Combinatorial enzymes, enzymatic reaction reagents, kits, and their use in high-throughput sequencing library construction

Abstract

The present application relates to the field of biotechnology, and more particularly to a combination enzyme, an enzymatic reaction reagent, a kit and application thereof in high-throughput sequencing library construction. The present application optimizes the enzymatic reaction reagent for the first amplification product in the process of high-throughput sequencing library construction, and optimizes the enzymatic reaction procedure, thereby obtaining an efficient enzymatic reaction reagent and reaction system. The enzymatic reaction reagent and reaction system are used for the construction of high-throughput sequencing library, which simplifies the steps, reduces the risk of contamination, improves the library construction efficiency and shortens the experimental time, and has important popularization and application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to combinatorial enzymes, enzyme digestion reagents, kits, and their applications in the construction of high-throughput sequencing libraries. Background Technology

[0002] Targeted NGS (tNGS) involves the enrichment of target pathogens in patient samples based on multiplex PCR amplification or targeted capture, exhibiting excellent sensitivity. Due to its practicality and efficiency, tNGS is widely used in the clinical diagnosis of infectious diseases across various fields. Multiplex PCR targeted amplification technology is a targeted detection technique that combines multiplex PCR with next-generation sequencing. Its principle is relatively simple, obtaining a library of the target region through two rounds of PCR and two rounds of purification. Multiplex PCR library preparation offers high sequencing depth, and its library preparation process is simpler, faster, more flexible, and less costly than hybridization capture, thus it is widely used in the detection of various infectious diseases. Library construction is a critical step in the entire sequencing process, significantly affecting the quality of sequencing data and the accuracy of downstream analysis. Even with continuous optimization efforts from researchers, targeted library preparation typically still requires more than 30 steps and at least two hours of manual operation time. During library preparation, nucleotide incorporation errors often introduce sequencing noise, leading to harmful false positives and false negatives for some pathogens.

[0003] However, regardless of optimization, high-sensitivity tNGS technology is highly susceptible to cross-contamination. Traditional primer design and multiplex amplification strategies often require multiple purification processes and complex operations, making them prone to aerosol contamination and increasing operational complexity and time consumption. Currently, although some improvements have been made to simplify the library construction process and reduce operational complexity, the following main problems remain: First, the cumbersome traditional purification steps increase the risk of human error and cross-contamination; second, the formation of primer dimers during multiplex amplification affects the specificity of the amplified products; and third, the high operational complexity of the library construction process leads to low efficiency, especially when processing large numbers of samples.

[0004] Therefore, how to simplify the tNGS library construction process, reduce operational complexity, improve efficiency, and reduce the risk of contamination and primer dimer problems caused by operation, while ensuring library construction quality, remains a pressing problem to be solved in the current technical field. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a combination enzyme, an enzymatic digestion reaction reagent, a kit, and its application in the construction of high-throughput sequencing libraries.

[0006] This invention provides a combination enzyme comprising: exonuclease VII, exonuclease I, fast alkaline phosphatase FastAP, RecJf exonuclease, T7 endonuclease I, and exonuclease T.

[0007] Furthermore, the combined enzymes include: 8U~12U exonuclease VII, 8U~12U exonuclease I, 0.5U~1.5U fast alkaline phosphatase FastAP, 8U~12U RecJf exonuclease, 1U~20U T7 endonuclease I, and 5U~7U exonuclease T.

[0008] In a specific embodiment of the present invention, the combined enzyme comprises: 10 U exonuclease VII, 10 U exonuclease I, 1 U fast alkaline phosphatase FastAP, 10 U RecJf exonuclease, 10 U T7 endonuclease I, and 6 U exonuclease T.

[0009] The combined enzymes described in this invention are the optimal combinations obtained through screening; in a specific embodiment of this invention, the combined enzymes have been optimized, specifically, the combined enzymes participating in the screening include:

[0010] 10U exonuclease VII, 10U exonuclease I, 1U fast alkaline phosphatase FastAP, 10U RecJf exonuclease, 10U T7 endonuclease I and 6U exonuclease T (Group A1);

[0011] 10U exonuclease I, 1U fast alkaline phosphatase FastAP, and 10U T7 endonuclease I (Group A2);

[0012] 10U exonuclease VII and 10U exonuclease T (Group A3);

[0013] 10U RecJf exonuclease and 10U T7 endonuclease (Group A4);

[0014] 10U T7 endonuclease I and 6U exonuclease T (A5 group);

