Construction method and application of sequencing library without human nucleic acid

Abstract

The invention discloses a sequencing library for removing human nucleic acid and a construction method and application thereof, and the construction method comprises the following steps: fragmenting nucleic acid, adding a sequencing joint, constructing a DNA or RNA library, or co-constructing a library by RNA and DNA, taking the library added with the sequencing joint as a template, carrying out denaturation annealing on the library and a specific human hybridization probe, digesting by using DSN enzyme, and carrying out sequencing on the human nucleic acid. In the renaturation process, the human nucleic acid with higher content is firstly annealed with the human hybridization probe to form double strands, the double strands are degraded by DSN enzyme, the pathogenic microorganisms with low content and slow renaturation still keep a single strand state and cannot be digested by the DSN enzyme, the human nucleic acid is effectively removed, and the pathogenic microorganism nucleic acid is further enriched through subsequent PCR (Polymerase Chain Reaction). According to the method, the proportion of human nucleic acid in the library can be effectively reduced, and nucleic acid information of pathogenic microorganisms can be reserved to the maximum extent.

Description

Technical Field

[0001] The present invention relates to the field of gene technology, and in particular to a method for constructing a sequencing library for removing human nucleic acid and an application thereof. Background Art

[0002] Infectious diseases are one of the leading causes of death in the world. The number of deaths directly caused by pathogenic microorganisms is always among the highest in the world, which places a heavy burden on the public health system. Rapid identification of pathogens can greatly improve the efficiency of treatment and prevention and control, but traditional microbial testing has greatly limited its clinical use due to its many steps, complex operations, and single targets. Literature reports that more than 20% to 60% of infections cannot identify the pathogen. Metagenomic testing provides new solutions for difficult, critical or special infections with its advantages of rapid identification, comprehensive coverage, no need for culture, and no need for presets. However, due to the high human background in the sample, the sensitivity of metagenomic testing is low, and the amount of sequencing data is high, requiring a high cost for the entire test, which imposes a relatively large cost burden on patients.

[0003] At present, the differential lysis method is mainly used to reduce the interference of human nucleic acids. Based on the fact that human cell membranes are more fragile than the outer walls of microorganisms, human cells are first lysed with mild detergents (such as saponins) before nucleic acid extraction to release host nucleic acids, and then deoxyribonuclease I is used to degrade host DNA. Although this method can reduce the interference of human nucleic acids to a certain extent, it has obvious disadvantages. It cannot avoid the loss of microorganisms with fragile cell membranes such as mycoplasmas and chlamydia, as well as microorganisms without cell structures such as viruses. At the same time, this method will also remove free nucleic acids in the sample, resulting in the loss of possible pathogenic nucleic acid information, which in turn affects the accuracy and comprehensiveness of the test results.

[0004] In summary, the current detection of infectious diseases faces many challenges, and a sequencing library construction method that removes human nucleic acids is urgently needed to minimize the loss of pathogenic microorganisms in the process of removing human nucleic acids and provide stronger support for the diagnosis and treatment of infectious diseases. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for constructing a sequencing library that removes human nucleic acids and its application. A library construction method that can remove human nucleic acids of the present invention includes library preparation: fragmenting nucleic acids and adding sequencing adapters; dividing into DNA libraries, RNA libraries, and RNA and DNA co-construction libraries according to nucleic acid types; then using the library with the sequencing adapters added as a template, the library is denatured and annealed with a specific human hybridization probe, and then digested with a thermostable double-stranded specific nuclease (DSN). During the renaturation process, the human nucleic acid content is high and preferentially anneals with the human hybridization probe to form a double strand, which is degraded by the DSN enzyme. The pathogenic microorganism content is low and renatures slowly, and still remains in a single-stranded state and cannot be digested by the DSN enzyme. Therefore, the human nucleic acid is effectively removed, and the pathogenic microorganism nucleic acid is further enriched by subsequent PCR. The present invention also provides a method for preparing a human probe, which specifically binds to the dispersed repetitive sequences, rRNA sequences, and gene sequences widely distributed in the human genome. The method of the present invention can effectively reduce the proportion of human nucleic acids in the library and maximize the retention of nucleic acid information of pathogenic microorganisms.

