A kit for circularizing a high gc content library and uses and methods thereof

Abstract

The present application relates to the field of high-throughput sequencing detection, in particular to a kit for high GC content library circularization and its use and method, the kit comprises a single-strand circularization reagent, the single-strand circularization reagent comprises a sequencing platform connecting primer, a ligase and a single-strand stabilizing additive, the single-strand stabilizing additive is selected from any one or more of betaine, formamide, DMSO or glycerol. The kit can be used to prepare a single-strand circularization DNA product. The kit of the present application has high circularization efficiency for high GC content library, sufficient sequencing depth and does not cause missed detection.

Description

Technical Field

[0001] This invention relates to the field of high-throughput sequencing detection, and in particular to a kit for circularizing high-GC-content libraries, its uses, and methods. Background Technology

[0002] In the life sciences field, with the development of sequencing technology, sequencing tools are constantly being innovated. BGI Genomics, with its unique new DNBseq... TM Sequencing technology, with its high accuracy, low error accumulation, low repetitive sequences, and low tag skipping, is widely used.

[0003] The overall workflow of the BGISEQ sequencing platform consists of three main steps: single-stranded circular DNA preparation, DNB preparation / loading, and sequencing analysis. Single-stranded circular DNA preparation is the foundation of this workflow. The core technology of single-stranded DNA circularization is the ligation reaction assisted by an oligonucleotide chain. Specifically, single-stranded DNA circularization involves denaturing double-stranded DNA (dsDNA) with adapter sequences at high temperature to form single-stranded DNA (ssDNA). Under the catalysis of ligase, splint oligo primers are complementary to the two ends of the ssDNA. The two ends of the ssDNA are ligated to form a single-stranded circular DNA molecule, which is subsequently used to construct a single-stranded circular DNA library specifically for the MGI high-throughput sequencer.

[0004] Genomic regions with high GC content often possess important biological functions, and variations or changes in the epigenetic status of these regions are often directly related to the occurrence and development of diseases such as tumors. Therefore, the detection of variations in high-GC genomic regions has significant clinical application value. However, several existing single-stranded circularized DNA preparation kits on the market are not ideal for circularizing library fragments containing high GC content, resulting in sequencing data performance indicators that do not meet the requirements of routine gene detection. In particular, the sequencing coverage depth is significantly lower than in other regions, easily leading to missed detections of gene variations. Therefore, there is an urgent need to develop a method to improve the circularization of high-GC content libraries and overcome the above problems. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a kit for high GC content library cyclization, its uses and methods, to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides a kit for circularization of high GC content libraries, the kit comprising a single-strand circularization reagent, wherein the single-strand circularization reagent comprises sequencing platform ligation primers, ligase, and single-strand stabilizing additives.

[0007] The single-chain stabilizing additive is selected from one or more of betaine, formamide, DMSO, or glycerol.

[0008] The present invention also provides the use of the kit for high GC content library circularization in the preparation of single-stranded circularized DNA products.

[0009] The present invention also provides a method for cyclizing a high GC content library, the method comprising cyclizing using the kit described above for cyclizing a high GC content library.

[0010] As described above, the high GC content library circularization kit, its uses, and methods of the present invention have the following beneficial effects: high circularization efficiency for high GC content libraries, sufficient sequencing depth, and no missed detections; at the same time, it does not affect the sequencing depth of other GC content libraries, thus effectively improving the uniformity between libraries and enhancing the overall performance of the panel for multi-primer mixed libraries. Attached Figure Description

[0011] Figure 1 The image shown is an agarose gel electrophoresis diagram of library fragments of different lengths according to the present invention.

[0012] Figure 2 The graph shows a comparison of the circularization efficiency of different library fragments in this invention.

[0013] Figure 3 The graph shows a comparison of the cyclization efficiency of different concentrations of betaine.

[0014] Figure 4 The graph shows a comparison of the cyclization efficiency of formamide at different concentrations. Detailed Implementation

[0015] This invention provides a kit for circularizing high GC content libraries, the kit comprising a single-stranded circularization reagent, which includes sequencing platform ligation primers, ligase, and single-stranded stabilizing additives.

[0016] In one embodiment, the high GC content refers to a GC content of 50-85%. For example, GC contents of 50-60%, 60-70%, 70-75%, 75-80%, and 80-85%.

