Second-generation sequencing excision reaction liquid

Abstract

The invention provides a next-generation sequencing excision reaction solution which is characterized by comprising a buffer solution, a reducing agent, a sodium salt and a surfactant, and the buffer solution comprises ethanolamine, ethanolamine hydrochloride and / or tris (hydroxymethyl) aminomethane and tris (hydroxymethyl) aminomethane hydrochloride. The method is used in the second-generation sequencing excision reaction process, the activity of DNA polymerase is not affected in the reaction, the probability of connecting chain residues in the excision process is reduced, the stability of the excision reaction is improved, and the sequencing quality is guaranteed.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene sequencing, and particularly relates to a second-generation sequencing excision reaction solution. Background Art

[0002] Next-generation sequencing (NGS), also known as high-throughput sequencing, is a DNA sequencing technology developed based on PCR and gene chips. In the process of DNA replication, NGS determines the DNA sequence by capturing the special markers (generally fluorescent molecule markers) carried by the newly added bases. The existing NGS technologies, such as sequencing by synthesis (SBS) represented by the bridge amplification technology of Illumina and the DNBSEQ technology of BGI, all identify and distinguish the four bases (A, C, G, and T / U) in the DNA sequence by detecting fluorescence signals. Specifically, these methods use dNTPs (dATP, dCTP, dGTP, dTTP) with fluorescent groups and blocking groups, and dATP, dCTP, dGTP, and dTTP carry different fluorescent groups respectively. Currently, the sequencing methods based on SBS include the following three steps: 1. Synthesis reaction: Under the action of polymerase, dNTPs with fluorescent groups and blocking groups are added to the 3'-end of the sequencing synthesis strand; 2. Signal acquisition: After the polymerization reaction is completed, the optical imaging system collects its fluorescence signal; 3. Excision reaction: After the signal acquisition is completed, a suitable excision reagent is added to cut the fluorescent group and the blocking group from the dNTPs, so that the dNTPs return to the natural state and enter the next cycle.

[0003] In the currently most popular Illumina's Miseq / Hiseq platform, different fluorescent groups are respectively labeled on 4 kinds of reversible termination nucleotides (NRT) and added to the reaction to distinguish different base sequences. These fluorescent groups are connected to the bases through cleavable chains, and an azide group is added at the 3'-OH end for protection. Currently, the fluorescent dye labels modified on NRT are connected to the bases through cleavable chains, and in the excision process, there may be a phenomenon of partial residual connection chains. These residual chemical bonds will affect the extension of polymerase and greatly increase the probability of base complementary mismatch, reducing the flexibility of the new strand, thus affecting the sequencing quality and interfering with the judgment of the results. Summary of the Invention

[0004] To solve the problems in the prior art, the present invention provides a second-generation sequencing excision reaction solution, which is used in the second-generation sequencing excision reaction process. During the reaction, it will not affect the activity of DNA polymerase, reduce the probability of residual ligation chains during excision, improve the stability of the excision reaction, and ensure the sequencing quality.

[0005] The present invention solves its technical problems by adopting the following technical solutions:

[0006] The first object of the present invention is to provide a second-generation sequencing excision reaction solution, which includes a buffer solution, a reducing agent, a sodium salt, and a surfactant. The buffer solution includes ethanolamine, ethanolamine hydrochloride, and / or tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride.

[0007] Furthermore, the concentration ratio of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride is 1:1:1:1.

[0008] A second-generation sequencing excision reaction solution includes a buffer solution, a reducing agent, a sodium salt, and a surfactant. The buffer solution includes ethanolamine and ethanolamine hydrochloride.

[0009] Furthermore, the concentration ratio of ethanolamine and ethanolamine hydrochloride is 1:1.

[0010] A second-generation sequencing excision reaction solution includes a buffer solution, a reducing agent, a sodium salt, and a surfactant. The buffer solution includes tris(hydroxymethyl)aminomethane and tris(hydroxymethyl)aminomethane hydrochloride.

