Cleaning solution, kit and method for removing primer dimers in targeted high-throughput sequencing library

Abstract

The invention discloses a cleaning solution, a kit and a method for removing primer dimers in a targeted high-throughput sequencing library, and belongs to the technical field of high-throughput sequencing. And the cleaning solution comprises polyethylene glycol, NaCl and MgCl2. By utilizing the cleaning solution, the kit and the method provided by the invention, the primer dimer generated by the super-multiplex PCR amplification can be efficiently removed under the condition of not depending on special equipment and special operation, the compatibility of the multiplex PCR amplification to the primer which is easy to generate the primer dimer can be improved, the design optimization cost of a primer pool is saved, and the detection efficiency is improved. The application scene of the ultra-multiplex PCR amplification in the field of molecular diagnosis is widened.

Description

Technical Field

[0001] The present application relates to the technical field of high-throughput sequencing, and in particular to a cleaning solution, a kit, and a method for removing primer dimers in a targeted high-throughput sequencing library. Background Art

[0002] In recent years, with the maturity and application of high-throughput sequencing (NGS) technology in disease diagnosis, its high throughput, high sensitivity, and high specificity have demonstrated significant advantages in the diagnosis of infectious diseases. In this application, targeted high-throughput sequencing (tNGS) can detect hundreds of pathogens with less than 1M sequencing reads. Compared to metagenomics next-generation sequencing (mNGS), tNGS offers higher throughput and lower costs.

[0003] Super-multiplex PCR technology is one of the important methodological foundations of tNGS technology. Its principle is to add hundreds to thousands of pairs of targeted amplification primers to the PCR reaction system, which can achieve amplification and enrichment of hundreds to thousands of targets in a single-tube reaction. The tNGS technology developed based on this methodology has the advantages of convenient operation and low reagent cost. However, the high concentration and multiple sequence types of primers in the super-multiplex PCR reaction system are very easy to form primer dimers during the PCR reaction. Once primer dimers are introduced into the subsequent library construction system to form a library, they will reduce the sequencing quality and generate a large number of meaningless reads. These meaningless reads will squeeze the detection data throughput of the target target, resulting in a decrease in the sensitivity of the detection method. In summary, super-multiplex PCR technology has the advantages of convenient operation and low reagent cost, but the problem of easy formation of primer dimers limits its application in tNGS disease diagnosis.

[0004] The existing methods for reducing primer dimers in the reaction products of super-multiplex PCR include enzymatic removal and magnetic bead purification. The enzymatic removal method introduces special nucleotides or special sequences into the target amplification primers, and after the super-multiplex PCR reaction, an endonuclease that can recognize the special nucleotides or special sequences is used to cut the primer dimers, so that the dimers lose the double-stranded structure or the linker sequence, preventing the primer dimers from forming a complete library structure and entering the sequencing reaction, thereby reducing the influence of primer dimers on sequencing. However, the introduction of special nucleotides and modifications on the primers increases the cost of primer synthesis, and the enzymatic reaction also increases the complexity of the operation and the detection time, which cannot take advantage of the convenience and low cost of super-multiplex PCR operation. Magnetic bead purification is a method of nucleic acid purification based on the Solid Phase Reversible Immobilization (SPRI) technology. The principle is that the magnetic beads modified with carboxyl groups reversibly bind to nucleic acid fragments in a polyethylene glycol and sodium chloride environment, and by adjusting the concentrations of magnetic beads, polyethylene glycol and sodium chloride, selective adsorption and elution of nucleic acid fragments of different molecular weights can be achieved. This method does not require biochemical reactions or special instruments and equipment, and can purify the products after super-multiplex PCR to selectively enrich the target products and remove primer dimers. However, when detecting trace amounts of targets using super-multiplex PCR, the proportion of primer dimers in the products will be much higher than that of the target products. In this scenario, although the classic magnetic bead purification method can selectively preferentially adsorb larger target product fragments, a large amount of primer dimers will still be non-specifically adsorbed on the magnetic beads and ultimately introduced into the library construction reaction system.

[0005] In summary, although there are methods such as enzymatic removal and magnetic bead purification to reduce the proportion of primer dimers in super-multiplex PCR products, both methods have limitations and cannot be widely used in various application scenarios of tNGS diagnosis. SUMMARY

[0006] To solve at least one of the above technical problems, the technical solution adopted by the present application is as follows.