[0006] Specifically, the purpose of the present invention is achieved through the following technical solutions: <First aspect> The technical solution adopted by the present invention is as follows: A method for constructing a sequencing library by removing human nucleic acid comprises the following steps: S1. Library preparation: fragment the nucleic acid and add sequencing adapters; divided into DNA library, RNA library, and RNA and DNA co-constructed library according to the nucleic acid type; S2, human nucleic acid hybridization: adding a human hybridization probe to the library constructed in step 1, and performing a denaturation annealing treatment to allow the human hybridization probe to specifically bind to the human sequence library; the human hybridization probe specifically binds to the dispersed repetitive sequences, rRNA sequences and housekeeping gene sequences widely distributed in the human genome; S3, heat-stable double-stranded specific nuclease digestion: on the basis of step S3, heat-stable double-stranded specific nuclease (DSN) is added to perform digestion while keeping the annealing temperature unchanged. After the digestion is completed, a stop solution is added to terminate the reaction; the DSN enzyme can specifically degrade double-stranded DNA and can maintain stable activity at a higher annealing temperature; S4, amplifying the digested products, using library universal primers to perform PCR amplification on the products digested by the thermostable double-stranded specific nuclease in step S3, and performing library enrichment to obtain a sequencing library from which human nucleic acids have been removed; the PCR amplification uses the products digested by the thermostable double-stranded specific nuclease S3 as a template, the library universal primers are universal sequences of sequencing adapters, and matching sequencing adapters and library universal primers are selected based on different sequencing platforms.

[0007] In S2, the method for preparing the human hybridization probe comprises the following steps: A. RNA extraction: Extract total RNA from Hela cells; B. RNA fragmentation: Take an appropriate amount of total RNA and mix it with fragmentation buffer. Under certain reaction conditions, the RNA will be broken into fragments of about 100-150bp; C. Reverse transcription reaction: using the fragmented RNA as substrate, mixing it with reverse transcription reaction buffer and reverse transcriptase, performing reverse transcription reaction, synthesizing cDNA, and realizing the conversion from RNA to cDNA; D. Preparation of single-stranded cDNA by RNA digestion: Mix the cDNA product with digestion reaction buffer and RNase H enzyme, react, recover, and prepare single-stranded cDNA; E. Preparation of human hybridization probes: Mix equal amounts of Human Cot-1 DNA and the single-stranded cDNA prepared in step D to prepare a probe mixture that can specifically bind to different types of human nucleic acid sequences, thereby completing the preparation of human hybridization probes.

[0008] In step B, the fragmentation buffer includes 50-250 mM Tris-HCl (PH 8-8.5), 2.5 μM-15 μM MgCl2, and 100 mM-400 mM KCl. The fragmentation reaction procedure is: 94°C for 10 min, 1 cycle.

[0009] In step C, the reverse transcription reaction buffer includes 250~750 mM Tris-HCl (pH 8-8.5), 375~900 mM KCl, 15~50 mM MgCl2, 50~150 mM DTT, 1~10 mM dNTPs, and 2.5~25 mM Random Hexamers. Random Haxamers are random N6 primers (NNNNNN).

[0010] The reverse transcriptase is M-MLV Reverse Transcriptase.

[0011] Reverse transcription reaction procedure: react at 25°C for 10 min, 42°C for 15 min, and 70°C for 15 min, each for 1 cycle, to synthesize cDNA.

[0012] In step D, the digestion reaction buffer includes: 200 mM Tris-HCl (pH 7.8), 400 mM KCl, 80 mM MgCl2, 10 mM DTT.

[0013] Digestion reaction procedure: react at 37°C for 30 min, perform 1 cycle, prepare single-stranded cDNA, and recover it for later use.

[0014] In step E, Human Cot-1 DNA (Thermo Fisher Scientific Catalog No. 15279, derived from human placental genomic DNA, with a maximum length of approximately 50-300bp and rich in interspersed repetitive sequences); Human Cot-1 DNA can bind to human DNA sequences in the DNA library, and cDNA products can bind to human rRNA sequences and commonly expressed gene sequences (such as housekeeping gene sequences) in the RNA library.

[0015] The human hybridization probe comprises a single-stranded DNA probe with a length of 100-150 bp and a supplementary 50-300 bp human Cot-1 DNA.

[0016] In step S1, the DNA library is prepared by physical shearing, fragmentation enzyme shearing or Tn5 transposase shearing to fragment the DNA, then end-repair and A addition, and then ligation of sequencing adapters.

[0017] Generally, the DNA template is mechanically or enzymatically fragmented, and the fragmented DNA is end-repaired and A-added. Then, the sequencing adapters are connected to the two ends of the DNA fragments by T4 DNA ligase, and finally, the library is PCR amplified and enriched by universal primers of the library that match the sequencing adapters. Alternatively, the DNA template is fragmented with a transposase and adapters are added, and then the sequencing adapters are connected to the fragments by PCR amplification to prepare a complete DNA library.

[0018] In step S1, the RNA library is prepared by thermally cleaving the fragmented RNA in a divalent metal ion buffer, followed by reverse transcription and second-strand synthesis, followed by end repair and A addition, and ligation of sequencing adapters.