[0015] 10U exonuclease I, 1U fast alkaline phosphatase FastAP, 10U RecJf exonuclease, 10U T7 endonuclease I and 6U exonuclease T (A6 group);

[0016] 10U exonuclease VII, 10U exonuclease I, 1U fast alkaline phosphatase FastAP, 10U T7 endonuclease I and 6U exonuclease T (A7 group);

[0017] 10U exonuclease I, 1U fast alkaline phosphatase FastAP, 10U T7 endonuclease I and 6U exonuclease T (A8 group);

[0018] 10U exonuclease I, 1U fast alkaline phosphatase FastAP, and 10U RecJf exonuclease (A10 group);

[0019] 10U exonuclease VII, 10U T7 endonuclease I and 6U exonuclease T (A11 group);

[0020] 10U exonuclease VII, 10U exonuclease I, 1U FastAP, 10U RecJf exonuclease, 10U U4 endonuclease VII and 6U exonuclease T (A12 group);

[0021] 10U exonuclease VII, 10U exonuclease III, 1U FastAP, 10U RecJf exonuclease, 10U T4 endonuclease VII and 6U exonuclease T (A13 group);

[0022] 10U exonuclease VII, 10U exonuclease I, 1U FastAP, 10U RecJf exonuclease, 10U T7 endonuclease I, 6U exonuclease T and 10U T4 endonuclease VII (Group A14);

[0023] The experimental results showed that the dedimerization results of groups A1, A6, and A7 were comparable to or better than those of the control procedure, and the data output was relatively stable. Among them, group A1 had the best dedimerization effect. The dimer ratio increased after adding, reducing, or replacing the enzyme type based on A1.

[0024] Meanwhile, the present invention optimized the concentration of T7 endonuclease I in the enzyme combination. The experimental results show that the combination enzyme is optimal when the concentration of T7 endonuclease I is 10U.

[0025] This invention provides a combination enzyme preparation comprising the combination enzyme described in this invention, glycerol, and Tris-HCl; specifically, the combination enzyme preparation comprises the combination enzyme shown in group A1, 50 vt% glycerol, and 10 mM Tris-HCl (pH 8.0).

[0026] This invention provides an enzymatic hydrolysis reaction reagent, comprising:

[0027] Enzymatic hydrolysis reaction buffer and the combined enzyme described in this invention; or

[0028] Enzymatic hydrolysis reaction buffer and the combined enzyme preparation described in this invention;

[0029] The enzymatic hydrolysis reaction buffer includes: Tris-HCl, NaCl, MgCl2, ZnCl2, DTT, Triton X-100 and / or glycine-KOH.

[0030] Furthermore, the enzymatic hydrolysis reaction buffer comprises: 80mM~120mM Tris-HCl, 230mM~270mM NaCl, 60mM~80mM MgCl2, 8mM~12mM ZnCl2, 8mM~12mM DTT, 0.01vt%~0.25vt% Triton X-100 and 650mM~690mM glycine-KOH.

[0031] In a specific embodiment of the present invention, the optimal composition of the enzymatic hydrolysis buffer is: 100mM Tris-HCl (pH 8.5), 250mM NaCl, 70mM MgCl2, 10mM ZnCl2, 10mM DTT, 0.15% (v / v) Triton X-100 and 670mM glycine-KOH.

[0032] In this invention, glycine-KOH is an initial solution containing glycine, and the pH is adjusted to 8.5 using KOH, which is a commonly used conventional reagent in the field.

[0033] This invention provides a kit comprising:

[0034] At least one of amplification reagents, purification reagents, and / or quantitative detection reagents, and the combination enzyme described in this invention; or

[0035] At least one of amplification reagents, purification reagents, and / or quantitative detection reagents, and the combined enzyme preparation described in this invention; or

[0036] At least one of amplification reagents, purification reagents, and quantitative detection reagents, and the enzymatic reaction reagents described in this invention.

[0037] Furthermore, the amplification reagents include primers, buffer solutions, enzymes, and / or dNTPs, which are not limited in this invention;

[0038] The purification reagents include specific adsorption magnetic beads, washing solution and / or elution solution, which are not limited in this invention.

[0039] This invention provides at least one of the following applications in high-throughput sequencing library construction:

[0040] i) The combined enzyme described in this invention;

[0041] ii) The combined enzyme preparation described in this invention;

[0042] iii) The enzymatic hydrolysis reaction reagents described in this invention;

[0043] iv) The reagent kit described in this invention.