[0007] The first aspect of the present application provides a cleaning solution for removing primer dimers in a targeted high-throughput sequencing library, which comprises polyethylene glycol, NaCl and MgCl2, wherein the molar concentration of the polyethylene glycol is 20-30 mM, the molar concentration of the NaCl is 1-2 M, and the molar concentration of the MgCl2 is 1-2 M.

[0008] In the present application, when the magnetic beads adsorbed with the high-throughput sequencing library are cleaned with the cleaning solution, the primer dimers adsorbed on the magnetic beads can be cleaned away without affecting the library fragments.

[0009] In some embodiments of the present application, the molecular weight of the polyethylene glycol is 8000, i.e., PEG8000.

[0010] The second aspect of the present application provides a kit for removing primer dimers in a targeted high-throughput sequencing library, comprising any cleaning solution described in the first aspect of the present application.

[0011] In some embodiments of the present application, the kit further comprises DNA purification magnetic beads and / or ethanol solution.

[0012] In some embodiments of the present application, the concentration of the ethanol solution is 70% to 85%. In some specific embodiments of the present application, the concentration of the ethanol solution is 80%.

[0013] A third aspect of the present application provides a method for removing primer dimers in a targeted high-throughput sequencing library, comprising the following steps: S1, using DNA purification magnetic beads to adsorb the targeted high-throughput sequencing library; S2, performing a first alcohol washing on the magnetic beads using an ethanol solution; S3, washing the magnetic beads using any of the washing solutions described in the first aspect of the present application; S4, performing a second alcohol wash on the magnetic beads using an ethanol solution; S5, using an elution solution to elute the targeted high-throughput sequencing library adsorbed on the magnetic beads.

[0014] In some embodiments of the present application, the targeted high-throughput sequencing library is amplified using super-multiplex PCR technology.

[0015] Multiplex polymerase chain reaction (MPCR) is a technique that simultaneously amplifies multiple targets through a single PCR reaction and uses specific detection methods to detect the amplified products, thereby enabling diagnosis of multiple targets. Hypermultiplex PCR generally refers to multiplex PCR with more than 20 targets.

[0016] In some embodiments of the present application, the fragment size in the targeted high-throughput sequencing library is greater than 400 bp.

[0017] In some specific embodiments of the present application, the fragment size in the targeted high-throughput sequencing library is 400 bp to 700 bp.

[0018] Compared with the prior art, this application has the following beneficial effects: The cleaning solution of the present application has simple ingredients and is easy to prepare. Combined, it can improve the selective adsorption of large-fragment target products by DNA purification magnetic beads and reduce the adsorption of primer dimers, thereby effectively removing the proportion of primer dimers in super-multiplex PCR amplification products and obtaining higher quality sequencing libraries.

[0019] The cleaning solution, kit, and method of the present application can efficiently remove primer dimers produced by super-multiplex PCR amplification without relying on special equipment and special operations, thereby improving the compatibility of multiplex PCR amplification with primers that are prone to producing primer dimers, saving the cost of primer pool design optimization, and broadening the application scenarios of super-multiplex PCR amplification in the field of molecular diagnosis.

[0020] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and other objects, features and advantages of the exemplary embodiments of the present application will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present application are shown in an illustrative and non-limiting manner, in which: Figure 1 The results of fragment analysis of the purified library in Example 1 of the present application are shown; Figure 2 The results of fragment analysis of the purified library in Comparative Example 1 of this application are shown; Figure 3 The results of fragment analysis of the purified library in Comparative Example 2 of this application are shown; Figure 4 The results of fragment analysis of the purified library in Comparative Example 3 of this application are shown; Figure 5 The results of fragment analysis of the purified library in Comparative Example 4 of this application are shown; Figure 6 The results of fragment analysis of the purified library in Comparative Example 5 of this application are shown; Figure 7 The results of fragment analysis of the purified library in Comparative Example 6 of this application are shown; Figure 8 The results of fragment analysis of the purified library in Comparative Example 7 of the present application are shown. DETAILED DESCRIPTION

[0022] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, particularly for definitions of relevant terms in the art disclosed therein. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0023] The numerical ranges in this application are approximate, so unless otherwise stated, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For ranges containing values ​​less than 1 or containing fractions greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For ranges containing single-digit numbers less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0024] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination thereof.