[0019] The RNA library construction process generally involves fragmenting the RNA template by thermal lysis, then reverse transcribing and synthesizing the second strand of the fragmented RNA to generate double-stranded DNA, then performing end repair and A addition, connecting the sequencing adapters to the two ends of the DNA fragments by T4 DNA ligase, and finally amplifying and enriching the library by PCR using universal primers for the library that match the sequencing adapters; or reverse transcribing and synthesizing the second strand of the RNA template to generate double-stranded DNA, then breaking it with a transposase and adding adapters, and connecting the sequencing adapters to the fragments by PCR amplification to prepare a complete DNA library.

[0020] The preparation of the RNA and DNA co-constructed library in step S1 is to reverse transcribe and synthesize the second strand of RNA, then to fragment it together with DNA by enzyme method, then to perform end repair and add A, and then to connect the sequencing adapter.

[0021] In the RNA and DNA co-library construction process, the input template is the total nucleic acid containing both DNA and RNA. The nucleic acid is reverse transcribed and double-stranded synthesized to generate double-stranded DNA, which is then mechanically or enzymatically sheared. The fragmented DNA is then end-repaired and A-added. The sequencing adapters are connected to the two ends of the DNA fragments by T4 DNA ligase. Finally, the library is PCR amplified and enriched using universal primers for the library that match the sequencing adapters. Alternatively, the generated double-stranded DNA is sheared with a transposase and adapters are added, and the sequencing adapters are connected to the fragments by PCR amplification to prepare a complete DNA library.

[0022] In the above-mentioned library construction process, the library universal primer is a universal sequence of a sequencing adapter, and matching sequencing adapters and library universal primers are selected based on different sequencing platforms.

[0023] Preferably, the universal primer sequence of the library is as follows: Upstream primer 5'-CAAGCAGAAGACGGCAT-3', as SEQ ID No. 1; The downstream primer is 5'-AATGATACGGCGACCAC-3', such as SEQ ID No.2.

[0024] In step S2, the human hybridization probe includes a single-stranded DNA probe with a length of 100-150 bp and a supplementary 50-300 bp of human Cot-1 DNA.

[0025] Human nucleic acid hybridization is to add human hybridization probes to the library (DNA library, RNA library, RNA and DNA co-constructed library), denature and anneal: prepare the reaction system in a 200µl PCR tube: 5-10µl library, 1-2µl human hybridization probe, 2-5µl hybridization buffer.

[0026] The library input amount is 10~500 ng; The input amount of the human hybridization probe is 0.1~5μg.

[0027] The hybridization buffer contains 50~400 mM HEPES (pH 7.5 @ 25°C), 0.5~2 M NaCl, and 0.2~2 mM EDTA.

[0028] The hybridization reaction procedure is as follows: Step 1: 98°C for 3 min, 1 cycle; Step 2: 68℃ for 1-7h, 1 cycle.

[0029] In S3, the thermostable double-stranded specific nuclease (DSN enzyme) digestion is performed by adding DSN enzyme to the step S3 while keeping the annealing temperature unchanged, and then adding the stop solution to terminate the reaction: specifically, preheat the DSN enzyme reaction buffer at 68°C, then add 1µL to the hybridization product, mix well and quickly return to the 65-70°C thermostat, then add 0.5-1.5µL DSN enzyme, mix well and quickly return to 65-70°C for digestion for 7-30 min; after the reaction is completed, add 3-7µL stop solution and terminate the reaction at 65-70°C for 5-10 min.

[0030] The DSN enzyme reaction buffer contains 100-500 mM Tris-HCl (pH 8.0 @ 25°C), 10-50 mM MgCl2, and 2-10 mM DTT; The usage amount of the thermostable double-stranded specific nuclease is 0.2~1U.

[0031] The stop solution can be SDS solution or EDTA solution.

[0032] Amplification of digestion products in step S4: Perform PCR amplification of the products digested by DSN enzyme in the previous step with universal primers for library enrichment: prepare a 50-100µl reaction system in a 200µl PCR tube: 5-10µl universal library primer pair, 25-50µl nucleic acid amplification reaction solution, 10-20µL of the purified digestion product in the previous step, and make up to a total volume of 50-100µl with water; The universal library primer pair is a primer that matches the universal sequence of the library sequencing adapter; The amount of the universal library primer is 5-80 pmol; The nucleic acid amplification reaction solution contains 150~300 mM Tris HCl (pH 8.4), 200~500 mM KCl, 5~15 mM MgCl2, 0.5~2 mM dNTPs, 0.1~1 U / μL pfu DNA polymerase, and 5~20% glycerol (v / v).

[0033] The PCR amplification procedure is as follows: Step 1: 95°C for 3 min, 1 cycle; Step 2: 95℃ 20s, 60℃ 15s, 72℃ 30s, 3~18 cycles; Step 3: 72°C for 3 min, 1 cycle.

