplex PCR targeted sequencing library molecules is provided, comprising the following steps:

[0048] S100: Acquire multiplex PCR amplification products, wherein the multiplex PCR amplification products include one or more of a first round of multiplex PCR amplification products of target sequence amplification and a second round of multiplex PCR amplification products with added sequence adapters;

[0049] S200: purifying the multiplex PCR amplification products using magnetic beads and purification solution in sequence, wherein the purification solution includes magnetic bead supernatant and enzyme-free water, and the magnetic bead supernatant includes polyethylene glycol, disodium ethylenediaminetetraacetic acid and sodium chloride.

[0050] In some embodiments, in step S100, the multiplex PCR amplification product may be only the first round multiplex PCR amplification product of the targeted sequence amplification or the second round multiplex PCR amplification product with the sequence linker added, or may be the first round multiplex PCR amplification product of the targeted sequence amplification and the second round multiplex PCR amplification product with the sequence linker added.

[0051] In some embodiments, in step S200, the volume proportion of the magnetic bead supernatant in the purified solution may be 60% to 100%. Further, the volume proportion of the magnetic bead supernatant in the purified solution may be 67% to 80%. Non-limitingly, the volume proportion of the magnetic bead supernatant in the purified solution may be, but is not limited to, 60%, 67%, 70%, 80%, 90%, 100%, or a value or range between any two of the above values.

[0052] In some embodiments, in step S200, the steps of sequentially purifying using magnetic beads and purification solution include:

[0053] S210: adding magnetic beads to the multiplex PCR amplification products for purification;

[0054] S220: The multiplex PCR amplification products purified by magnetic beads are directly mixed with the purification solution for purification.

[0055] In some embodiments, the volume ratio of the magnetic beads to the purified solution may be 1:(1.5-2). In a non-limiting manner, the volume ratio of the magnetic beads to the purified solution may be, but is not limited to, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, or a ratio or range between any two of the above ratios.

[0056] In some embodiments, in step S210, the multiplex PCR amplification products are not washed with alcohol after being purified by magnetic beads.

[0057] In some embodiments, the noise reduction results of the multiplex PCR targeted sequencing library molecules are judged by the results of the mapping ratio and / or the on-target ratio.

[0058] In this application, unless otherwise specified, "Mapping ratio" refers to the ratio of the number of successfully mapped reads to the total number of reads after the short sequences (reads) obtained by sequencing are aligned with the reference genome (reference genome).

[0059] In this application, unless otherwise specified, "On-target ratio" refers to the proportion of sequenced reads that can be mapped to a pre-set target region.

[0060] In some implementations, the value of the Mapping ratio is greater than or equal to 95%, and the value of the On-target ratio is greater than or equal to 85%.

[0061] In a second aspect of the present application, a method for constructing a multiplex PCR targeted sequencing library is provided, comprising purifying and reducing the noise of the multiplex PCR targeted sequencing library using the noise reduction method of the first aspect of the present application.

[0062] In some embodiments, the method for constructing a multiplex PCR targeted sequencing library comprises the following steps:

[0063] S1000: Extract genomic DNA and perform the first round of multiplex PCR amplification using upstream and downstream specific primers containing specific sequences complementary to the target sequence and universal sequences;

[0064] S2000: Purify the products of the first round of multiplex PCR amplification using magnetic beads and purification solution in sequence;

[0065] S3000: adding adapter primers to the products purified by magnetic beads and purification solution in sequence to perform a second round of PCR amplification;

[0066] S4000: The products of the second round of PCR amplification are purified using magnetic beads and purification solution in turn to obtain a multiplex PCR targeted sequencing library.

[0067] In a third aspect of the present application, a kit for constructing a multiplex PCR targeted sequencing library is provided, the kit comprising magnetic beads and a purification solution, wherein the purification solution comprises magnetic bead supernatant and enzyme-free water, and the magnetic bead supernatant comprises polyethylene glycol, disodium ethylenediaminetetraacetic acid and sodium chloride.

[0068] In some embodiments, the volume proportion of the magnetic bead supernatant in the purified solution is 60% to 100%, and further, the volume proportion of the magnetic bead supernatant in the purified solution is 67% to 80%.

[0069] In some embodiments, the kit further comprises one or more of multiple specific primers, label primers, PCR reaction solution and ethanol.

[0070] Some examples are provided below.

[0071] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0072] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0073] In the following examples, the magnetic beads are DNA purification magnetic beads purchased from Suzhou Weidu Biotechnology Co., Ltd.