[0017] The size of the high GC content library fragment is in the range of 100-600 bp. Preferably, the size of the high GC content library fragment is 200-400 bp.

[0018] In one embodiment, the sequencing platform ligation primers are 18-30 bp in length. In another embodiment, the sequencing platform ligation primers are ligation primers adapted to the BGISEQ sequencing platform, i.e., the 5' end sequence of the ligation primers is reverse complementary to the 3' end of the library constructed on the BGI platform, and the 3' end sequence is reverse complementary to the 5' end of the library constructed on the BGI platform.

[0019] In one embodiment, the ligation primer sequence is as shown in SEQ ID NO: 25: 5'-GGAACCGAGTGTCTTGCTGTACCG-3'.

[0020] In one embodiment, the ligase is selected from T4 ligase, Escherichia coli DNA ligase, or thermostable DNA ligase.

[0021] The single-chain stabilizing additive is selected from one or more of betaine, formamide, DMSO, or glycerol.

[0022] In one embodiment, the single-chain cyclization reagent further includes a cyclization buffer. The cyclization buffer provides a cyclization environment for the reaction. Those skilled in the art can select a suitable cyclization buffer according to the actual situation. The cyclization buffer includes Tris-HCl, MgCl2, DTT, and ATP. For example, based on the total volume of the cyclization buffer, it includes 300–500 mM Tris-HCl, 50–150 mM MgCl2, 50–150 mM DTT, and 3–8 mM ATP. In one embodiment, the cyclization buffer is, for example, T4 DNA ligase buffer, brand: Invitrogen, catalog number: 46300018.

[0023] The single-stranded cyclization reagent is used to prepare a DNA cyclization intermediate with sequencing adapters at both ends of a library into a DNA cyclization intermediate with connecting primers.

[0024] Each reagent in the single-chain cyclization reagent can be a separately packaged reagent or a mixture formed by mixing the reagents.

[0025] In one embodiment, the kit further includes an enzyme digestion reagent. The enzyme digestion reagent is used to digest the DNA circularization intermediate.

[0026] The enzyme digestion reagent includes a nuclease and a digestion buffer. The nuclease is selected from exonuclease I or exonuclease VII. The digestion buffer provides the digestion environment for the reaction. Those skilled in the art can select a suitable digestion buffer according to the actual situation. The digestion buffer includes glycine-KOH, MgCl2, and DTT. For example, the digestion buffer contains 600-700 mM glycine-KOH, 60-75 mM MgCl2, and 5-15 mM DTT, based on the total volume of the digestion buffer. In one embodiment, the digestion buffer is, for example, exonuclease I buffer, brand: Ruijing Biotechnology, catalog number RJ001T-A.

[0027] In one embodiment, the enzymatic digestion reagent further includes a digestion termination reagent. The digestion termination reagent is a metal chelating agent. Those skilled in the art can select precipitating chelating agents such as sodium carbonate and sodium orthophosphate, complexing chelating agents such as amino acid derivatives (e.g., EDTA) and hydroxy acids (e.g., citric acid), or phosphate chelating agents (e.g., sodium tripolyphosphate) as needed.

[0028] In one embodiment, the kit further includes a purification reagent. The purification reagent is used to purify the DNA circularization product.

[0029] The purification reagents include magnetic beads, ethanol, and TE buffer.

[0030] In one embodiment, the kit may also include consumables for cyclization, such as magnetic racks, centrifuge tubes, PCR tubes, etc.

[0031] The present invention also provides the use of the kit for high GC content library circularization in the preparation of single-stranded circularized DNA products or in gene sequencing.

[0032] The gene sequencing is for non-disease diagnostic purposes. Examples of non-disease diagnostic purposes include observing or detecting a specific gene to study the mechanisms of disease development.

[0033] The present invention also provides a method for cyclizing a high GC content library, the method comprising cyclizing using the kit for cyclizing a high GC content library.

[0034] In one embodiment, the method for circularizing the high GC content library includes the following steps:

[0035] 1) A single-stranded DNA library with sequencing adapters at both ends is mixed with a single-stranded circularization reagent and reacted to obtain a DNA circularization intermediate with linking primers.

