Library construction method

Abstract

The method for constructing the library comprises the following steps: a pretreatment step: in the same reaction system, carrying out terminal repair and 'A' adding reaction on a nucleic acid sample to obtain a reacted sample; linker connection and purification: carrying out linker connection on the reacted sample, and carrying out magnetic bead purification on the sample connected with the linker to obtain a purified sample; and library amplification and purification: carrying out PCR amplification on the purified sample, and carrying out magnetic bead purification on the amplified sample to obtain the library. According to the library construction method provided by the invention, a reaction system and a purification mode are improved, and the library quality is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a library. Background Art

[0002] Infectious diseases are a general term for diseases caused by pathogenic microorganisms (bacteria, viruses, fungi, parasites, etc.). According to statistics, infectious diseases account for more than 25% of all deaths and are a major disease that seriously threatens human health in the world today. The accurate identification of pathogenic microorganisms is of great significance for the medication and treatment of patients. The diagnosis of conventional infectious diseases has always relied on morphology, immunology, pathogen isolation and culture, and molecular biology methods, which cannot effectively identify new, difficult, and complex infections. The pathogen metagenomic next-generation sequencing (mNGS) technology that has emerged in recent years can perform unbiased sequencing of all nucleic acids in the sample. The number of pathogens detected has expanded to more than 10,000, which can effectively make up for the shortcomings of traditional detection methods and has important clinical significance for difficult and critical infections.

[0003] Metagenomic technology can detect different syndrome infections, so the types of samples tested are wide, including blood, cerebrospinal fluid, alveolar lavage fluid, sputum, throat swabs, pleural effusion, tissue, pus, etc. The human content in different sample types is different, resulting in large fluctuations in the yield of extracted nucleic acid. The concentration of extracted nucleic acid in some samples is lower than the quantitative detection limit, and in some samples it is as high as several micrograms. The scope of application of commercial kits is the construction of second-generation sequencing DNA libraries, and it is not a special kit for metagenomic detection. The commercial kit is suitable for nucleic acid starting amounts of 100pg to 1000ng, but due to the excessively high content of human nucleic acid in samples such as the respiratory tract and sputum, the sample positive rate will be reduced. Therefore, the metagenome experimental process generally adds a host removal step before nucleic acid extraction. After the host removal sample is extracted, the amount of nucleic acid is lower than the quantitative detection limit, and the measured concentration value is too low. The concentration of some plasma and cerebrospinal fluid samples is also too low. In order to meet the library construction requirements of different samples, commercial kits generally adjust the amount of adapters added and the number of PCR cycles according to the amount of nucleic acid input to reduce the proportion of library adapters and increase library concentration. However, in actual production and delivery, too many dilution steps and the use of multiple PCR instruments will increase the complexity of the production process and fail to meet production needs. In addition, conventional metagenomic testing of blood samples is mainly to detect free nucleic acids in plasma samples, but for viruses, when they exist as virus particles, they may be lost during the library construction process.

[0004] In response to the above problems, it is necessary to provide a rapid and standardized detection process for metagenomic sample testing that can meet the needs of different samples and simplify the product delivery production process. Summary of the invention

[0005] According to the first aspect, in one embodiment, a method for constructing a library is provided, comprising:

[0006] The pretreatment step includes performing end repair and adding "A" reaction on the nucleic acid sample in the same reaction system to obtain a reacted sample;

[0007] The connector connection and purification step includes connecting the connector to the sample after the reaction, and purifying the sample after the connector connection by magnetic beads to obtain a purified sample;

[0008] The library amplification and purification steps include performing PCR amplification on the purified sample and performing magnetic bead purification on the amplified sample to obtain the library.

[0009] According to the second aspect, in one embodiment, a library is provided, which is constructed by any method described in the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A schematic diagram showing a comparison between a method of an embodiment of the invention and an existing library building process;

[0011] Figure 2 to Figure 4 These are the quality control results of the libraries of samples S1, S2, and S5;

[0012] Figure 5 The detection of pathogens with and without nucleic acid fragmentation in plasma samples;

[0013] Figure 6 The Qseq100 quality control peak diagram of Comparative Example 1 and Example 1;

[0014] Figure 7 This is the Qseq100 quality control peak diagram of Comparative Example 2 and Example 4. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0016] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0017] The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0018] According to the first aspect, in one embodiment, a method for constructing a library is provided, comprising:

[0019] The pretreatment step includes performing end repair and adding "A" reaction on the nucleic acid sample in the same reaction system to obtain a reacted sample;

[0020] The connector connection and purification step includes connecting the connector to the sample after the reaction, and purifying the sample after the connector connection by magnetic beads to obtain a purified sample;

[0021] The library amplification and purification steps include performing PCR amplification on the purified sample and performing magnetic bead purification on the amplified sample to obtain the library.