[0011] Furthermore, the concentration ratio of tris(hydroxymethyl)aminomethane and tris(hydroxymethyl)aminomethane hydrochloride is 1:1.

[0012] Through the above technical solutions, the two buffer systems can be combined with each other or used alone, and good sequencing effects can be obtained. They have a very good effect on stabilizing the pH of the reaction environment. Under their combined action, higher stability of the excision reaction reagent can be obtained under the same conditions compared with the excision reaction solution on the market, thereby obtaining higher sequencing quality.

[0013] Furthermore, the sodium salt is selected from sodium chloride. By adding the sodium salt, a corresponding ionic environment is provided for the unexcised reaction.

[0014] Furthermore, the surfactant is selected from Tween 20.

[0015] By adding the surfactant, the surface tension of the solution is reduced, making the solution easier to wet and penetrate. It can increase the contact area between the liquid and the flow cell, which helps the progress of the excision reaction.

[0016] Further, the concentrations of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride are 1 to 100 mM.

[0017] Further, the concentrations of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride are 40 to 60 mM.

[0018] Further, the concentrations of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride are any one of 40 mM, 50 mM, and 60 mM.

[0019] Further, the concentrations of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride are 50 mM.

[0020] Further, the reducing agent includes tris(3-hydroxypropyl)phosphine and / or tris(2-carboxyethyl)phosphine. By increasing the reducing agent, the sequencing quality is improved and the probability of residual ligation chains during excision is reduced. When tris(3-hydroxypropyl)phosphine or tris(2-carboxyethyl)phosphine is used alone, the above two reducing agents have the best excision effect on fluorescent dye labeling, and the probability of residual ligation chains during excision is the lowest. When tris(3-hydroxypropyl)phosphine and tris(2-carboxyethyl)phosphine act together under the same conditions, higher sequencing quality can be obtained than that of the excision reaction solution on the market.

[0021] Further, the reducing agent is tris(3-hydroxypropyl)phosphine and tris(2-carboxyethyl)phosphine, and the concentration ratio of tris(3-hydroxypropyl)phosphine to tris(2-carboxyethyl)phosphine is 1:1.

[0022] Further, the concentrations of tris(3-hydroxypropyl)phosphine and tris(2-carboxyethyl)phosphine are 1 to 500 mM.

[0023] Further, the concentrations of tris(3-hydroxypropyl)phosphine and tris(2-carboxyethyl)phosphine are 80 to 200 mM.

[0024] Further, the concentrations of tris(3-hydroxypropyl)phosphine and tris(2-carboxyethyl)phosphine are any one of 80 mM, 90 mM, 100 mM, and 200 mM.

[0025] Further, the concentration of the sodium salt is 10 to 1000 mM.

[0026] Further, the concentration of the sodium salt is 300 to 700 mM.

[0027] Further, the concentration of the sodium salt is any one of 300 mM, 400 mM, 500 mM, 600 mM, and 700 mM.

[0028] Further, the concentration of the sodium salt is 500 mM.

[0029] Further, the volume fraction of the surfactant is 0.01 - 1%.

[0030] Further, the volume fraction of the surfactant is 0.03 - 0.1%.

[0031] Further, the volume fraction of the surfactant is any value among 0.03%, 0.05%, 0.07%, 0.08%, and 0.1%.

[0032] Further, the volume fraction of the surfactant is 0.05%.

[0033] Further, a second-generation sequencing excision reaction solution includes 50 mM ethanolamine, 50 mM ethanolamine hydrochloride, 50 mM tris(hydroxymethyl)aminomethane, 50 mM tris(hydroxymethyl)aminomethane hydrochloride, 100 mM tris(3-hydroxypropyl)phosphine, 100 mM tris(2-carboxyethyl)phosphine, 500 mM sodium chloride, and Tween 20 with a volume fraction of 0.05%.