[0025] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer and more understandable, the present application is further described in detail below in conjunction with the embodiments.

[0026] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the application, and are not intended to limit the scope of what the inventors regard as their application. One skilled in the art will recognize many methods and materials as equivalent to those described herein and such

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials described herein will be referred to by the citation of the reference that first introduced such term into the art. As used and provided herein "exemplary" is used to mean, "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0028] Those of ordinary skill in the art will realize and appreciate that many embodiments of the application can be adapted to different applications, and that the application is not limited to the embodiments described herein. Accordingly, many modifications can be made by those skilled in the art without departing from the spirit and scope of the application.

[0029] Unless otherwise indicated, experimental methods in the following examples were carried out by conventional methods. Unless otherwise indicated, the apparatuses used in the following examples were conventional laboratory apparatuses; and unless otherwise indicated, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0030] Example 1 Removal of primer-dimers and library construction from multiplex PCR amplification products 1. Multiplex PCR reaction Multiplex PCR reactions were performed using UltraClean Pathogen Multiplex PCR Mix for DNA reagents, following the reagent instruction for reaction system configuration and PCR reaction. The target amplicon length in the reaction system used for testing was between 400-700 bp.

[0031] 2. Wash solution configuration Accurately weigh 20 g of PEG8000, 87.75 g of NaCl, and 136.4 g of MgCl2, and dissolve with deionized water and make up to 1000 mL.

[0032] The prepared wash solution was autoclaved at high temperature and high pressure, and stored in a medical refrigerator.

[0033] 3. Product purification and primer-dimer removal The multiplex PCR reaction products were purified and primer-dimer removed using the wash solution according to the following steps.

[0034] (1) DNA adsorption: Add 22.5 μL of DNA purification magnetic beads (VAHTS DNAClean Beads) to the super-multiplex PCR reaction system, vortex to mix, and let it stand for 5 minutes; (2) Alcohol washing: After adsorption, separate the magnetic beads using a magnetic stand, discard the supernatant and retain the magnetic beads. Wash the magnetic beads with 80% ethanol solution, discard the ethanol after washing and dry the magnetic beads.

[0035] (3) Dimer cleaning: Add deionized water to resuspend the magnetic beads and let it stand at room temperature for 3 minutes. After the standing period, add a cleaning solution equal to the volume of the magnetic bead suspension in (1) to the system, vortex to mix, and let it stand for 5 minutes to clean the primer dimers; (4) Alcohol washing: After dimer washing, separate the magnetic beads using a magnetic stand, discard the supernatant and retain the magnetic beads, wash the magnetic beads with 80% ethanol solution, discard the ethanol after washing and dry the magnetic beads; (5) Elution: Add enzyme-free water to resuspend the magnetic beads and let it stand at room temperature for 5 minutes to elute the product.

[0036] 3. Library Construction Use the VAHTS HiFi Amplification Mix to construct a library from the purified product in the previous step. Follow the instructions for library construction and PCR. After the reaction is complete, purify the library using magnetic beads.

[0037] 4. Library Fragment Analysis The purified library was analyzed using Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 1 shown.

[0038] Depend on Figure 1 It can be seen that by using the cleaning solution of this example and performing primer dimer removal according to the method of this example, the primer dimer peak in the library constructed by the amplification product is significantly reduced.

[0039] Comparison Example 1 Different from Example 1, during the product purification and primer dimer removal process, no cleaning solution is used for cleaning, that is, step (3) is omitted, and only two alcohol washes are performed.

[0040] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 2 shown.

[0041] Comparative Example 2 The difference from Example 1 is that the configuration of the cleaning liquid is replaced as follows: Accurately weigh 20 g of PEG8000 and 136.4 g of MgCl2, dissolve them in deionized water and dilute to 1000 mL, i.e., no NaCl is used in the cleaning solution.

[0042] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 3 shown.

[0043] Comparative Example 3 The difference from Example 1 is that the configuration of the cleaning liquid is replaced as follows: Accurately weigh 20 g of PEG8000 and 87.75 g of NaCl, dissolve them in deionized water and adjust the volume to 1000 mL, i.e., no MgCl2 is used in the cleaning solution.

[0044] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 4 shown.

[0045] Comparative Example 4 Different from Example 1, during the product purification and primer dimer removal process, the order of step (2) and step (3) is reversed, i.e., dimer washing is performed first, followed by two alcohol washes.