[0036] 2) Mix the DNA circularization intermediate with the enzyme digestion reagent and perform enzyme digestion;

[0037] 3) After the enzymatic digestion is completed, mix with the digestion termination reagent to terminate the enzymatic digestion and obtain the cyclized product.

[0038] In one embodiment, the single-stranded DNA library with sequencing adapters at both ends in step 1) is obtained by annealing and denaturing a double-stranded DNA library with sequencing adapters at both ends. The double-stranded DNA library with sequencing adapters at both ends can be obtained using existing techniques. For example, the double-stranded DNA library with sequencing adapters at both ends can be obtained by amplifying and purifying the target DNA using 2xKAPA 2G Fast MultiplexMix, followed by mixing the purified product with adapter primers for a second round of amplification and purification.

[0039] In step 1), if there are special requirements for the library during library construction, obtain the required amount of DNA according to those requirements. If there are no special requirements, based on the circularization system, the amount of single-stranded DNA library in step 1) is 0.5 pmol to 1 pmol.

[0040] Different DNA fragment sizes correspond to different amounts per pmol molecule. The required amount of single-stranded DNA for the library can be calculated using the following formula:

[0041] Conversion formula between moles and mass:

[0042]

[0043] The single-stranded DNA library can be a single sample consisting of a single DNA library, or a mixture of samples consisting of multiple DNA libraries with different barcodes.

[0044] The total amount of single-stranded DNA library in the pooled sample should be 0.5 pmol to 1 pmol. If the required amount of data for each sample is the same, then the same amount should be pooled. For multiple DNA libraries with significant fragment differences, it is recommended to circularize them separately rather than circularizing them together as a pooled sample.

[0045] The single-stranded DNA library is 200-400 bp in length. In one embodiment, the single-stranded DNA library has BGI sequencing adapters at both ends. In another embodiment, the single-stranded DNA library has adapters adapted to the BGISEQ sequencing platform at both ends.

[0046] Based on the total volume of the circularized system, the final concentrations of the sequencing platform ligation primers and ligase are 0.2–0.8 μM and 0.2–1 U / μL, respectively. Preferably, the final concentrations of the sequencing platform ligation primers are 0.2–0.4 μM, 0.4–0.6 μM, and 0.6–0.8 μM. Preferably, the final concentrations of the ligase are 0.2–0.4 U / μL, 0.4–0.6 U / μL, 0.6–0.8 U / μL, and 0.8–1 U / μL.

[0047] When the single-chain stabilizing additive is betaine, the final concentration of betaine is 0.4–1.2 mol / L, based on the total volume of the cyclized system. Preferably, the final concentration is selected from 0.4–0.6 mol / L, 0.6–0.8 mol / L, 0.8–1.0 mol / L, and 1.0–1.2 mol / L.

[0048] In one embodiment, the cyclization conditions are 35–38°C for 12–18 min. In one embodiment, cyclization is performed on a PCR instrument. In one embodiment, the cyclization conditions also include maintaining the temperature at 4–20°C after enzyme digestion.

[0049] In step 2), the final concentration of nuclease is 0.5–2 U / μL, based on the total volume of the enzymatic digestion system.

[0050] In step 3), the final concentration of the digestion termination reagent is 0.05–0.1 mol / L, based on the total volume of the enzyme digestion system.

[0051] In one embodiment, the enzyme digestion conditions are 35–38°C for 8–12 minutes. In one embodiment, the enzyme digestion is performed on a PCR instrument. In one embodiment, the enzyme digestion conditions also include maintaining the temperature at 4–20°C after digestion.

[0052] In one embodiment, the cyclized product obtained in step 3) is further purified using a purification reagent. The purification step can be performed using techniques conventional in the art.

[0053] The present invention also provides a gene sequencing method, comprising circularizing the gene using the kit for circularizing high GC content libraries and then sequencing it using a sequencing platform.

[0054] The gene sequencing method of this invention is particularly suitable for sequencing high-GC-content libraries. In one embodiment, the sequencing platform is selected from BGI sequencing platforms, such as the BGISEQ sequencing platform.