[0022] The method for constructing a library provided by the present invention improves the quality of the library and shortens the library construction cycle to 2 hours.

[0023] In one embodiment, at least one of the magnetic bead purification after linker ligation and the magnetic bead purification after library amplification is double-selection purification.

[0024] In one embodiment, when the magnetic bead purification after the connector connection is double-selection purification, the double-selection purification after the connector connection includes: first adding M1 times the volume of magnetic beads to the reaction solution, removing the supernatant, and then adding N1 times the volume of magnetic beads, removing the supernatant, and obtaining a purified library.

[0025] In a preferred embodiment, the double-selection purification after the connector connection includes: first adding M1 times the volume of magnetic beads to the reaction solution, mixing and letting it stand, then placing it on a magnetic rack until the liquid is clarified, and removing the supernatant; then adding N1 times the volume of magnetic beads, mixing and letting it stand, then placing it on a magnetic rack until the liquid is clarified, removing the supernatant, and obtaining a purified sample.

[0026] In a preferred embodiment, the purification step further comprises: eluting the sample after double-selection purification with ethanol to obtain a purified sample.

[0027] In one embodiment, M1 is 0.5-0.7, preferably 0.6.

[0028] In one embodiment, N1 is 0.4-0.6, preferably 0.5.

[0029] In one embodiment, when the magnetic bead purification after the library amplification is double-selection purification, the double-selection purification after the library amplification includes: first adding M2 times the volume of magnetic beads to the reaction solution, collecting the supernatant, and then adding N2 times the volume of magnetic beads to the supernatant, and after removing the supernatant, obtaining the purified library.

[0030] In a preferred embodiment, the double selection purification after library amplification includes: first adding M2 times the volume of magnetic beads to the reaction solution, mixing and letting stand, then placing on a magnetic stand until the liquid is clarified, collecting the supernatant, adding N2 times the volume of magnetic beads to the supernatant, mixing and letting stand, then placing on a magnetic stand until the liquid is clarified, and removing the supernatant to obtain a purified library.

[0031] In a preferred embodiment, the purification step further comprises: eluting the library after double selection purification with ethanol to obtain a purified library.

[0032] In one embodiment, M2 is 0.5-0.7, preferably 0.6.

[0033] In one embodiment, N2 is 0.3-0.5, preferably 0.4.

[0034] In one embodiment, the pretreatment step includes fragmenting, end-repairing and adding "A" reaction to the nucleic acid sample in the same reaction system to obtain a post-reaction sample.

[0035] In one embodiment, the nucleic acid sample is extracted from at least one of a tissue sample and a body fluid sample.

[0036] In one embodiment, the nucleic acid sample is extracted from whole blood, pleural effusion, ascites, cerebrospinal fluid, bronchoalveolar lavage fluid or sputum.

[0037] In one embodiment, the nucleic acid sample is extracted from plasma in whole blood.

[0038] The present invention also fragments plasma sample nucleic acid to build a library, which improves the detection of viruses in plasma samples. In addition, the library building process of all sample types is unified, which is conducive to the realization of product automation in the later stage.

[0039] The library construction method provided by the present invention is compatible with samples with extremely low nucleic acid starting amounts, such as plasma, cerebrospinal fluid, and host-free samples, for library construction.

[0040] In one embodiment, in the pretreatment step, the reaction solution contains at least one of a fragmentation enzyme, a fragmentation enzyme buffer, T4 DNA polymerase, rTaq polymerase, T4 polynucleotide kinase (T4 PNK), dATP, and dNTP.

[0041] In one embodiment, the nucleic acid sample is a metagenomic sample.

[0042] According to the second aspect, in one embodiment, a library is provided, which is constructed by any method described in the first aspect.