[0046] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 5 shown.

[0047] Comparative Example 5 Different from Example 1, during the product purification and primer dimer removal process, the order of step (3) and step (4) is reversed, i.e., two alcohol washes are performed first, and then dimer cleaning is performed.

[0048] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 6 shown.

[0049] Comparative Example 6 The difference from Example 1 is that the configuration of the cleaning liquid is replaced as follows: Accurately weigh 20 g of PEG2000, 87.75 g of NaCl, and 136.4 g of MgCl2, dissolve them in deionized water, and adjust the volume to 1000 mL.

[0050] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 7 shown.

[0051] Comparative Example 7 The difference from Example 1 is that the configuration of the cleaning liquid is replaced as follows: Accurately weigh 20 g of PEG600, 87.75 g of NaCl, and 136.4 g of MgCl2, dissolve them in deionized water, and adjust the volume to 1000 mL.

[0052] The purified library was also analyzed using the Qsep100 biological fragment analyzer. The analysis results are as follows: Figure 8 shown.

[0053] By comparing the library construction analysis results of Example 1 with those of Comparative Example 1, it can be seen that without using a cleaning solution for cleaning, the peak of primer dimers in the library constructed by the amplified product is very high ( Figure 2 ), while the cleaning solution of Example 1 was used for cleaning between the two alcohol washes, and the peak of primer dimers in the constructed library decreased significantly ( Figure 1 ).

[0054] By comparing the library construction analysis results of Example 1 with those of Comparative Examples 2 and 3, it can be seen that if the cleaning solution does not include NaCl or MgCl, the peak of primer dimers in the library constructed by the purified product will not only not decrease, but will be higher than that without using the cleaning solution ( Figure 3 and Figure 4 ).

[0055] By comparing the library construction analysis results of Example 1 with those of Comparative Examples 4 and 5, it can be seen that the peak of primer dimers in the library constructed by the purified product is still relatively high, whether the cleaning solution is used before the two alcohol washes or after the second alcohol wash ( Figure 5 and Figure 6 ), failed to achieve the effect of removing primer dimers.

[0056] By comparing the library construction analysis results of Example 1 with those of Comparative Examples 6 and 7, it can be seen that when the PEG molecular weight in the cleaning solution is reduced to PEG2000 (Comparative Example 6), the peak of primer dimers in the library constructed by the purified product is still high. Under the condition of PEG600 (Comparative Example 7), the enrichment of the target amplicon in the library is affected, and the peak of the target amplicon is significantly reduced ( Figure 7 and Figure 8 ).

[0057] In addition, it should be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A cleaning solution for removing primer dimers in a targeted high-throughput sequencing library, characterized in that: The invention comprises polyethylene glycol, NaCl and MgCl2, wherein the molar concentration of the polyethylene glycol is 20-30 mM, the molar concentration of the NaCl is 1-2 M, and the molar concentration of the MgCl2 is 1-2 M.

2. The cleaning solution according to claim 1, characterized in that The molecular weight of the polyethylene glycol is 8000.

3. A kit for removing primer dimers in a targeted high-throughput sequencing library, characterized in that: The cleaning solution comprises the cleaning solution according to claim 1 or 2.

4. The kit according to claim 3, wherein Also included are DNA purification magnetic beads and / or an ethanol solution.

5. The kit according to claim 4, characterized in that The concentration of the ethanol solution is 70% to 85%.

6. A method for removing primer dimers in a targeted high-throughput sequencing library, characterized in that: The following steps are involved: S1, using DNA purification magnetic beads to adsorb the targeted high-throughput sequencing library; S2, performing a first alcohol washing on the magnetic beads using an ethanol solution; S3, washing the magnetic beads using the washing solution according to claim 1 or 2; S4, performing a second alcohol wash on the magnetic beads using an ethanol solution; S5, using an elution solution to elute the targeted high-throughput sequencing library adsorbed on the magnetic beads.

7. The method according to claim 6, characterized in that The targeted high-throughput sequencing library is amplified using super-multiplex PCR technology.

8. The method according to claim 6 or 7, characterized in that The fragment size in the targeted high-throughput sequencing library is greater than 400 bp.

9. The method according to claim 8, characterized in that The fragment size in the targeted high-throughput sequencing library is 400 bp to 700 bp.

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