[0074] Example 1

[0075] 1. Preparation of purified solution

[0076] After the magnetic bead reagent used for library construction is left to stand or adsorbed on a magnetic rack, the supernatant solution is transferred to a clean tube and the volume of the supernatant is measured. The supernatant includes polyethylene glycol, disodium ethylenediaminetetraacetic acid and sodium chloride. Then, the magnetic bead supernatant is mixed with enzyme-free water in a certain volume ratio (the magnetic bead supernatant accounts for 67%) and fully shaken and mixed. Then, it is divided into corresponding test tubes according to the actual dosage requirements and placed in a 4°C refrigerator for standby use. Before use, it needs to be placed at room temperature for half an hour in advance for equilibrium before use.

[0077] The raw materials required for the preparation of the purified solution do not increase in conventional multiplex PCR library construction, and the preparation operation is quick and easy. However, the ratio of magnetic bead supernatant to enzyme-free water required for the preparation of the purified solution needs to be specifically optimized. Different preparation ratios will produce different effects, mainly in the Mapping ratio and On-target ratio. The final required ratio was determined through repeated experiments. The specific results are shown in Table 1 below, that is, the total proportion of magnetic bead supernatant is about 67%, the Mapping ratio (greater than 98%) and the On-target ratio (about 90%) are both the highest, the effective utilization rate of data is the highest, and the effect is the best.

[0078] Table 1: Effect of different ratios of magnetic beads supernatant in purification solution preparation on data quality

[0079]

[0080] 2. Multiplex PCR library construction method with purification solution operation

[0081] This example uses clinical samples for testing. The clinical samples come from the Obstetrics and Gynecology Hospital Affiliated to Fudan University. Sample 1 and sample 2 are whole blood samples, and sample 3 and sample 4 are dried blood spot samples. Multiple primers (primer sequences refer to patent application CN116287198A, paragraphs

[149] to

[169] ) select a specific primer set of about 200, covering a 38kb region of the human genome. The specific experimental steps are as follows:

[0082] 1. Whole genome DNA extraction: Use a nucleic acid extraction kit (purchased from Guangzhou Meiji Biotechnology Co., Ltd.) to extract whole genome DNA from blood or blood card;

[0083] 2. First round of PCR: targeted sequence amplification

[0084] For each sample, a reaction system is prepared in a PCR tube according to Table 2, and the prepared system is placed on a PCR instrument to react according to the reaction conditions in Table 3.

[0085] Table 2: Reaction system

[0086]

[0087] [1] Determine the input volume based on the DNA Qubit quantitative results, and the required starting amount is 5-100 ng / reaction tube.

[0088] Table 3: Reaction conditions

[0089]

[0090] 3. The first round of magnetic beads and purification solution purification

[0091] a) Place the purified magnetic beads and purification solution at room temperature for at least 30 minutes to allow the temperature to equilibrate to room temperature, and vortex to mix.

[0092] b) Mix anhydrous ethanol and nuclease-free water (or ultrapure water, prepared by yourself) in a ratio of 4:1 to prepare an ethanol solution with a concentration of 80%.

[0093] c) Add 27 μL (0.9X) of purified magnetic beads to each centrifuge tube that completed the first round of PCR reaction, vortex to mix, and incubate at room temperature for 5 minutes.

[0094] d) Briefly centrifuge the centrifuge tube that has completed the above steps and place it on a magnetic rack to separate the magnetic beads and liquid. Let it stand for 2 to 5 minutes. After the solution is clarified, discard the supernatant and retain the magnetic beads.

[0095] e) Keep the centrifuge tubes on the magnetic rack, add 50 μL of purification solution, then remove the centrifuge tubes from the magnetic rack, shake to mix, and incubate at room temperature for 5 minutes.

[0096] f) Briefly centrifuge the centrifuge tube that has completed the above steps and place it on a magnetic rack to separate the magnetic beads and liquid. Let it stand for 2 to 5 minutes. After the solution is clear, discard the supernatant and retain the magnetic beads.

[0097] g) Keep the centrifuge tubes on the magnetic rack and add 200 μL of freshly prepared 80% ethanol solution to wash them. Incubate at room temperature for about 30 seconds, discard the supernatant, and retain the magnetic beads.

[0098] h) Repeat step g for a total of two washes.

[0099] i) Centrifuge briefly to remove the residual ethanol at the bottom, keep the centrifuge tube on the magnetic rack, add 13 μL of nuclease-free water, then remove the centrifuge tube from the magnetic rack, shake to mix and centrifuge briefly for subsequent library construction.

[0100] 4. Second round of PCR: adding adapter sequence

[0101] A linker was added to the PCR amplification product obtained in step 3. The reaction system was shown in Table 4, and the reaction conditions were shown in Table 5.

[0102] Table 4: Second round PCR reaction system

[0103]

[0104] Table 5: Second round PCR reaction program

[0105]

[0106] 5. Second round of magnetic beads and purification solution purification

[0107] The specific operation is the same as step 3.