[0055] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0056] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0057] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0058] Example 1: A High-GC Library Circularization Method Based on Fragment Size

[0059] In tumor cells, there are many driving factors for abnormal TERT gene expression, including transcription activators, TERT copy number variations, TERT promoter mutations, and TERT promoter region hypermethylation. The two most prominent mechanisms are TERT promoter region methylation (53%) and TERT promoter region mutations (31%). However, the GC content in the TERT promoter region is as high as 70%, posing a significant challenge to the detection of tumor-related genes.

[0060] The embodiments take the TERT boot sector as an example, and further illustrate the technical means and effects of the present invention with reference to the accompanying drawings and embodiments. The technical solution of the present invention will be further explained through specific implementation methods.

[0061]

Material

[0062] 1. Universal cyclization reaction kit, Ruijing Biotechnology, catalog number RJ001T-A;

[0063] 2.2×KAPA 2G Fast Multiplex Mix, Kapa biosystems, part number KK5802;

[0064] 3. TERT C250T low-frequency DNA (5%), Ruijing Biotechnology, catalog number RJ005Y;

[0065] 4. Agencourt AMPure XP (purified magnetic beads), Beckman, catalog number A63881;

[0066] 5. Qubit instrument and matching reagents (the reagent is Thermo, catalog number: Q10212);

[0067] 6. MGISEQ-200 sequencer and matching reagents (MGI Tech Manufacturing, reagent catalog number: 1000019932)

[0068] 7. Betaine, Ruijing Biotechnology, Product No. RJ003H;

[0069] 8. PCR instrument.

[0070] The library construction method is as follows:

[0071] 1. Sample types: TERT promoter region double-stranded DNA library containing BGI sequencing adapter primers, with GC content of 75% and lengths of 100bp, 200bp, 300bp, 400bp, 500bp, and 600bp; GAPDH internal reference gene double-stranded DNA library containing BGI sequencing adapter primers, with GC content of 50% and lengths of 100bp, 200bp, 300bp, 400bp, 500bp, and 600bp.

[0072] 1.1 Sample Preparation

[0073] 1.1.1 Primer sequences of different lengths were designed based on the TERT promoter region. The primer sequences are shown in Table 1 below:

[0074] Table 1 Primer sequences of different lengths for the TERT starter region

[0075]

[0076]

[0077] 1.1.2 Primer sequences of different lengths were designed based on the GAPDH exons, and their primer sequences are shown in Table 2 below:

[0078] Table 2 Primer sequences of different lengths for GAPDH exons

[0079]

[0080] 1.1.3 Construction of primer library containing BGI sequencing adapters

[0081] 1.1.3.1 DNA was amplified using TERT and GAPDH amplicon primers of different lengths (Table 1, Table 2) and the amplification reagent 2xKAPA2G Fast Multiplex Mix. The reaction solution system and reaction conditions are as follows (Table 3, Table 4):

[0082] Table 3 Preparation of reaction solution

[0083]

[0084] Table 4 shows the PCR instrument settings for the following reactions:

[0085]

[0086] 1.1.3.2 Purification of the first round of PCR products

[0087] 1) After the reaction is complete, the sample is briefly centrifuged, and then 20 μL of the sample is made up with nucleic acid-free water and transferred to a container containing 14 μL (0.7×) XP magnetic beads. The mixture is vortexed and incubated at room temperature for 2 min.

[0088] 2) Place the sample on a magnetic rack and wait for the solution to clarify. Then transfer the supernatant to a container containing 10 μL (0.5×) XP magnetic beads, vortex to mix, and incubate at room temperature for 5 min.

[0089] 3) Place the sample on the magnetic rack and wait for the solution to clarify before discarding the supernatant;

[0090] 4) Add 200 μL of freshly prepared 80% ethanol to the sample, rotate it once on the magnetic rack, and discard the ethanol after 30 seconds; repeat this step once, for a total of 2 times.

[0091] 5) Centrifuge the sample, discard the residual liquid on the magnetic rack, and open the cap to dry the magnetic beads for 1-2 minutes until the magnetic beads no longer reflect light;

[0092] 6) Add 9 μL of Nuclease-Free Water to the magnetic beads, vortex to mix, and centrifuge briefly;

[0093] 7) Transfer the sample with magnetic beads to a new PCR tube. Do not remove the magnetic beads. Proceed directly to the second round of amplification.