[0034] Step S4 also includes a step of purifying the PCR amplification.

[0035] The step of purifying the amplified product comprises: 1) Equilibrate the magnetic beads to room temperature; 2) Take 45-50µl of magnetic beads and add them to the PCR product. Mix thoroughly and let it react for 5-10 minutes. 3) Place the centrifuge tube on the magnetic rack and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed, then discard the supernatant with a pipette; 4) Add 500-600µl of 75-80% (v / v) ethanol solution, let stand for 30-60s, and then discard the supernatant; 5) Repeat step 4) once; 6) Keep the centrifuge tube in the magnetic rack at all times, open the cover and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; 7) Add 20-50µl sterile ultrapure water for elution, use a pipette to gently blow and mix thoroughly, and place at room temperature for 5-10 minutes. Centrifuge the centrifuge tube instantaneously (usually 2-5 seconds) and place it on a magnetic rack to stand. After the solution becomes clear (2-5 minutes), collect the supernatant to obtain the purified amplified product.

[0036] Compared with the prior art, the present invention has the following significant beneficial effects: 1. Accurately remove human nucleic acids and retain pathogen information completely. In the detection of infectious diseases, accurately obtaining nucleic acid information of pathogenic microorganisms is the key to diagnosis and treatment. In the process of removing human nucleic acids, the existing technology often cannot avoid the loss of pathogenic microorganism nucleic acids. For example, the traditional differential lysis method, although it aims to reduce the interference of human nucleic acids through specific steps, cannot accurately distinguish between human cells and some fragile pathogenic microorganisms, resulting in a large number of microorganisms with fragile cell membranes such as mycoplasmas and chlamydia, and cell-free microorganisms such as viruses being lost during the treatment process, which seriously affects the integrity of pathogenic nucleic acid information. The method of the present invention uses a unique technical path, using a thermostable double-stranded specific nuclease (DSN) and a specific human hybridization probe to efficiently and accurately remove human nucleic acids during the library construction process. During the renaturation process, human nucleic acids anneal with human hybridization probes to form double strands due to their high content, and are then specifically degraded by the DSN enzyme, while pathogenic microorganism nucleic acids remain single-stranded due to their low content and slow renaturation, thereby effectively avoiding the risk of digestion. This process not only effectively removes human nucleic acids, but more importantly, it maximizes the retention of nucleic acid information of all pathogenic microorganisms in the sample, providing a comprehensive and accurate data basis for subsequent testing and analysis, greatly improving the reliability of test results and the accuracy of diagnosis.

[0037] 2. Simplify the process and reduce costs. The process of existing detection technology is usually complicated, involving multiple cumbersome steps and links. Taking the traditional microbial detection method as an example, from sample collection, culture, separation to identification, each step requires strict operation and a long time period, which not only consumes a lot of manpower and material resources, but also easily introduces errors during the operation. Although metagenomics detection technology has certain advantages in detection range and speed, it requires a large amount of sequencing data to be processed, and the performance requirements of the equipment are extremely high. It not only needs to be equipped with advanced sequencing instruments, but also requires powerful data analysis software and professional technicians to operate and interpret, which greatly increases the detection cost and limits its application in some resource-limited areas. The method of the present invention greatly simplifies the entire detection process. The removal of human nucleic acids is directly realized during the library construction process, which avoids multiple complex pretreatment steps in traditional methods and reduces sample processing time and possible errors. At the same time, since there is no need to rely on expensive special equipment and complex data analysis systems, the method of the present invention significantly reduces the detection cost while ensuring the detection effect. This is of great significance for improving detection efficiency and reducing the economic burden on medical institutions and patients, and is particularly suitable for areas with relatively scarce resources and large-scale detection needs.

[0038] 3. Easy to operate and easy to promote. The existing technology often requires high professional skills of operators in actual operation. For example, the concentration of detergent, action time and the amount of deoxyribonuclease I used in the differential lysis method need to be precisely controlled, and the slightest carelessness may lead to deviations in experimental results. Metagenomics detection technology involves complex instrument operation and data analysis, which requires professional technicians to undergo long-term training to master. The method of the present invention is simple and convenient to operate and easy to master. Each step in the entire process has clear operating guidelines and parameter ranges, and even operators without rich experience can quickly get started. Moreover, the reagents and equipment used in the present invention are relatively common in the market and easy to obtain, which further reduces the difficulty and threshold of operation. This convenience in operation enables the method of the present invention to be widely used in medical institutions and scientific research units at different levels, providing strong technical support for promoting the rapid diagnosis and prevention and control of infectious diseases, and has extremely high promotion value.