[0034] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0035] The present invention adds a buffer solution. Ethanolamine, ethanolamine hydrochloride, and / or tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride stabilize the environmental pH value, enabling the excision reaction to have better stability; a reducing agent is added and used in combination with the buffer solution, which has a good excision effect on fluorescent dye labeling and almost no residue in the ligation chain; the sodium salt provides the required ionic environment for the excision reaction, and the surfactant reduces the surface expansion force of the solution, making the solution easier to wet and penetrate, increasing the contact area between the liquid and the flow cell, and contributing to the progress of the excision reaction. The excision reaction solution of the present invention increases the contact area between the liquid and the flow cell, reduces the probability of ligation chain residue during excision, improves the stability of the excision reaction, and ensures the sequencing quality; moreover, the excision reaction will not affect the activity of DNA polymerase, and has higher stability of the excision reaction reagent compared with the excision reaction solutions on the market, obtaining higher sequencing quality.

[0036] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above and other objects, features, and advantages of the present invention more obvious and understandable, the following specifically illustrates the embodiments of the present invention. Detailed Embodiments

[0037] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0038] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or prepared by existing methods.

[0039] The sequencing platform used in the embodiments of the present invention is Illumina Nextseq 550.

[0040] Example 1

[0041] An optimized second-generation sequencing excision reaction solution includes a buffer, a reducing agent, a sodium salt, and a surfactant. The buffer is ethanolamine (50 mM), ethanolamine hydrochloride (50 mM), tris(hydroxymethyl)aminomethane (50 mM), and tris(hydroxymethyl)aminomethane hydrochloride (50 mM); the reducing agents are tris(3-hydroxypropyl)phosphine (100 mM) and tris(2-carboxyethyl)phosphine (100 mM); the sodium salt is sodium chloride (500 mM), and the volume fraction of the surfactant Tween 20 is 0.05%.

[0042] In order to detect the advantages of the second-generation sequencing excision reaction solution of the present invention over the excision reaction solution in the prior art in sequencing, a commercially available Illumina sequencing kit (NextSeq 500 / 550 v2.5 Kit) was purchased, and the excision reaction solution in the kit was replaced with the excision reaction solution of each embodiment of the present invention, and sequencing comparison was carried out with the original Illumina sequencing kit (NextSeq 500 / 550 v2.5 Kit). When sequencing, the experimental conditions were the same, and two tests were carried out respectively. The sequencing methods used are as follows:

[0043] A sequencing method for human fetal free DNA includes the following steps:

[0044] 1. Sequencing library construction

[0045] The extraction and purification of free DNA from the sample were completed using a magnetic bead method free DNA extraction kit (Magen, IVD5432-TL-06A), and a universal library construction kit, Novoprotein VAHTS Universal Plus DNA Library Prep Kit for Illumina (product number: ND617-02), was used to construct the library. The concentrated library was subjected to concentration detection and fragment length quality control.

[0046] After the above operation, a library sample of about 50 nM with a length of about 1 to 400 bp was obtained.

[0047] 2. Preparation of sequencing chips

[0048] Illumina's Nextseq550 sequencer and its matching sequencing kit (NextSeq 500 / 550v2.5Kit) were used to perform library denaturation, library loading, and chip surface amplification to obtain DNA amplification clusters, and sequencing primers were added. After hybridization, the DNA amplification clusters hybridized with sequencing primers were fixed in the flow cell of the sequencing chip, waiting for the next sequencing reaction.

[0049] 3. Preparation of Sequencing Reagents and Sequencing

[0050] After signal acquisition is completed, the excision reaction solution is pumped in for next-generation sequencing.

[0051] After Example 1 and the comparative example were sequenced according to the above method, the sequencing results and analysis are shown in Table 1. According to Table 1, it can be seen that the use of the sequencing reagent containing the excision reaction solution can significantly improve the amount and quality of sequencing data of the high GC content library.

[0052] Table 1 Comparison of sequencing results

[0053]

[0054] Example 2

[0055] An optimized second-generation sequencing excision reaction solution comprises a buffer, a reducing agent, a sodium salt and a surfactant, wherein the buffer comprises ethanolamine (60 mM), ethanolamine hydrochloride (60 mM), tris(hydroxymethyl)aminomethane (60 mM) and tris(hydroxymethyl)aminomethane hydrochloride (60 mM); the reducing agent comprises tris(3-hydroxypropyl)phosphine (200 mM) and tris(2-carboxyethyl)phosphine (200 mM); the sodium salt comprises sodium chloride (500 mM), and the volume fraction of the surfactant Tween 20 is 0.05%.