[0094] 1.1.3.3 Second Round of Amplification

[0095] The product from the previous round was amplified using 2xKAPA 2G Fast Multiplex Mix amplification reagent and adapter primers (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: NM108). The reaction solution system and reaction conditions are as follows (Table 5, Table 6):

[0096] Table 5 Preparation of reaction solution

[0097]

[0098] *The adapter primers were purchased from Nanjing Novizan Biotechnology Co., Ltd., product code: NM108.

[0099] Table 6 shows the reaction conditions set on the PCR instrument.

[0100]

[0101] 1.1.3.4 Purification of Second-Round PCR Products

[0102] 1) Transfer 20 μL of the sample with magnetic beads to a container containing 20 μL of (1×) XP magnetic beads, vortex to mix, and incubate at room temperature for 5 min;

[0103] 2) Place the sample on the magnetic rack and wait for the solution to clarify before discarding the supernatant;

[0104] 3) Add 200 μL of freshly prepared 80% ethanol to the sample, rotate it once on the magnetic rack, and discard the ethanol after 30 seconds; repeat this step once, for a total of 2 times.

[0105] 4) Centrifuge the sample tube, discard the residual liquid on the magnetic rack, open the cap and dry the magnetic beads for 1-3 minutes until the surface of the magnetic beads is no longer reflective;

[0106] 5) Add 15 μL of nuclease-free water to the magnetic beads, vortex to mix, and incubate at room temperature for 5 min;

[0107] 6) Place the sample tube on a magnetic rack and wait for it to clarify. Then transfer the supernatant to a new, clearly labeled 1.5 mL EP tube. The purified product is a double-stranded DNA library with sequencing adapters at both ends.

[0108] 1.1.3.5 Library verification:

[0109] The constructed library was accurately quantified using Qubit, and the library fragments were quality checked using agarose gel electrophoresis. The library fragments are as follows: Figure 1 As shown.

[0110] [Circulation Operation Procedure]

[0111] 1. Transgender

[0112] 1.1 Based on the length of the DNA fragment to be added, take 1 pmol and add it to a new 0.2 ml PCR tube, then add TE buffer to make up to 50 μL.

[0113] 1.2 After mixing and centrifuging, place on a PCR machine and set the denaturation reaction conditions: 98℃, 3 min; heat-covered 105℃.

[0114] 1.3 After the reaction is complete, immediately place on ice, in an ice bath for 2 minutes, then centrifuge briefly and place on ice for later use.

[0115] 2 Single-chain cyclosing

[0116] 2.1 Prepare the single-chain cyclization reaction solution on ice (Table 7)

[0117] Table 7 Preparation of Single-Chain Cycling Reaction Solution

[0118]

[0119] Note: The primer sequence in Table 7 is 5'-GGAACCGAGTGTCTTGCTGTACCG-3'.

[0120] 2.2 Add 20 μL of single-chain cyclization reaction solution to the product from the previous step, making a total volume of 70 μL. Gently vortex to mix, and then centrifuge briefly.

[0121] 2.3 Place the above PCR tubes on a PCR instrument for circularization and set the circularization reaction conditions as follows: 37℃, 15 min; 10℃, hold; no heat cap.

[0122] 2.4 After the reaction is complete, remove the PCR tube, centrifuge briefly, and immediately proceed to the next reaction.

[0123] 3. Enzymatic digestion process

[0124] 3.1 Prepare the enzyme digestion reaction solution on ice (Table 8)

[0125] Table 8 Preparation of Enzyme Digestion Reaction Solution

[0126]

[0127] 3.2 Add 10 μL of enzyme digestion reaction solution to the product from the previous step, gently vortex to mix, and then centrifuge briefly.

[0128] 3.3 Place the enzyme digestion device on a PCR instrument and set the enzyme digestion reaction conditions as follows: 37℃, 10 min; 10℃, hold; no heat cap.

[0129] 3.4 After the reaction program is completed, centrifuge briefly, add 8 μL of digestion stop solution (0.5 M EDTA), vortex to mix, centrifuge briefly, and immediately proceed to the next purification step.

[0130] Purification of 4-cyclized products

[0131] 4.1 Remove the purified magnetic beads 30 minutes in advance and bring them to room temperature. Vortex thoroughly before use.