[0039] 4. Since the method of the present invention can completely retain the nucleic acid information of pathogenic microorganisms while removing human nucleic acids, and has the advantages of simple process, low cost and convenient operation, it has broad application prospects in many fields. In clinical diagnosis, it can quickly and accurately detect pathogenic microorganisms, provide an important basis for doctors to formulate personalized treatment plans, help improve the treatment effect, and shorten the patient's recovery period. In the field of public health, it can be used for large-scale disease monitoring and prevention and control, timely detect potential pathogens, and take effective prevention and control measures to prevent the spread and spread of diseases. In the field of scientific research, it also provides a powerful technical means for studying the diversity, evolution and pathogenic mechanisms of pathogenic microorganisms, and promotes the continuous in-depth development of research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 It is a schematic diagram of the process of the present invention; Figure 2 This is the distribution diagram of library fragments. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0042] Figure 1 It is a schematic diagram of the process of the present invention.

[0043] Example 1 Human total RNA extraction Take Hela cells 10 7 cells, and total RNA was extracted using the Tiangen Biotechnology Cell Total RNA Extraction Kit (DP451): A. Centrifuge Hela cells at 300×g for 5 minutes, discard the supernatant, and retain the cell pellet; use low-speed centrifugation to precipitate the cells from the culture medium for subsequent processing and to remove impurities such as serum and culture medium components that may interfere with RNA extraction; B. Add 600µl of Lysis Buffer RLA and 10µl of Proteinase K to the cell pellet and vortex to mix. Lysis Buffer RLA destroys the cell membrane and nuclear membrane structure and releases RNA in the cell. Proteinase K can degrade proteins and reduce the interference of protein impurities on RNA extraction. Vortexing helps to fully mix the reagents and cell pellet to ensure complete lysis reaction. C. Centrifuge at 12000rpm for 5min, transfer the supernatant to a new centrifuge tube, add 600µl 70v / v% ethanol, mix well, and transfer all the solution including the precipitate to the RNA adsorption column CR4; centrifuge at high speed to precipitate cell debris and insoluble matter, take the supernatant to ensure the purity of the extracted RNA, and use 70% ethanol to precipitate RNA to reduce its solubility, which is conducive to subsequent capture in the adsorption column; D. Centrifuge at 12000rpm for 30s, discard the filtrate, and put the adsorption column back into the collection tube; this operation allows the RNA-containing solution to pass through the adsorption column, the RNA binds to the medium in the adsorption column, and the filtrate containing unbound impurities is discarded; E. Add 80 µl of DNase I enzyme working solution to the center of the adsorption column and let it stand at room temperature for 15 minutes. DNase I enzyme specifically degrades DNA, removes possible genomic DNA contamination in the sample, and ensures that the extracted RNA does not contain DNA impurities. F. Add 350µl of deproteinized solution RW3 to the center of the adsorption column, centrifuge at 12000rpm for 30s, discard the filtrate, and put the adsorption column back into the collection tube; the deproteinized solution removes residual protein and enzyme impurities in the lysate, and the filtrate containing protein impurities is removed by centrifugation to further purify RNA; G. Add 500µl of rinse solution RW to the center of the adsorption column, let it stand at room temperature for 2 minutes, then centrifuge at 12000rpm for 30 seconds, discard the filtrate, and put the adsorption column back into the collection tube; the rinse solution removes residual salt ions, buffer components and a small amount of unbound impurities on the adsorption column to further purify the RNA.

[0044] H. Repeat step G; I. Centrifuge at 12000rpm for 30s, discard the filtrate, and let it stand at room temperature to dry for 2min; remove the residual rinsing liquid in the adsorption column by high-speed centrifugation to prevent it from affecting the subsequent RNA elution and quality; dry at room temperature to remove water to avoid water dilution of the eluted RNA or introduction of impurities, thereby ensuring the purity and stability of the RNA; J. Place the adsorption column in a new centrifuge tube, add 30µl of enzyme-free water in the center of the adsorption column, let it stand at room temperature for 2 minutes, and then centrifuge at 12000rpm for 2 minutes to obtain RNA solution.