[0043] In one embodiment, the present invention provides a method for library construction, comprising the following steps:

[0044] 1. Sample pretreatment:

[0045] Homogenize tissue samples; liquefy viscous samples such as sputum and bronchoalveolar lavage fluid; remove the host from samples with high human content such as sputum, bronchoalveolar lavage fluid, pleural effusion, etc.; and perform a combined enzymatic hydrolysis and bead milling treatment to break the sample wall in order to improve the detection of Gram-positive bacteria and fungi in the sample.

[0046] 2. Nucleic acid extraction and concentration determination:

[0047] The magnetic bead method was used to extract nucleic acids from the samples. After extraction, the concentration of nucleic acids was measured using a Qubit fluorescence quantifier.

[0048] 3. Library construction:

[0049] This process combines fragmentation, end repair, and "A" addition processes. Compared with the original mechanical fragmentation method or enzyme fragmentation followed by end repair and "A" addition process, the time and operation steps are shortened, which has obvious advantages. Figure 1 .

[0050] 3.1 Fragmentation, end repair and "A" addition reaction: This step simultaneously fragments, end repairs and adds A to the extracted DNA.

[0051] 3.2 Adapter ligation and purification: In this step, the adapter ligation is first completed under the action of the ligation reaction solution and the ligase. Then the ligation product is purified using magnetic beads. During the purification, the double selection purification method is used to remove the residual adapter, which has a higher purification efficiency.

[0052] 3.3 Library amplification and purification: In this step, the library is first amplified by PCR, and then the library is purified using magnetic beads. This step uses a double-selection purification method, which can effectively remove large and small fragments and make the main peak of the library more concentrated.

[0053] 4. Determination of library concentration.

[0054] 5. Library quality control: Use Qseq100 according to the instructions.

[0055] 5. On-machine sequencing: On-machine sequencing was performed according to the MGISEQ-2000 platform operation.

[0056] 6. Off-line data analysis: Perform data quality control on the data obtained by sequencing, split the qualified data according to the label sequence on the connector, and obtain the sequencing data corresponding to each sample. Remove the human sequences in the sequencing data, compare the remaining sequences with the pathogen sequence library, filter the data according to the set comparison parameters, and finally obtain the pathogen comparison results. Count the single sample data volume and the number of pathogen-specific detection sequences respectively.

[0057] In one embodiment, the method provided by the present invention can effectively shorten the cycle of metagenomic library construction to 2 hours, and the method optimizes the purification method, which can effectively improve the library quality of the sample. This method is not only suitable for library construction of high starting amount sample nucleic acid, but also compatible with plasma, cerebrospinal fluid and host-free samples with extremely low starting amount of nucleic acid for library construction. This method is different from commercial kits and does not use cumbersome linker dilution and different PCR cycle numbers, which can reduce the probability of errors in production delivery and ensure product stability and extreme delivery. Moreover, the present invention innovatively fragments the plasma sample nucleic acid, which can effectively improve the detection of viruses in plasma samples.

[0058] Example 1

[0059] The reagent information used in this example is as follows:

[0060] Table 1

[0061]

[0062]

[0063] A method for rapid library construction of metagenomic samples, comprising the following steps:

[0064] 1. Sample pretreatment

[0065] Experimental samples S1 to S8 are clinical samples. The sample types, pretreatment methods, and pathogen detection are shown in the following table.

[0066] Table 2

[0067] Sample No. Sample Type Pre-treatment method Pathogens S1 Whole blood Plasma separation Candida albicans S2 Whole blood Plasma separation Human alphaherpesvirus type 1 (HSV1) S3 Cerebrospinal fluid Wall breaking treatment Human alphaherpesvirus type 1 (HSV1) S4 Cerebrospinal fluid Wall breaking treatment Enterococcus faecium S5 Bronchoalveolar lavage fluid Host removal + wall breaking Aspergillus fumigatus S6 Sputum Wall breaking treatment Staphylococcus aureus S7 Bronchoalveolar lavage fluid Wall breaking treatment Staphylococcus aureus, Enterococcus faecium, Aspergillus fumigatus, Parvovirus S8 Bronchoalveolar lavage fluid Host removal + wall breaking Enterococcus faecium, Candida albicans, human gammaherpesvirus 4 (EBV)

[0068] 2. Nucleic Acid Extraction and Concentration Determination

[0069] Take 300 μL of the pre-treated sample and use the "Nucleic Acid Extraction or Purification Reagent" (Medical Device Registration Certificate No.: E-Han Xiebei 20220609) produced by BGI Biotechnology (Wuhan) Co., Ltd. to extract nucleic acid. After extraction, use Qubit fluorescence quantification instrument to measure the nucleic acid concentration according to the Qubit dsDNA HS Assay kit2.0Fluorometer instructions.