[0132] 4.2 Transfer 143 μL of purified magnetic beads to a new 1.5 ml centrifuge tube. Transfer all digestion products to the corresponding numbered 1.5 ml centrifuge tube containing the purified magnetic beads. Gently pipette at least 10 times until completely mixed. Incubate at room temperature for 10 min.

[0133] 4.3 Place the centrifuge tube on the magnetic rack and discard the supernatant after the solution has clarified;

[0134] 4.4 Keep the centrifuge tubes on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, discard the ethanol after 30 seconds; repeat this step once, for a total of 2 times.

[0135] 4.5 Briefly centrifuge the centrifuge tubes, discard any remaining liquid on the magnetic rack, and open the cap to dry the magnetic beads for 5-10 minutes until the magnetic beads no longer reflect light;

[0136] 4.6 Add 22 μL of TE buffer to the magnetic beads, vortex to mix, and incubate at room temperature for 10 min;

[0137] 4.7 Place the centrifuge tube on a magnetic rack and wait for it to clarify. Then transfer the supernatant to a new, clearly labeled 1.5 ml centrifuge tube.

[0138] 5. Circulating product testing

[0139] 5.1 The cyclized purified product was quantified using the Qubits sDNA fluorescence quantitative kit according to the kit's instructions. The cyclization efficiency was calculated based on the ratio of yield to input. Results are as follows: Figure 2 The results showed that the circularization efficiency of libraries of different lengths in the TERT promoter region was significantly lower than that of the internal reference gene library, but the circularization efficiency was greater than 10% when the library fragment was between 200-400 bp.

[0140] 6. Amplicon Deep Bioinformatics Analysis

[0141] After the library that passed the previous step is circularized, high-throughput sequencing is performed using a sequencing platform.

[0142] First, the sequencing quality of the bases was evaluated using Q30, and then the depth libraries of each amplicon were analyzed, as shown in Table 9:

[0143] Table 9. Comparison of sequencing depth for amplicon lengths.

[0144] 100bp 1102 0.3％ 10300 200bp 11141 4.3％ 19759 300bp 11561 5.1％ 19128 400bp 11036 5.5％ 18077 500bp 6306 2.9％ 16606 600bp 5255 3.0％ 15555

[0145] Compared to different amplicones of the GAPDH gene, the coverage depth of the TERT promoter region amplicones is significantly different, with the sequencing coverage depth and mutation abundance of 100bp, 500bp, and 600bp regions being significantly lower than those of other regions.

[0146] Combination Figure 2 Analysis showed that the amplicon coverage depth and circularization efficiency in the TERT promoter region were positively correlated.

[0147] Example 2: Cycloning method with added single-chain stabilizing additives

[0148] The experimental steps in this embodiment are exactly the same as in Example 1, except that a 300bp library is used and betaine or formamide is added to the single-chain cyclization reaction system. The preparation of the single-chain cyclization reaction solution containing betaine is shown in Table 10 below. The preparation of the single-chain cyclization reaction solution containing formamide is simply to replace betaine with formamide in Table 10. The final mass concentration of formamide is 1%-4%.

[0149] Table 10 Preparation of Single-Chain Cyclation Reaction Solution

[0150]

[0151] Note: In Table 10, the **ligation primer sequence is 5'-GGAACCGAGTGTCTTGCTGTACCG-3'

[0152] like Figure 3 As shown, compared to the control group without betaine, the addition of betaine significantly improved cyclization efficiency, especially at a final concentration of 0.4-0.8 M, where the cyclization efficiency was optimal. Furthermore, the cyclization efficiency of the TERT promoter region library was comparable to that of the internal reference gene library. Compared to the control group without single-chain stabilizers, the cyclization efficiency of the formamide group showed no significant change. Figure 4 ).

[0153] The addition of betaine significantly increased the TERT amplicon depth, as shown in Table 11.

[0154] Table 11 Comparison of sequencing depths for betaine-added amplicon assays

[0155] 0 11561 5.1％ 19128 0.4M 16578 5.3％ 18790 0.8M 16004 5.2％ 17998 1.2M 11289 4.7％ 18320

[0156] In summary, when constructing libraries with high GC content, it is recommended that the length of the library fragments be 200-400 bp. At the same time, adding 0.4-0.8 M of betaine to the circularization reaction system can effectively improve the circularization efficiency and amplicon sequencing depth, with a circularization efficiency of up to 21%.