[0045] Example 2 Preparation of human hybridization probes (1) RNA fragmentation Prepare the reaction system according to Table 1 and break the RNA into fragments of about 100-150 bp: Table 1

[0046] Fragmentation reaction program: 94°C for 10 min, 1 cycle; (2) Reverse transcription reaction Prepare the reaction system according to Table 2, and reverse transcribe the total RNA to synthesize cDNA: Table 2

[0047] The reverse transcription reaction procedure is as follows: Step 1: 25°C for 10 min, 1 cycle; Step 2: 42°C for 15 min, 1 cycle; Step 3: 70°C for 15 min, 1 cycle; (3) RNA digestion reaction Prepare the reaction system according to Table 3, digest RNA with RNase H, and prepare single-stranded cDNA: Table 3

[0048] The digestion reaction procedure is as follows: Step 1: 37°C for 30 min, 1 cycle; (4) Recovery of single-stranded cDNA products (using Tiangen Biotechnology's general DNA product purification kit (Cat. No. DP204)): cDNA was recovered using the centrifugal adsorption column method A. Place the adsorption column in the collection tube, then add 500µl of balancing solution BL, centrifuge at 12000rpm for 1min, then pour out the liquid in the collection tube, and put the adsorption column back into the collection tube for later use; B. Add 150µl of binding solution PB to 30µl of product and mix thoroughly; C. Transfer the mixed solution from the previous step to the adsorption column, let it stand at room temperature for 5 minutes, then centrifuge it at 12000 rpm for 30 seconds, pour out the liquid in the collection tube, and put the adsorption column back into the collection tube; D. Add 600 µl of rinse solution PW to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, pour out the liquid in the collection tube, and put the adsorption column back into the collection tube; E. Repeat step D. F. Centrifuge at 12000 rpm for 2 min, remove the rinse solution, and then stand and dry at room temperature for 3 min; G. Take out the adsorption column and place it in a clean centrifuge tube. Add 20µl of elution buffer EB to the middle of the silica matrix adsorption membrane. Let it stand at room temperature for 5 minutes, then centrifuge at 12000 rpm for 2 minutes to collect the DNA liquid.

[0049] (5) Product quantification: The ssDNA concentration was detected using qubit 2.0 and diluted to 100 ng / uL for later use.

[0050] Purity detection: Use NanoDrop to detect the absorbance (OD value) of the product at 260nm and 280nm. OD260 / OD280 should be between 1.7 and 1.9.

[0051] Fragment detection: The Agilent 2100 instrument was used to detect the fragments of the product, and the fragments were distributed between 100 and 150 bp.

[0052] (6) Preparation of human hybridization probes Human Cot-1 DNA (Thermo Fisher Scientific Catalog No. 15279) is derived from genomic DNA extracted from human placenta. The maximum length is approximately 50 to 300 bp and is rich in interspersed repetitive sequences (IRS) such as SINEs (small interspersed repetitive sequences, such as Alu sequences) and LINEs (large interspersed repetitive sequences, such as L1 sequences) that are widely distributed in the human genome.

[0053] The Human Cot-1 DNA and the cDNA product prepared in step (5) are mixed in equal masses to prepare a human hybridization probe mixture, wherein the Human Cot-1 DNA can bind to the human DNA sequence in the DNA library, and the cDNA product can bind to the human rRNA sequence and the commonly expressed gene sequence (such as the housekeeping gene sequence) in the RNA library.

[0054] Fragment detection: The Agilent 2100 instrument was used to detect the fragments of the product, and the fragments were distributed between 50 and 300 bp.

[0055] Example 3: Removal of human nucleic acid (1) Preparation of reference products: ZymoBIOMICS™ microbial community standard (Zymo, product number D6300) and influenza A pseudovirus (H1N1 nucleic acid reference, Jingliang Biotechnology, product number GW-IAF110) were added to 2×105 cells / mL human Hela cell line at different concentration gradients, and the matrix solution was PBS buffer, as shown in Table 4 below: Table 4

[0056] (1) Total nucleic acid extraction: A DNA and RNA co-extraction kit (NoviZan, magnetic bead-based pathogenic microorganism DNA / RNA co-extraction kit, catalog number RM601) was used to co-extract nucleic acids from the reference sample to obtain total nucleic acids (RNA & DNA).

[0057] (2) DNA and RNA co-construction library The co-extracted nucleic acids included DNA and RNA. A co-library construction scheme was adopted, and three libraries were constructed in parallel for each reference sample.

[0058] A. Nucleic acid denaturation Prepare the reaction system according to Table 5 and mix thoroughly by gently pipetting 10 times: Table 5

[0059] The denaturation reaction procedure is as follows: Step 1: 70°C for 5 min, 1 cycle; Step 2: Immediately place in ice bath for 3 minutes to obtain denatured total nucleic acid (RNA & DNA).

[0060] B. Reverse transcription reaction Prepare the first-strand cDNA synthesis reaction system according to Table 6: Table 6

[0061] The reverse transcription reaction procedure is as follows: Step 1: 25°C for 5 min, 1 cycle; Step 2: 42°C for 15 min, 1 cycle; Step 3: 85℃ for 5min, 1 cycle to obtain the first-strand cDNA.

[0062] C. Double-stranded DNA synthesis Prepare the double-strand cDNA synthesis reaction system according to the following table: Table 7

[0063] The second-strand synthesis reaction procedure is as follows: Step 1: 16°C for 30 min, 1 cycle.