[0070] 3. Library construction:

[0071] This process combines fragmentation, end repair, and "A" addition processes. Compared with the existing mechanical fragmentation method or enzyme fragmentation followed by end repair and "A" addition process, the time and operation steps are shortened, which has obvious advantages. Figure 1 .

[0072] 3.1 The fragmentation, end repair and "A" addition reaction system is as follows:

[0073] Table 3

[0074] Reagent name A reaction standard Nucleic Acids ≤1000ng Segmentase (1.3 U / μl) 2.0μL 10x Segmentase buffer 5.0μL T4 DNA poly(3U / μl) 0.4μL rTaq (5U / μl) 0.2μL T4 PNK (10U / μl) 0.2μL dATP (100 mM) 0.3μL dNTP(25mM) 0.06μL Total volume Refill to 50 μL

[0075] The reaction conditions were as follows: 4°C, 1 min; 37°C, 20 min; 65°C, 10 min; 4°C hold.

[0076] 3.2 Connector connection and purification reaction system:

[0077] Table 4

[0078]

[0079]

[0080] The reaction conditions were as follows: 23°C, 15 min, 4°C hold.

[0081] Purification after connector connection: add 48 μL (0.6x) magnetic beads to 80 μL reaction solution, mix thoroughly, let stand at room temperature for 5 min, place on a magnetic stand for 3 min until the solution is clear, and discard the supernatant; add 50 μL nuclease-free water and 40 μL (0.5x) magnetic beads to the magnetic beads, mix well and let stand for 5 min, let stand on a magnetic stand for 2 min until the liquid is clear, and carefully remove the supernatant; add 200 μL 80% ethanol to elute twice, dry until the surface of the magnetic beads is matte, and add library amplification mix for library amplification.

[0082] 3.3 Library amplification and purification reaction system is as follows:

[0083] Table 5

[0084] Reagents A reaction standard PCR reaction solution 25.0μL PCR primers 4.0μL Nuclease-free water 21.0μL Total volume 50.0μL

[0085] The reaction conditions were as follows: 98°C for 2 min; 20 cycles (98°C for 15 s, 56°C for 15 s, 72°C for 30 s); 72°C for 5 min; 4°C hold.

[0086] Library purification after PCR amplification: add 30 μL (0.6 times) magnetic beads to 50 μL reaction solution, mix thoroughly, let stand at room temperature for 5 minutes, place on a magnetic stand for 3 minutes until the solution is clear, and carefully pipette the supernatant into a new centrifuge tube; add 20 μL (0.4 times) magnetic beads to the supernatant, mix well and let stand for 5 minutes, let stand on a magnetic stand for 2 minutes until the liquid is clear, and carefully remove the supernatant; add 200 μL 80% ethanol to elute twice, dry until the surface of the magnetic beads is matte, and add nuclease-free water to recover the nucleic acid.

[0087] 4. Determination of library concentration: follow the instructions of Qubit dsDNA HS Assay kit 2.0 Fluorometer.

[0088] 5. Library quality control: Use Qseq100 according to the instructions.

[0089] 6. Sequencing: Perform sequencing on the MGISEQ-2000 platform.

[0090] 7. Off-line data analysis: Perform data quality control on the data obtained by sequencing, split the qualified data according to the label sequence on the connector, and obtain the sequencing data corresponding to each sample. Remove the human sequences in the sequencing data, compare the remaining sequences with the pathogen sequence library, filter the data according to the set comparison parameters, and finally obtain the pathogen comparison results. Count the single sample data volume and the number of pathogen-specific detection sequences respectively.

[0091] 8. Result analysis.

[0092] The test results are shown in the table below. When the nucleic acid input is too low, the library can still be successfully built. The single sample data volume is greater than 20M, the proportion of adapters is less than 5%, and the pathogen detection is qualified. This shows that the inventive method is suitable for the construction of metagenomic sample libraries with different sample types and different nucleic acid starting concentrations. This method avoids adapter dilution and different PCR cycle numbers in library construction, which is conducive to the realization of standard processes and automation in production.

[0093] Table 6

[0094]

[0095]

[0096] Example 2

[0097] A method for constructing a metagenomic library, the difference being that a conventional nucleic acid purification method is used. Purification is performed after linker ligation without using a double-selection purification method; library purification is performed after PCR amplification without using a double-selection purification method. Other steps are the same as in Example 1.