[0044] Furthermore, the high-throughput sequencing library is a tNGS library; meanwhile, the combination enzymes and reagents described in this invention can also be used in other high-throughput libraries such as metagenomic sequencing, and this invention does not limit them.

[0045] This invention provides a method for constructing a high-throughput sequencing library, which includes constructing a high-throughput sequencing library using at least one of the following methods A) to D):

[0046] A) The combined enzyme described in this invention;

[0047] B) The combined enzyme preparation described in this invention;

[0048] C) The enzymatic hydrolysis reaction reagent described in this invention;

[0049] D) The reagent kit described in this invention.

[0050] Furthermore, the construction method described in this invention includes the following steps:

[0051] Step 1: Perform multiplex PCR amplification on the sample to obtain the first round of amplification products;

[0052] Step 2: The first round of amplification products are mixed with the mixed enzyme preparation, enzyme digestion reaction buffer and water, and then enzymatically digested.

[0053] Step 3: Amplify the enzymatic digestion product, obtain a second round of amplification product, and then purify it to obtain the high-throughput sequencing library.

[0054] In the construction method described in this invention, the sample is DNA or cDNA, and this invention does not limit the type of sample. In this invention, extracting DNA samples, or extracting, reverse transcribing, and purifying RNA samples to obtain sample cDNA, are conventional techniques used by those skilled in the art, and this invention will not elaborate on them.

[0055] In the construction method described in this invention, after obtaining the second round of amplification products, purification is performed, followed by quantification and quality control steps. Quantification and quality control of the library are necessary steps taken by those skilled in the art to ensure that a high-quality library that can be used for the accuracy of subsequent sequencing results is obtained, and this invention will not elaborate on these steps.

[0056] In this invention, the purification aims to obtain a purer target product. Magnetic beads can be used for purification, or other purification methods can be used. This invention does not limit the specific method used.

[0057] Furthermore, in step 2, the enzymatic hydrolysis procedure is as follows: 37℃ for 10 min ~ 20 min, 85℃ for 3 min;

[0058] In this invention, the processing involves processing according to the above procedure and then storing at 0~4℃ for subsequent use; however, this invention does not limit this process.

[0059] The present invention optimizes the processing procedure, and experimental results show that the optimal time is 37℃ for 10-20 minutes and 85℃ for 3 minutes.

[0060] In this invention, the volume ratio of the first round amplification product, the mixed enzyme preparation, the enzymatic reaction buffer, and water is 30:3:4:3.

[0061] This invention optimizes the enzymatic digestion reagents and the enzymatic digestion procedure for the first amplification product in high-throughput sequencing, resulting in highly efficient enzymatic digestion reagents and reaction systems. These reagents and systems are used in the construction of high-throughput sequencing libraries, simplifying the steps, reducing the risk of contamination, improving library construction efficiency, and shortening experimental time, thus possessing significant value for widespread application. Attached Figure Description

[0062] Figure 1 This demonstrates a commonly used multiplex PCR library preparation process;

[0063] Figure 2 This invention demonstrates a multiplex PCR library construction process similar to that of the present invention;

[0064] Figure 3 The flowchart of this invention is shown. Detailed Implementation

[0065] This invention provides combinatorial enzymes, enzymatic digestion reagents, kits, and their applications in high-throughput sequencing library construction. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0066] In this invention, vt% is equivalent to %(v / v).

[0067] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0068] Example 1: Simplified tNGS Database Construction Method

[0069] The simplified tNGS library construction method in this invention includes the following steps:

[0070] I. Sample DNA Extraction:

[0071] DNA is extracted from the biological sample to be analyzed; for RNA samples, reverse transcription is required to obtain sample cDNA for subsequent detection.

[0072] II. First round of PCR

[0073] The amplification system is a 30µL reaction system. Add 15µL of DNA, 6µL of multiplex primers, 0.8µL of nuclease-free water, 1.2µL of Enhancer Buffer, 1µL of Uracil-DNA Glycosylase (UDG) enzyme, and 6µL of multiplex PCR enzyme mix as required. Mix well and place the reaction tube in a PCR instrument. Run the following program: 25℃ for 15min; 95℃ for 3min; 95℃ for 30s → 60℃ for 1min → 72℃ for 30s, cycle 30 times; 72℃ for 3min, and hold at 4℃.