[0064] D. Fragmentation and end repair plus A Table 8 Preparation of reaction system: Table 8

[0065] The fragmentation and end-repair plus A reaction procedures are as follows: Step 1: 37°C for 20 min, 1 cycle; Step 2: 65°C for 30 min, 1 cycle; E. Connector connection Prepare the reaction system according to Table 9: Table 9

[0066] The adapter ligation reaction procedure is as follows: Step 1: 20℃ for 15min, 1 cycle.

[0067] F. Purification of the Connector Ligation Product F1, DNA purification magnetic beads are balanced to room temperature; F2. Take 60µl of DNA purification magnetic beads and add them to the adapter ligation product in step E. Mix thoroughly and let it react for 5 minutes. F3. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed. Then, carefully discard the supernatant with a pipette. F4, add 500 µl of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant; F5, repeat F4; F6. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; F7. Add 22 µl of sterile ultrapure water for elution. Use a pipette to gently pipette and mix thoroughly. Place at room temperature for 5 minutes. Centrifuge the tube for 3 seconds and place it on a magnetic rack. After the solution becomes clear (about 2 minutes), carefully transfer 20 µl of the supernatant to a new centrifuge tube. Do not touch the magnetic beads.

[0068] (3) Human Probe Hybridization Prepare the reaction system according to Table 10: Table 10

[0069] The hybridization reaction procedure is as follows: Step 1: 98°C for 3 min, 1 cycle; Step 2: 68°C for 1 h, 1 cycle; Step 3: 68℃ hold, 1 cycle.

[0070] (4) DSN enzyme digestion Preheat the DSN enzyme reaction buffer at 68°C, then add 1µL to the hybridization product of step (3), mix well and quickly return to the 68°C thermostat, add another 1µL DSN enzyme, mix well and quickly return to 68°C for digestion for 10 min; after the reaction is completed, add 5µL of stop solution and terminate the reaction at 68°C for 5 min.

[0071] (5) Library amplification after digestion Prepare the reaction system according to Table 11: Table 11

[0072] The PCR amplification procedure is as follows: Step 1: 95°C for 3 min, 1 cycle; Step 2: 95°C 20s, 60°C 15s, 72°C 30s, 18 cycles; Step 3: 72°C for 3 min, 1 cycle.

[0073] (6) Purification of amplified products A. DNA purification magnetic beads are balanced to room temperature; B. Take 45 µl of DNA purification magnetic beads and add them to the product of step (5). Mix thoroughly and let it react for 5 minutes. C. Place the centrifuge tube on the magnetic stand and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed. Then, carefully discard the supernatant with a pipette. D. Add 500 µl of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant; E. Repeat step D. F. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; G. Add 22 µl of sterile ultrapure water for elution, use a pipette to gently pipette to mix thoroughly, and place at room temperature for 5 min. Centrifuge the tube briefly and place it on a magnetic rack to stand. After the solution becomes clear (about 2 min), carefully transfer 20 µl of the supernatant to a new centrifuge tube without touching the magnetic beads.

[0074] (7) Library quality control: Qseq100 was used to detect the size of the product, and qubit 2.0 was used to check the concentration of the library. Figure 2 shown.

[0075] (8) Sequencing All products were standardized and mixed in equal amounts, and the mixed libraries were sequenced in parallel using the Illumina NextSeq550Dx sequencing platform and SE75 sequencing type.

[0076] (9) Data analysis Fastp software was used to filter low-quality sequences and remove adapter primer sequences. Bowtie2 was used to count and remove the read ratios aligned to the human reference genome hg19. Kraken2 was used for species annotation and abundance statistics.

[0077] Analysis steps A to E; including filtering adapter primer sequences, host ratio statistics and host removal, species annotation and abundance statistics.

[0078] Results: After DNA and RNA libraries were constructed for the three reference samples, the samples that were directly sequenced without human-source removal were used as control groups (S1_control, S2_control, S3_control), and the samples that were subjected to human-source removal using the method of the present invention were used as experimental groups (S1_HD, S2_HD, S3_HD). The proportions of human-source sequences in the control groups were 98.51%, 99.04%, and 99.38%, respectively, and the proportions of human-source sequences in the experimental groups were 76.69%, 82.96%, and 85.54%, respectively. After human-source removal using this method, the proportion of human-source sequences decreased by 13% to 21%, as shown in Table 12: Table 12

[0079] The RPM values ​​detected by species were statistically analyzed.

[0080] Among them: RPM value = specific reads number / measured effective reads number (per million)), Fastp software was used to filter low-quality sequences and remove adapter primer sequences to obtain the filtered effective reads number, Kraken2 performed species annotation and abundance analysis and output the output file, and the optimal count corresponding to the species was the specific reads number.