[0098] Purification method after connector connection: add 40 μL (0.5 times) magnetic beads to 80 μL reaction solution, mix thoroughly, let stand at room temperature for 5 minutes, place on a magnetic stand for 3 minutes until the solution is clear, and discard the supernatant; add 200 μL 80% ethanol to elute twice, dry until the surface of the magnetic beads is matte, and add library amplification mix for library amplification.

[0099] Library purification method after PCR amplification: add 50 μL (1x) magnetic beads to 50 μL reaction solution, mix thoroughly, let stand at room temperature for 5 minutes, place on a magnetic stand for 3 minutes until the solution is clear, and carefully pipette the supernatant into a new centrifuge tube; add 200 μL 80% ethanol to elute twice, dry until the surface of the magnetic beads is matte, and add nuclease-free water to recover the nucleic acid.

[0100] The test results are as follows:

[0101] Table 7

[0102]

[0103] The results are shown in the table above. When using conventional purification methods, for samples with too low nucleic acid content, connector contamination is prone to occur, resulting in the single sample data volume of samples S1, S2, and S5 being less than 20M. The reasons are: 1. The nucleic acid content in the sample is low. 2. The following problems exist in the library construction reaction process: 2.1 The enzyme cutting, end repair, and "A" addition reaction performance are poor; 2.2 Poor connection performance; 2.3 Too many connectors are added to the sample or the purification method is unreasonable. The CV value of the single sample data volume in Example 2 is 75%, while the CV value of the single sample data volume in Example 1 is 12%, indicating that the use of the purification method of Example 1 of the present invention can effectively improve the quality of the library, reduce database volatility, increase uniformity, and thus improve the quality control pass rate. The detection of pathogens in Example 1 is also relatively lower than that in Example 2, indicating that the library quality is higher and the detection of pathogens can also be improved. The library quality control results of samples S1, S2, and S5 in Example 1 and Example 2 are shown in Figure 2. Figure 2 to Figure 4 .

[0104] Figure 2 In the upper figure, the values ​​of the vertical axis are 1.13, 24.71, 48.30, 71.89, 95.48, and 119.06 from small to large; in the lower figure, the values ​​of the vertical axis are 0.92, 25.79, 50.66, 75.52, 100.39, and 125.26 from small to large.

[0105] Figure 3 In the upper figure, the values ​​of the vertical axis are -0.73, 23.17, 47.07, 70.97, 94.87, and 118.77 from small to large; in the lower figure, the values ​​of the vertical axis are 0.98, 26.35, 51.72, 77.08, 102.46, and 127.82 from small to large.

[0106] Figure 4 In the upper figure, the values ​​of the vertical axis are 0.81, 26.19, 51.57, 76.95, 102.33, and 127.71 from small to large; in the lower figure, the values ​​of the vertical axis are 3.16, 27.17, 51.19, 75.20, 99.21, and 123.23 from small to large.

[0107] Example 3

[0108] Metagenomics tests whole blood samples, generally to detect free nucleic acids in plasma. In this embodiment, the library construction methods of different sample types are unified to facilitate the unification of production processes and the realization of product automation in the later stage. The nucleic acids in plasma samples are also fragmented to build libraries. After fragmentation, the detection of some viruses in plasma is improved, and there is no significant difference in the detection of bacteria or fungi. The results are as follows Figure 5 The fragmentation library was constructed using the method of Example 1. The unfragmented experimental process is as follows:

[0109] 1. Take 300 μL of plasma sample and use the "Nucleic Acid Extraction or Purification Reagent" produced by BGI Biotech (Wuhan) Co., Ltd. (Medical Device Registration Certificate Number: E-Han Xiebei 20220609) for nucleic acid extraction.

[0110] 2. Library construction: No fragmentation is performed during library construction, and the steps include end repair and "A" addition reaction, adapter ligation and purification, PCR amplification and purification.

[0111] 2.1 DNA end repair and "A" addition reaction system is as follows:

[0112] Table 8

[0113] Reagents Dosage DNA 43μL 10×PNK Buffer 5μL 20:1dATP dNTP 1.2μL T4 DNA Polymerase 0.4μL T4 PNK 0.2μL rTaq 0.2μL Reaction volume 50μL

[0114] Reaction conditions: 37°C, 10 min; 65°C, 15 min; 4°C hold.