[0074] III. Enzymatic digestion of first-round PCR products

[0075] 1. Preparation of the enzymatic hydrolysis reaction system for the product

[0076] Prepare a mixed enzyme and an enzyme preparation containing the mixed enzyme; the mixed enzyme comprises: 10 U exonuclease VII, 10 U exonuclease I (Exo I), 1 U FastAP (rapid alkaline phosphatase), 10 U RecJf exonuclease, 10 U T7 endonuclease I, and 6 U exonuclease T (Beyotime D6003S); add 10 mM Tris-HCl (pH 8.0) and 50 vt% glycerol to the mixed enzyme to obtain the mixed enzyme preparation.

[0077] The enzyme digestion reaction buffer consists of: 100mM Tris-HCl (pH 8.5), 250mM NaCl, 70mM MgCl2, 10mM ZnCl2, 10mM DTT, 0.15% (v / v) Triton X-100, and 670mM glycine-KOH.

[0078] 2. Enzymatic hydrolysis of the product

[0079] The enzymatic digestion reaction system is 40 µL. Add 3 µL of mixed enzyme preparation, 4 µL of enzyme digestion reaction buffer, and 3 µL of nuclease-free water to the product after the first round of PCR as required. Mix well by pipetting and briefly centrifugation. Place the reaction tube in the PCR instrument and run the following program: 37 °C for 15 min, 85 °C for 3 min, and hold at 4 °C.

[0080] IV. Second Round of Amplification

[0081] 1. Second round PCR reaction system

[0082] The amplification system is a 60µL reaction system. Add 15µL of PCR Mix and 5µL of adapter primer to the product from the previous step as required. After mixing well, briefly centrifuge and place the reaction tube in the PCR instrument. Run the following program: 98℃ for 3 min; 98℃ for 20s → 60℃ for 15s → 72℃ for 30s, cycle 12 times, 72℃ for 5 min, and hold at 4℃.

[0083] V. Purification

[0084] 1. Magnetic bead purification

[0085] (1) Add 48µL of magnetic beads to the second round of PCR products, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, magnetically hold it for 2min, discard the supernatant, and keep the magnetic beads.

[0086] (2) Then, add 50µL of BB Buffer to the reaction tube, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, let stand for 2min, discard the supernatant, and keep the magnetic beads.

[0087] (3) Then, wash twice with 200µL of 80% ethanol each time, and air dry for 5 minutes.

[0088] (4) Finally, add 25µL of nuclease-free water to the reaction tube to wash the magnetic beads, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, let stand magnetically for 2min, and transfer 23µL of the library to a new centrifuge tube.

[0089] VI. Quantitative and Quality Control of the Library

[0090] Quantitative method: Library quantification was performed using the Qubit dsDNA HS Assay.

[0091] Quality assessment: The Bioanalyzer analyzes fragments to ensure that the fragment size meets sequencing requirements.

[0092] Example 2 Related Optimizations

[0093] I. Enzyme Screening and Optimization

[0094] (a) Enzyme combination setup

[0095] To improve the removal efficiency of residual primers and dimers in the PCR amplification system, the mixed enzymes for primer and dimer removal were optimized. Four different mixed enzyme groups (A1-A14) were prepared for experimental testing. The specific formulations are as follows (— in the table indicates no addition):

[0096] Table 1. Mixed enzyme (preparation) formulations of A1~A11

[0097]

[0098] A12 consists of: 10 mM Tris-HCl (pH 8.0), 50% (v / v) glycerol, 10 U exonuclease VII, 10 U exonuclease I, 1 U FastAP, 10 U RecJf exonuclease, 10 U T4 endonuclease VII, and 6 U exonuclease T;

[0099] A13 includes: 10 mM Tris-HCl (pH 8.0), 50% (v / v) glycerol, 10 U exonuclease VII, 10 U exonuclease III, 1 U FastAP, 10 U RecJf exonuclease, 10 U T4 endonuclease VII, and 6 U exonuclease T;

[0100] Group A14 includes: 10 mM Tris-HCl (pH 8.0), 50% (v / v) glycerol, 10 U exonuclease VII, 10 U exonuclease I, 1 U FastAP, 10 U RecJf exonuclease, 10 U T7 endonuclease I, 6 U exonuclease T, and 10 U T4 endonuclease VII;

[0101] (II) Experimental Procedure

[0102] Step 1: Use the following 4 reference samples as the test samples (pure nucleic acid);

[0103] Step 2: Amplify the test sample according to the first round of PCR in Example 1;

[0104] Step 3: The products from the first round of PCR were subjected to enzymatic digestion. Specifically, 14 groups were set up, with each group containing 40 µL of enzyme. 3 µL of the mixed enzyme (preparation) shown in Table 1 (A1-A14, each containing 10 mM Tris-HCl (pH 8.0) and 50 vt% glycerol), 4 µL of the enzyme digestion buffer shown in Example 1, and 3 µL of nuclease-free water were added to the products after the first round of PCR. After mixing by pipetting, the mixture was briefly centrifuged. The reaction tubes were placed in a PCR instrument, and the following program was run: 37°C for 15 min, 85°C for 3 min, and held at 4°C to obtain the digested products.