[0108] 6. Library quantification and quality control

[0109] Take 1 μL of library and use Qubit to measure the library concentration and record the library concentration; take 1 μL of library and use Agilent DNA 1000 kit or equivalent instrument reagents to measure the library fragment length.

[0110] 7. High-throughput sequencing and data analysis

[0111] The library obtained in step 6 is subjected to high-throughput sequencing using a second-generation sequencing platform such as Illumina Novaseq or Nextseq CN500. The sequencing steps are strictly performed according to the operation provided by the sequencing platform. The sequencing data is analyzed by professionals according to certain requirements.

[0112] The library construction method of the present application can significantly improve the on-target rate, reduce data waste, and thus reduce sequencing costs. Some test data are shown in Table 6 and Figure 3 ,in, Figure 3 A in the figure is the library constructed without treatment with purification solution. Figure 3 B in the figure is the library constructed after adding the purification solution, "RFU" indicates the fluorescence intensity, and "length" indicates the fragment length. The library molecules used in the two methods are obtained by connecting the same sample to the adapters with different indexes. The library enrichment products are loaded on the machine in the same batch, and the data off the machine are compared and analyzed after down-sampling.

[0113] Table 6: Comparison of on-target rates of different DNA library molecules

[0114]

[0115] Example 2

[0116] Under the premise that other quality control parameters are not affected, in order to pursue a higher on-target rate and to further achieve economic savings, the amount of purification solution was further studied, and the results are shown in Table 7. The library construction method in this example is the same as that in Example 1, and the only difference is the amount of purification solution used (the amount before optimization is 15 microliters, and the amount after optimization is 50 microliters).

[0117] Table 7: Comparison of on-target rate before and after optimization of purified solution dosage

[0118]

[0119] Example 3

[0120] The method containing the purification solution operation step remained unchanged, and three groups of tests were performed (the amount of purification solution was 50 μL): the purification solution operation was performed only after PCR1; the purification solution operation was performed only after PCR2; the purification solution operation was performed after both PCR1 and PCR2. The results are shown in Table 8 below.

[0121] Table 8: Comparison of target hit rates with different purification solution operation times

[0122]

[0123] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0124] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A noise reduction method based on multiplex PCR targeted sequencing library molecules, characterized in that: The following steps are involved: Obtaining multiplex PCR amplification products, wherein the multiplex PCR amplification products include one or more of a first round of multiplex PCR amplification products of targeted sequence amplification and a second round of multiplex PCR amplification products with added sequence adapters; The multiplex PCR amplification products are purified using magnetic beads and purification solution in sequence, wherein the purification solution includes magnetic bead supernatant and enzyme-free water, and the magnetic bead supernatant includes polyethylene glycol, disodium ethylenediaminetetraacetic acid and sodium chloride.

2. The noise reduction method according to claim 1, characterized in that: The volume proportion of the magnetic bead supernatant in the purified solution is 60% to 100%.

3. The noise reduction method according to claim 2, characterized in that: The volume proportion of the magnetic bead supernatant in the purified solution is 67% to 80%.

4. The noise reduction method according to claim 1, wherein: The steps of sequentially purifying using magnetic beads and purification solution include: adding magnetic beads to the multiplex PCR amplification products for purification; The multiplex PCR amplification products purified by magnetic beads were directly mixed with the purification solution for purification.

5. The noise reduction method according to claim 4, characterized in that: The volume ratio of the magnetic beads to the purification solution is 1:(1.5~2).

6. A method for constructing a multiplex PCR targeted sequencing library, characterized in that: The method comprises purifying and denoising the multiplex PCR targeted sequencing library using the denoising method according to any one of claims 1 to 5.

7. The construction method according to claim 6, characterized in that: The following steps are involved: The genomic DNA was extracted and the first round of multiplex PCR amplification was performed using upstream and downstream specific primers containing specific sequences complementary to the target sequence and universal sequences; Purifying the products of the first round of multiplex PCR amplification using magnetic beads and purification solution in sequence; Adding adapter primers to the products purified by magnetic beads and purification solution in sequence to perform a second round of PCR amplification; The products of the second round of PCR amplification were purified using magnetic beads and purification solution in turn to obtain a multiplex PCR targeted sequencing library.

8. A kit for constructing a multiplex PCR targeted sequencing library, characterized in that: The kit comprises magnetic beads and a purification solution, wherein the purification solution comprises a magnetic bead supernatant and enzyme-free water, and the magnetic bead supernatant comprises polyethylene glycol, disodium ethylenediaminetetraacetate and sodium chloride.

9. The kit according to claim 8, characterized in that The volume proportion of the magnetic bead supernatant in the purified solution is 60% to 100%. Optionally, the volume proportion of the magnetic bead supernatant in the purified solution is 67% to 80%.

10. The kit according to claim 9, characterized in that The kit also includes one or more of multiple specific primers, label primers, PCR reaction solution and ethanol.

Images