[0056] The sequencing was performed using the same sequencing method as in Example 1. The detection in this example used a commercially available Illumina sequencing kit (NextSeq 500 / 550v2.5 Kit), and the excision reaction solution in the kit was replaced with the excision reaction solution in this example; the comparative example also used the original Illumina sequencing kit (NextSeq 500 / 550v2.5 Kit) for sequencing comparison. When performing sequencing, the experimental conditions were consistent, and two tests were performed respectively.

[0057] After Example 2 and the comparative example were sequenced according to the above method, the sequencing results and analysis are shown in Table 2.

[0058] Table 2 Comparison Table of Sequencing Results

[0059]

[0060]

[0061] Example 3

[0062] An optimized second-generation sequencing excision reaction solution, comprising a buffer, a reducing agent, a sodium salt, and a surfactant, wherein the buffer is ethanolamine (50 mM), ethanolamine hydrochloride (50 mM); the reducing agent is tris(2-carboxyethyl)phosphine (100 mM); the sodium salt is sodium chloride (500 mM), and the volume fraction of the surfactant Tween 20 is 0.05%.

[0063] Sequencing was performed using the same sequencing method as in Example 1. In this example, a commercially available sequencing kit from Illumina (NextSeq 500 / 550 v2.5 Kit) was used for detection, and the excision reaction solution in the kit was replaced with the excision reaction solution of this example; the control also used the original Illumina sequencing kit (NextSeq 500 / 550 v2.5 Kit) for sequencing comparison. When sequencing, the experimental conditions were the same, and two tests were performed respectively.

[0064] After sequencing Example 3 and the control according to the above method, the sequencing results and analysis are shown in Table 3.

[0065] Table 3 Comparison Table of Sequencing Results

[0066]

[0067] Example 4

[0068] An optimized second-generation sequencing excision reaction solution, comprising a buffer, a reducing agent, a sodium salt, and a surfactant, wherein the buffer is ethanolamine (50 mM), ethanolamine hydrochloride (50 mM); the reducing agent is tris(3-hydroxypropyl)phosphine (100 mM); the sodium salt is sodium chloride (500 mM), and the volume fraction of the surfactant Tween 20 is 0.05%.

[0069] Sequencing was performed using the same sequencing method as in Example 1. In this example, a commercially available sequencing kit from Illumina (NextSeq 500 / 550 v2.5 Kit) was used for detection, and the excision reaction solution in the kit was replaced with the excision reaction solution of this example; the control also used the original Illumina sequencing kit (NextSeq 500 / 550 v2.5 Kit) for sequencing comparison. When sequencing, the experimental conditions were the same, and two tests were performed respectively.

[0070] After sequencing Example 4 and the comparative example according to the above method, the sequencing results and analysis are shown in Table 4.

[0071] Table 4 Comparison Table of Sequencing Results

[0072]

[0073] Example 5

[0074] An optimized second-generation sequencing excision reaction solution, comprising a buffer, a reducing agent, a sodium salt, and a surfactant, wherein the buffer is tris(hydroxymethyl)aminomethane (50 mM), tris(hydroxymethyl)aminomethane hydrochloride (50 mM); the reducing agent is tris(2-carboxyethyl)phosphine (100 mM); the sodium salt is sodium chloride (500 mM), and the volume fraction of the surfactant Tween 20 is 0.05%.

[0075] Sequencing was performed using the same sequencing method as in Example 1. In this example, a commercially available sequencing kit from Illumina (NextSeq 500 / 550 v2.5 Kit) was used for detection, and the excision reaction solution in the kit was replaced with the excision reaction solution of this example; the comparative example also used the original Illumina sequencing kit (NextSeq 500 / 550 v2.5 Kit) for sequencing comparison. When performing sequencing, the experimental conditions were the same, and two tests were performed respectively.