[0105] Step 4: The enzymatic hydrolysis products are amplified according to the system and procedure shown in the second round of amplification in Example 1;

[0106] Step 5: The product from the second round of amplification is purified according to the purification steps in Example 1;

[0107] Step 6: Purify the product to obtain a qualified gene library according to the library quantification and quality control in Example 1;

[0108] Step 7: Sequencing of the gene library;

[0109] (III) Control Group Procedure

[0110] Meanwhile, the steps of the control group were used as a reference for comparison; the specific steps of the control group are as follows:

[0111] Step 1: Use the following 4 reference samples as the test samples (pure nucleic acid);

[0112] Step 2: First round of PCR amplification. The amplification system is a 30µL reaction system. Add 15µL of sample DNA, 6µL of multiplex primers, 0.8µL of nuclease-free water, 1.2µL of Enhancer Buffer, 1µL of Uracyl-DNA Glycosylase (UDG) enzyme, and 6µL of multiplex PCR enzyme mix as required. Mix well and place the reaction tube in the PCR instrument. Run the following program: 25℃ for 15min; 95℃ for 3min; 95℃ for 30s → 60℃ for 1min → 72℃ for 30s, cycle 30 times; 72℃ for 3min, hold at 4℃.

[0113] Step 3: First round of purification, the specific steps are as follows:

[0114] (1) Add 24µL of magnetic beads to the first round of PCR products, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, magnetically hold it for 2min, discard the supernatant, and keep the magnetic beads.

[0115] (2) Subsequently, wash twice with 200µL of 80% ethanol each time, and air dry for 5 minutes.

[0116] (3) Finally, add 42µL of nuclease-free water to the reaction tube to wash the magnetic beads, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, let stand magnetically for 2min, and transfer 40µL of the library to a new centrifuge tube.

[0117] Step 4: Second round of PCR amplification. The amplification system is a 60µL reaction system. Add 15µL of PCR Mix and 5µL of adapter primer to the purified product from the previous step as required. Mix well and centrifuge briefly. Place the reaction tube in the PCR instrument and run the following program: 98℃ for 3 min; 98℃ for 20s → 60℃ for 15s → 72℃ for 30s, cycle 12 times, 72℃ for 5 min, and hold at 4℃.

[0118] Step 5: Second round of purification, the specific steps are as follows:

[0119] (1) Add 48µL of magnetic beads to the second round of PCR products, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, magnetically hold it for 2min, discard the supernatant, and keep the magnetic beads.

[0120] (2) Then, add 50µL of BB Buffer to the reaction tube, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, let stand for 2min, discard the supernatant, and keep the magnetic beads.

[0121] (3) Then, wash twice with 200µL of 80% ethanol each time, and air dry for 5 minutes.

[0122] (4) Finally, add 25µL of nuclease-free water to the reaction tube to wash the magnetic beads, vortex to mix, let stand at room temperature for 5min, place the reaction tube on a magnetic rack, let stand magnetically for 2min, and transfer 23µL of the library to a new centrifuge tube.

[0123] Step 6: Library Quantification and Quality Control

[0124] Quantitative method: Library quantification was performed using the Qubit dsDNA HS Assay.

[0125] Quality assessment: The Bioanalyzer analyzes fragments to ensure that the fragment size meets sequencing requirements.

[0126] Step 7: Perform gene library sequencing.

[0127] (iv) Sample information and test results

[0128] Information on the four reference samples is as follows:

[0129] Sample 1: Staphylococcus aureus SA (101 copies / μL), Pseudomonas aeruginosa PAE (10 copies / μL), 1 copies / μL), Haemophilus influenzae HIF (10 copies / μL), 1 copies / μL), Escherichia coli Eco (10 copies / μL), 1 (copies / μL)

[0130] Sample 2: Human herpesvirus 4 EB (10 1 copies / μL), Aspergillus fumigatus Afu (10 copies / μL), 1 copies / μL), Candida albicans CA (10 copies / μL), 1 copies / μL), Enterococcus faecalis Fium (10 copies / μL), 1 (copies / μL)