[0157] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A kit for cyclization of high GC content libraries, characterized in that, The kit includes a single-strand cyclization reagent, which includes sequencing platform ligation primers, ligase, and single-strand stabilizing additives. The sequence of the ligation primers is shown in SEQ ID NO:

25.

2. The reagent kit according to claim 1, characterized in that, The ligase is selected from T4 ligase, Escherichia coli DNA ligase or thermostable DNA ligase, and / or the single-chain stabilizing additive is selected from any one or more of betaine, formamide, DMSO or glycerol.

3. The reagent kit according to claim 1, characterized in that, The single-chain cyclizing reagent also includes a cyclization buffer.

4. The reagent kit according to claim 1, characterized in that, The kit also includes an enzyme digestion reagent.

5. The reagent kit according to claim 4, characterized in that, The enzyme digestion reagent includes nuclease and enzyme digestion buffer.

6. The reagent kit according to claim 5, characterized in that, The nuclease is selected from exonuclease I or exonuclease VII.

7. The reagent kit according to claim 5, characterized in that, The enzyme digestion reagent also includes a digestion termination reagent.

8. The reagent kit according to claim 7, characterized in that, The digestion termination agent is a metal chelating agent.

9. The reagent kit according to claim 8, characterized in that, The digestion termination reagent is EDTA.

10. The kit according to claim 1, characterized in that, The kit also includes purification reagents.

11. The reagent kit according to claim 10, characterized in that, The purification reagents include magnetic beads, ethanol, and TE buffer.

12. Use of the kit for high GC content library circularization according to any one of claims 1-11 in the preparation of single-stranded circularized DNA products or in gene sequencing.

13. A method for circularizing a library with high GC content, characterized in that, The method includes cyclization using the kit for cyclization of high GC content libraries according to any one of claims 1-11; the GC content of the high GC content library is 70-85%.

14. The method according to claim 13, characterized in that, The method for circularizing high-GC-content libraries includes the following steps: 1) A single-stranded DNA library with sequencing adapters at both ends is mixed with a single-stranded circularization reagent and reacted to obtain a DNA circularization intermediate with linking primers; 2) Mix the DNA circularization intermediate with the enzyme digestion reagent and perform enzyme digestion; 3) After the enzymatic digestion is completed, mix with the digestion termination reagent to terminate the enzymatic digestion and obtain the cyclized product.

15. The method according to claim 14, characterized in that, It also includes one or more of the following features: I) The single-stranded DNA library with sequencing adapters at both ends in step 1) is obtained by annealing and denaturing the double-stranded DNA library with sequencing adapters at both ends. II) In step 1), based on the circularization system, the amount of single-stranded DNA library is 0.5 pmol ~ 1 pmol; III) In step 1), the single-stranded DNA library has BGI sequencing adapters at both ends; IV) In step 1), based on the total volume of the circularized system, the final concentration of the primers used in the sequencing platform is 0.2~0.8 μM; V) In step 1), the final concentration of the ligase is 0.2~1U / μL, based on the total volume of the cyclization system; VI) In step 1), the single-chain stabilizing additive is betaine, and the final concentration of betaine is 0.4~1.2 mol / L based on the total volume of the cyclized system; VII) In step 1), the cyclization conditions are 35~38℃ for 12~18min; In step 2) of VIII), the final concentration of nuclease is 0.5~2 U / μL, based on the total volume of the enzymatic digestion system. In step 3), the final concentration of the digestion termination reagent is 0.05~0.1 mol / L, based on the total volume of the enzyme digestion system. X) In step 3), the enzymatic digestion conditions are 35~38℃ for 8~12 minutes; XI) The method further includes purifying the cyclized product obtained in step 3) with a purification reagent.

16. The method according to claim 15, characterized in that, In Feature III), the single-stranded DNA library has adapters at both ends that are compatible with the BGISEQ sequencing platform.

17. A gene sequencing method, characterized in that, This includes using the kit for circularizing high GC content libraries as described in any one of claims 1-11, followed by sequencing using a sequencing platform.

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