[0115] 2.2 The connector connection and purification reaction system are as follows:

[0116] Table 9

[0117] Reagents Dosage ATP(100mM) 0.8μL End-repair DNA 50μL 10X PNK Buffer 3μL T4 DNA Ligase 1μL 50% PEG 8000 12μL Ad153 2μL H20 11.2μL Reaction volume 80μL

[0118] Reaction conditions: 23℃, 20min, 4℃hold. Purification: Add 0.5 times volume (40μL) of magnetic beads to the ligation reaction solution, mix well and let stand for 5min, let stand on the magnetic rack for 2min until the liquid is clear, and carefully remove the supernatant; add 200μL 80% ethanol to elute twice, dry until the surface of the magnetic beads is matte, and add 23μl nuclease-free water to recover nucleic acids.

[0119] 2.3 PCR reaction and purification reaction system is as follows:

[0120] Table 10

[0121]

[0122]

[0123] The reaction conditions were as follows: 98°C for 2 min; 12 cycles (98°C for 15 s, 56°C for 15 s, 72°C for 30 s); 72°C for 5 min; 4°C hold.

[0124] Purification of PCR products: Add 0.5 times volume (40 μL) of magnetic beads to the ligation reaction solution, mix well and let stand for 5 minutes, let stand on the magnetic rack for 2 minutes until the liquid is clear, and carefully remove the supernatant; add 200 μL 80% ethanol to elute twice, dry until the surface of the magnetic beads is matte, and add 23 μl nuclease-free water to recover the nucleic acid.

[0125] 2.4 Library concentration determination: follow the instructions of Qubit dsDNA HS Assay kit 2.0 Fluorometer.

[0126] 2.5 Library quality control: Qseq100 was used according to the instructions.

[0127] 2.6 Sequencing: Sequencing was performed according to the MGISEQ-2000 platform operation.

[0128] 2.7 Off-line data analysis: The data obtained by sequencing is subjected to data quality control, and the qualified data is split according to the label sequence on the connector to obtain the sequencing data corresponding to each sample. The human sequences in the sequencing data are removed, and the remaining sequences are compared with the pathogen sequence library. The data is filtered according to the set comparison parameters, and finally the pathogen comparison results are obtained. The single sample data volume and the number of pathogen-specific detection sequences are counted separately.

[0129] Example 4

[0130] 1. The reaction system of step 3.1 in Example 1 can also use imported enzymes. The amount of fragmentation enzyme added, the amount of buffer added, and the enzyme cutting temperature are in accordance with the requirements of the reagent instructions, and the total volume is filled with water to 50 μL. The amount of other reagents added and the reaction conditions are the same as in Example 1. The reaction system of fragmentation, end repair, and addition of "A" using imported enzymes is as follows:

[0131] Table 11

[0132] Reagent name A reaction standard Nucleic Acids ≤1000ng Custom 5X DNA shearing enzyme premix 10.0μL Custom 10X DNA shearing buffer 5.0μL T4 DNA poly(3U / μl) 0.4μL rTaq (5U / μl) 0.2μL T4 PNK (10U / μl) 0.2μL dATP (100 mM) 0.3μL dNTP(25mM) 0.06μL Total volume Refill to 50 μL

[0133] The reaction conditions were as follows: 4°C, 1 min; 32°C, 20 min; 65°C, 10 min; 4°C hold.

[0134] 2. The test results are shown in the following table. Compared with Example 1, after replacing the fragmentase, buffer and reaction conditions with the imported enzyme adaptation conditions, the library can still be successfully built, the single sample data volume is greater than 20M, the proportion of connectors is less than 5%, and the pathogen detection is qualified. The process still has good performance, indicating that the process is also suitable for imported fragmentase.