[0076] After sequencing Example 5 and the comparative example according to the above method, the sequencing results and analysis are shown in Table 5.

[0077] Table 5 Comparison Table of Sequencing Results

[0078]

[0079] Example 6

[0080] An optimized second-generation sequencing excision reaction solution, comprising a buffer, a reducing agent, a sodium salt, and a surfactant, wherein the buffer is tris(hydroxymethyl)aminomethane (50 mM), tris(hydroxymethyl)aminomethane hydrochloride (50 mM); the reducing agent is tris(3-hydroxypropyl)phosphine (100 mM); the sodium salt is sodium chloride (500 mM), and the volume fraction of the surfactant Tween 20 is 0.05%.

[0081] Sequencing was performed using the same sequencing method as in Example 1. In this example, a commercially available sequencing kit from Illumina (NextSeq 500 / 550 v2.5 Kit) was used for detection, and the excision reaction solution in the kit was replaced with the excision reaction solution of this example; for the comparative example, the original Illumina sequencing kit (NextSeq 500 / 550 v2.5 Kit) was also used for sequencing comparison. When performing sequencing, the experimental conditions were the same for each, and two tests were carried out respectively.

[0082] After sequencing the samples of Example 6 and the comparative example according to the above method, the sequencing results and analysis are shown in Table 6.

[0083] Table 6 Comparison of sequencing results

[0084]

[0085] It can be seen that compared with the excision reaction solution in the prior art, the excision reaction solution in each embodiment of the present invention improves the sequencing throughput and data quality of high-GC-content libraries in next-generation sequencing.

[0086] The serial numbers of the embodiments of the present invention above are only for description and do not represent the superiority or inferiority of the embodiments.

[0087] The embodiments of the present invention have been described above, but the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention, and all of these fall within the protection scope of the present invention.

Claims

1. A second-generation sequencing excision reaction solution, characterized in that: It includes a buffer, a reducing agent, a sodium salt, and a surfactant. The buffer includes ethanolamine, ethanolamine hydrochloride, and / or tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride.

2. The second-generation sequencing excision reaction solution according to claim 1, wherein: The concentration ratio of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride is 1:1:1:

1.

3. The second-generation sequencing excision reaction solution according to claim 1, wherein: A second-generation sequencing excision reaction solution includes a buffer, a reducing agent, a sodium salt, and a surfactant. The buffer includes ethanolamine and ethanolamine hydrochloride.

4. The second-generation sequencing excision reaction solution according to claim 1, wherein: A second-generation sequencing excision reaction solution includes a buffer, a reducing agent, a sodium salt, and a surfactant. The buffer includes tris(hydroxymethyl)aminomethane and tris(hydroxymethyl)aminomethane hydrochloride.

5. The second-generation sequencing excision reaction solution according to claim 1, wherein: The surfactant is selected from Tween 20.

6. The second-generation sequencing excision reaction solution according to any one of claims 1-4, characterized in that: The concentrations of ethanolamine, ethanolamine hydrochloride, tris(hydroxymethyl)aminomethane, and tris(hydroxymethyl)aminomethane hydrochloride are 1 to 100 mM.

7. The second-generation sequencing excision reaction solution according to claim 1, wherein: The reducing agent includes tris(3-hydroxypropyl)phosphine and / or tris(2-carboxyethyl)phosphine.

8. The second-generation sequencing excision reaction solution according to claim 7, characterized in that: The concentrations of tris(3-hydroxypropyl)phosphine and tris(2-carboxyethyl)phosphine are 1 to 500 mM.

9. The second-generation sequencing excision reaction solution according to claim 1, wherein: It includes 50 mM ethanolamine, 50 mM ethanolamine hydrochloride, 50 mM tris(hydroxymethyl)aminomethane, 50 mM tris(hydroxymethyl)aminomethane hydrochloride, 100 mM tris(3-hydroxypropyl)phosphine, 100 mM tris(2-carboxyethyl)phosphine, 500 mM sodium chloride, and 0.05% (v / v) Tween 20.

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