[0131] Sample 3: Staphylococcus aureus SA (10 3 copies / μL), Pseudomonas aeruginosa PAE (10 copies / μL), 3 copies / μL), Haemophilus influenzae HIF (10 copies / μL), 3 copies / μL), Escherichia coli Eco (10 copies / μL), 3 (copies / μL)

[0132] Sample 4: Human herpesvirus type 4 EB (10 3 copies / μL), Aspergillus fumigatus Afu (10 copies / μL), 3 copies / μL), Candida albicans CA (10 copies / μL), 3 copies / μL), Enterococcus fium (10 copies / μL), 3 (copies / μL)

[0133] Table 2. Experimental results of enzyme reaction solutions for A1-A14 1

[0134]

[0135] Table 3. Experimental results of enzyme reaction solutions for A1-A14 2

[0136]

[0137] Table 4. Experimental results of enzyme reaction solutions for A1-A14 3

[0138]

[0139] Experimental conclusions: Comparing the experimental results of formulations A1-A14, the dedimerization results of A1, A6, and A7 were comparable to or better than those of the control process, and the data output was also relatively stable. Among them, A1 showed the best dedimerization effect.

[0140] II. Optimization of the amount or ratio of enzymes added to group A1

[0141] The concentration of T7 endonuclease I in group A1 was optimized, and the concentration gradient settings are shown in Table 5. The optimization results are shown in Table 6.

[0142] Table 5. Optimization conditions for the concentration of T7 endonuclease I

[0143]

[0144] Table 6. Results of enzyme concentration optimization experiments for B1-B5

[0145]

[0146] The B1 concentration has the lowest dimer content and produces more data than other concentrations, therefore B1 is the optimal concentration.

[0147] III. Optimization of Primer Removal and Dimerase System: Experimental Data - Optimization of Enzyme Reaction Buffer

[0148] Using the mixed enzyme (containing 10 mM Tris-HCl (pH 8.0) and 50 vt% glycerol) from group A1 in "I. Enzyme Screening and Optimization", the enzymatic digestion buffer for the primer-removed and dimerized systems was optimized. Enzymatic digestion buffers for C1-C9 were prepared for experimental testing, and the specific formulations are shown in the table below:

[0149] Table 7. Screening of enzyme digestion reaction buffers for C1-C9

[0150]

[0151] Experimental steps:

[0152] Step 1: Use the following 4 reference samples as the test samples (pure nucleic acid);

[0153] Step 2: Amplify the test sample according to the first round of PCR in Example 1;

[0154] Step 3: The products from the first round of PCR are subjected to enzymatic digestion. Nine groups are set up for the experiment, with each group having a 40µL digestion reaction system. Add 3µL of the mixed enzyme (preparation) shown in group A1, 4µL of the digestion reaction buffer shown in groups B1-B9 as shown in Table 7, and 3µL of nuclease-free water to the products after the first round of PCR. Mix well by pipetting and briefly centrifugation. Place the reaction tubes in a PCR instrument and run the following program: 37℃ for 15 min, 85℃ for 3 min, and hold at 4℃ to obtain the digestion products.

[0155] Step 4: The enzymatic hydrolysis products are amplified according to the system and procedure shown in the second round of amplification in Example 1;

[0156] Step 5: The product from the second round of amplification is purified according to the purification steps in Example 1;

[0157] Step 6: Purify the product to obtain a qualified gene library according to the library quantification and quality control in Example 1;

[0158] Step 7: Sequencing of the gene library;

[0159] Meanwhile, the steps of the control group were used as a reference for comparison; the steps of the control group were as above.

[0160] Information on the four reference samples is as follows:

[0161] Sample 1: Staphylococcus aureus SA (10 1 copies / μL), Pseudomonas aeruginosa PAE (10 copies / μL), 1 copies / μL), Haemophilus influenzae HIF (10 copies / μL), 1 copies / μL), Escherichia coli Eco (10 copies / μL), 1 (copies / μL)

[0162] Sample 2: Human herpesvirus 4 EB (10 1 copies / μL), Aspergillus fumigatus Afu (10 copies / μL), 1 copies / μL), Candida albicans CA (10 copies / μL), 1 copies / μL), Enterococcus faecalis Fium (10 copies / μL), 1 (copies / μL)

[0163] Sample 3: Staphylococcus aureus SA (10 3 copies / μL), Pseudomonas aeruginosa PAE (10 copies / μL), 3 copies / μL), Haemophilus influenzae HIF (10 copies / μL), 3 copies / μL), Escherichia coli Eco (10 copies / μL), 3 (copies / μL)