[0135] Table 12

[0136]

[0137]

[0138] Comparative Examples 1 and 2 were evaluated using simulated samples, and the concentrations of the simulated samples were as follows:

[0139] Table 13

[0140] Species name concentration HeLa cells 1E5 cells / mL Staphylococcus aureus 500CFU / mL Streptococcus pneumoniae 500CFU / mL Acinetobacter baumannii 1000CFU / mL Pseudomonas aeruginosa 500CFU / mL Candida albicans 500CFU / mL Aspergillus niger 1000CFU / mL Varicella-zoster virus (VZV) 10000copies / mL

[0141] Comparative Example 1

[0142] Add T4 PNK buffer to the reaction system in step 3.1 and add water to 50 μL. The rest is the same as in Example 1. The library concentration and pathogen detection of Comparative Example 1 are not significantly different from those of Example 1. However, the main peak of Qseq100 quality control is too large, resulting in less than 20M data for a single sample on the machine. The reason for this is that there is a low concentration of EDTA in T4 PNK buffer, which inhibits the enzyme activity of the fragmentase, resulting in a large main peak. The Qseq100 quality control peak diagram is shown in the figure below. Figure 6 . Figure 6 In the upper figure, the values ​​of the vertical axis are 8.31, 25.68, 43.05, 60.41, 77.78, and 95.15 from small to large; in the lower figure, the values ​​of the vertical axis are 3.56, 15.70, 27.84, 39.98, 52.11, and 64.25 from small to large.

[0143] The experimental results are as follows:

[0144] Table 14

[0145]

[0146] Comparative Example 2

[0147] Add T4 PNK buffer to the reaction system in step 3.1 and add water to 50 μL. The rest is the same as in Example 4. There is no significant difference between the library concentration and pathogen detection of Comparative Example 1 and Example 1. However, the main peak of Qseq100 quality control is too large, resulting in less than 20M data for a single sample on the machine. The reason for this is that there is a low concentration of EDTA in T4 PNK buffer, which inhibits the enzymatic activity of the fragmentase, resulting in a large main peak. The Qseq100 quality control peak diagram is shown in the figure below. Figure 7 . Figure 7 In the upper figure, the values ​​of the vertical axis are 10.06, 19.40, 28.74, 38.07, 47.41, and 56.74 from small to large; in the lower figure, the values ​​of the vertical axis are 3.42, 16.17, 28.92, 41.67, 54.42, and 67.17 from small to large.

[0148] The experimental results are as follows:

[0149] Table 15

[0150]

[0151] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A method for constructing a library, characterized in that: include: The pretreatment step includes performing end repair and "A" reaction on the nucleic acid sample in the same reaction system to obtain a sample after reaction; The connector connection and purification step includes connecting the connector to the sample after the reaction, and purifying the sample after the connector connection by magnetic beads to obtain a purified sample; The library amplification and purification steps include performing PCR amplification on the purified sample and performing magnetic bead purification on the amplified sample to obtain the library.

2. The method according to claim 1, characterized in that At least one of the magnetic bead purification after connector connection and the magnetic bead purification after library amplification is double selection purification.

3. The method according to claim 1 or 2, characterized in that When the magnetic bead purification after the connector connection is double-selection purification, the double-selection purification after the connector connection includes: first adding M1 times the volume of magnetic beads to the reaction solution, removing the supernatant, and then adding N1 times the volume of magnetic beads, removing the supernatant, and obtaining the purified library.

4. The method according to claim 3, characterized in that M1 is 0.5 to 0.7; Optionally, N1 is 0.4 to 0.

6.

5. The method according to claim 1 or 2, characterized in that: When the magnetic bead purification after the library amplification is double-selection purification, the double-selection purification after the library amplification includes: first adding M2 times the volume of magnetic beads to the reaction solution, collecting the supernatant, and then adding N2 times the volume of magnetic beads to the supernatant, removing the supernatant, and obtaining the purified library.

6. The method according to claim 5, characterized in that M2 is 0.5 to 0.7; Optionally, N2 is 0.3 to 0.

5.

7. The method according to claim 1, characterized in that The pretreatment step includes fragmenting, end-repairing and adding "A" reaction to the nucleic acid sample in the same reaction system to obtain a sample after reaction.

8. The method according to claim 1, characterized in that The nucleic acid sample is extracted from at least one of a tissue sample and a body fluid sample; Optionally, the nucleic acid sample is extracted from whole blood, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage fluid or sputum; Optionally, the nucleic acid sample is extracted from plasma in whole blood; Optionally, in the pretreatment step, the reaction system contains at least one of a fragmentation enzyme, a fragmentation enzyme buffer, T4 DNA polymerase, rTaq polymerase, T4 polynucleotide kinase, dATP, and dNTP.

9. The method according to claim 1, characterized in that The nucleic acid sample is a metagenomic sample.

10. A library, characterized in that The library is constructed by the method according to any one of claims 1 to 9.

Images