[0164] Sample 4: Human herpesvirus type 4 EB (10 3 copies / μL), Aspergillus fumigatus Afu (10 copies / μL), 3 copies / μL), Candida albicans CA (10 copies / μL), 3 copies / μL), Enterococcus fium (10 copies / μL), 3 (copies / μL)

[0165] Table 8. Screening results of enzyme reaction buffers for C1-C9 1

[0166]

[0167] Table 9. Screening results of enzyme reaction buffers for C1-C9 2

[0168]

[0169] Experimental conclusion: Comparing the experimental results of formulations B1-B9, B1 showed the best experimental effect.

[0170] IV. Optimization of Reaction Conditions

[0171] To obtain the best reaction results, the reaction program for the primer removal and dimerase system (enzymatic digestion of the first-round PCR products) was adjusted, and the following five different reaction programs were set, with the specific steps as follows:

[0172] Step 1: Use the following 4 reference samples as the test samples (pure nucleic acid);

[0173] Step 2: Amplify the test sample according to the first round of PCR in Example 1;

[0174] Step 3: The products from the first round of PCR are subjected to enzymatic digestion. Five groups are set up for the experiment, with each group having a 40µL digestion reaction system. Add 3µL of the mixed enzyme (preparation) shown in group A1, 4µL of the digestion reaction buffer shown in group B1, and 3µL of nuclease-free water to the products after the first round of PCR. Mix well by pipetting and aspirating, and then briefly centrifuge. Place the reaction tubes in a PCR instrument and run the programs shown in Table 10 below to obtain the digestion products.

[0175] Step 4: The enzymatic hydrolysis products are amplified according to the system and procedure shown in the second round of amplification in Example 1;

[0176] Step 5: The product from the second round of amplification is purified according to the purification steps in Example 1;

[0177] Step 6: Purify the product to obtain a qualified gene library according to the library quantification and quality control in Example 1;

[0178] Step 7: Sequencing of the gene library;

[0179] Meanwhile, the steps of the control group were used as a reference for comparison; the steps of the control group were as above.

[0180] Table 10. Program Optimization

[0181]

[0182] Information on the four reference samples is as follows:

[0183] Sample 1: Staphylococcus aureus SA (101 copies / μL), Pseudomonas aeruginosa PAE (10 copies / μL), 1 copies / μL), Haemophilus influenzae HIF (10 copies / μL), 1 copies / μL), Escherichia coli Eco (10 copies / μL), 1 (copies / μL)

[0184] Sample 2: Human herpesvirus 4 EB (10 1 copies / μL), Aspergillus fumigatus Afu (10 copies / μL), 1 copies / μL), Candida albicans CA (10 copies / μL), 1 copies / μL), Enterococcus faecalis Fium (10 copies / μL), 1 (copies / μL)

[0185] Sample 3: Staphylococcus aureus SA (10 3 copies / μL), Pseudomonas aeruginosa PAE (10 copies / μL), 3 copies / μL), Haemophilus influenzae HIF (10 copies / μL), 3 copies / μL), Escherichia coli Eco (10 copies / μL), 3 (copies / μL)

[0186] Sample 4: Human herpesvirus type 4 EB (10 3 copies / μL), Aspergillus fumigatus Afu (10 copies / μL), 3 copies / μL), Candida albicans CA (10 copies / μL), 3 copies / μL), Enterococcus fium (10 copies / μL), 3 (copies / μL)

[0187] Table 11. Results of Reaction Program Optimization

[0188]

[0189] Table 12. Results of Reaction Program Optimization

[0190]

[0191] Experimental conclusion: Comparing the experimental results of five different reaction procedures, the experimental effects of procedures 2, 3 and 4 were not significantly different. Considering factors such as reaction time, procedure 2 was selected as the optimal reaction procedure.

[0192] Example 3: Further comparison between the present invention and the control group

[0193] I. Comparison of 10 test samples using the methods of this invention and the control group

[0194] Experimental Design: To verify the effectiveness of the TNGS library construction procedure of this invention in controlling contamination, experiments were conducted under uncontrolled Haemophilus influenzae contamination conditions in the laboratory. The TNGS library construction procedure of this invention (as shown in Example 1) and the control group library construction procedure (see the control group steps in Example 2) were used to compare the detection results of Haemophilus influenzae. A total of 10 known positive reference samples were tested. The two library construction procedures were performed simultaneously by three different researchers. The final libraries were then sequenced.

[0195] Table 13. Information on 10 known positive reference samples

[0196]

[0197] Table 14. Experimental Results

[0198]

[0199] Experimental conclusions: In this experiment, the number of false positive sequences in the experimental group ranged from 1 to 14, with a frequency of 13; the number of false positive sequences in the control group ranged from 1 to 7021, with a frequency of 28; the contamination control effect of the experimental group was significantly better than that of the control group.

[0200] II. Clinical Cases and Control Group Comparison

[0201] This patented TNGS library construction process is applicable to various sample types, including bronchoalveolar lavage fluid, sputum, swabs, cerebrospinal fluid, urine, pleural and peritoneal fluid, and tissue samples. Using a two-step library construction process as a control, a total of 200 clinical samples were tested, including 120 bronchoalveolar lavage fluid samples, 45 sputum samples, 14 swabs, 10 cerebrospinal fluid samples, 5 urine samples, 3 pleural and peritoneal fluid samples, and 3 tissue samples. Combined with clinical and other validation methods (qPCR, mNGS), the results show that this patented library construction process can effectively control TNGS library contamination and dimer content while ensuring good detection results. Specific detection results are shown in Tables 15 and 16 below:

[0202] Table 15. Comparison Results of Clinical Cases and Control Groups 1

[0203]

[0204] Table 16. Comparison Results of Clinical Cases and Control Groups 2

[0205]

[0206] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination enzyme, characterized in that, The combined enzyme consists of 8U-12U exonuclease VII, 8U-12U exonuclease I, 0.5U-1.5U fast alkaline phosphatase FastAP, 8U-12U RecJf exonuclease, 5U-15U T7 endonuclease I, and 5U-7U exonuclease T.

2. A combination enzyme preparation, characterized in that, It includes the combined enzyme, glycerol, and Tris-HCl as described in claim 1.

3. An enzymatic hydrolysis reaction reagent, characterized in that, include: Enzymatic hydrolysis reaction buffer and the combined enzyme as described in claim 1; or Enzymatic hydrolysis reaction buffer and the combined enzyme preparation according to claim 2; The enzymatic hydrolysis reaction buffer includes: Tris-HCl, NaCl, MgCl2, ZnCl2, DTT, Triton X-100 and / or glycine-KOH.

4. The enzymatic hydrolysis reagent according to claim 3, characterized in that, The enzymatic hydrolysis reaction buffer comprises: 80mM~120mM Tris-HCl, 230mM~270mM NaCl, 60mM~80mM MgCl2, 8mM~12mM ZnCl2, 8mM~12mM DTT, 0.01vt%~0.25vt% Triton X-100 and 650mM~690mM glycine-KOH.

5. A reagent kit, characterized in that, include: At least one of amplification reagents, purification reagents, and / or quantitative detection reagents, and the combined enzyme as described in claim 1; or At least one of amplification reagents, purification reagents, and / or quantitative detection reagents, and the combined enzyme preparation of claim 2; or At least one of amplification reagent, purification reagent, and quantitative detection reagent, and the enzymatic reaction reagent as described in claim 3 or 4.

6. The application of at least one of the following (i) to (iv) in high-throughput sequencing library construction: i) The combined enzyme according to claim 1; ii) The combined enzyme preparation according to claim 2; iii) The enzymatic hydrolysis reagent as described in claim 3 or 4; iv) The kit according to claim 5.

7. A method for constructing a high-throughput sequencing library, characterized in that, This includes constructing high-throughput sequencing libraries using at least one of the methods shown in A) to D) below: A) The combined enzyme according to claim 1; B) The combined enzyme preparation according to claim 2; C) The enzymatic hydrolysis reagent as described in claim 3 or 4; D) The kit according to claim 5.

8. The construction method according to claim 7, characterized in that, Includes the following steps: Step 1: Perform multiplex PCR amplification on the sample to obtain the first round of amplification products; Step 2: The first round of amplification products are mixed with the combined enzyme preparation, enzyme digestion reaction buffer and water for enzymatic digestion; Step 3: Amplify the enzymatic digestion product, obtain a second round of amplification product, and then purify it to obtain the high-throughput sequencing library.

9. The construction method according to claim 8, characterized in that, In step 2, the enzymatic hydrolysis procedure is as follows: 37℃ for 10-20 minutes, 85℃ for 3 minutes; The volume ratio of the first-round amplification product, combined enzyme preparation, enzymatic reaction buffer, and water is 30:3:4:3.

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