[0081] All species were detected in the three reference samples of the experimental group, but some species were not detected in the S3 sample of the control group. The RPM values ​​detected in the experimental group increased by about 2 to 20 times, as shown in Table 13: Table 13

[0082] Conclusion: This method is used to construct a host-free library. The library construction process is simple, which can effectively reduce the proportion of host nucleic acid and improve the detection rate of microorganisms.

[0083] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing a sequencing library by removing human nucleic acid, characterized in that: The following steps are involved: S1. Library preparation: fragment the nucleic acid and add sequencing adapters, which are divided into DNA library, RNA library, and RNA and DNA co-constructed library according to the nucleic acid type; S2, human nucleic acid hybridization: adding the human hybridization probe to the library constructed in step S1, and performing denaturation annealing treatment to allow the human probe to specifically bind to the human sequence library; S3, heat-stable double-stranded specific nuclease digestion: add heat-stable double-stranded specific nuclease to digest while keeping the annealing temperature unchanged, and add stop solution to terminate the reaction after digestion is completed; S4, amplification of digestion products: the products digested by the thermostable double-stranded specific nuclease in step S3 are amplified by PCR using universal primers for library enrichment to obtain a sequencing library with human nucleic acid removed; The PCR amplification uses the product digested by S3 thermostable double-stranded specific nuclease as a template, and the library universal primer is a universal sequence of a sequencing adapter. Matching sequencing adapters and library universal primers are selected based on different sequencing platforms.

2. The method for constructing a sequencing library removing human nucleic acid according to claim 1, characterized in that: In S2, the method for preparing the human hybridization probe comprises the following steps: A. RNA extraction: Extract total RNA from Hela cells; B. RNA fragmentation: Take an appropriate amount of total RNA and mix it with fragmentation buffer. Under certain reaction conditions, the RNA will be broken into fragments of about 100-150bp; C. Reverse transcription reaction: using the fragmented RNA as substrate, mixing it with reverse transcription reaction buffer and reverse transcriptase, performing reverse transcription reaction, synthesizing cDNA, and realizing the conversion from RNA to cDNA; D. Preparation of single-stranded cDNA by RNA digestion: Mix the cDNA product with digestion reaction buffer and RNase H enzyme, react, recover, and prepare single-stranded cDNA; E. Preparation of human hybridization probes: Mix equal amounts of Human Cot-1 DNA and the single-stranded cDNA prepared in step D to prepare a probe mixture that can specifically bind to different types of human nucleic acid sequences, thereby completing the preparation of human hybridization probes.

3. The method for constructing a sequencing library removing human nucleic acid according to claim 1, characterized in that: The preparation of the DNA library in step S1 is performed by physical shearing, fragmentation enzyme shearing or Tn5 transposase shearing to fragment the DNA, followed by end repair and A addition, and then ligation of sequencing adapters.

4. The method for preparing a sequencing library by removing human nucleic acid according to claim 1, characterized in that: The RNA library in step S1 is prepared by thermally cleaving the fragmented RNA in a divalent metal ion buffer, performing reverse transcription and second-strand synthesis, and then performing end repair and A addition, and connecting sequencing adapters.

5. The method for preparing a sequencing library by removing human nucleic acid according to claim 1, characterized in that: The preparation of the RNA and DNA co-constructed library in step S1 is to reverse transcribe and synthesize the second strand of RNA, then to fragment it together with DNA by enzyme method, then to perform end repair and add A, and then to connect the sequencing adapter.

6. The method for preparing a sequencing library by removing human nucleic acid according to claim 1, characterized in that: In step S2, the input amount of the library is 10-500 ng, and the input amount of the human hybridization probe is 0.1-5 µg.

7. The method for preparing a sequencing library by removing human nucleic acid according to claim 1, characterized in that: In step S3, the usage amount of the thermostable double-stranded specific nuclease is 0.2-1 U.

8. The method for constructing a sequencing library removing human nucleic acid according to claim 1, characterized in that: Step S4 also includes a step of purifying the amplified product.

9. The method for constructing a sequencing library removing human nucleic acid according to claim 8, characterized in that: The step of purifying the amplified product comprises: 1) Equilibrate the magnetic beads to room temperature; 2) Take 45-50µl of magnetic beads and add them to the PCR product, mix well and let it react for 5-10 minutes; 3) Let the solution stand for 1-2 minutes until it becomes clear and the magnetic beads are completely adsorbed, then discard the supernatant; 4) Add 500µl ethanol solution, let stand for 30-60s, and discard the supernatant; 5) Repeat step 4) once; 6) Keep the centrifuge tube in the magnetic rack at all times, open the cover and dry the magnetic beads for 5-10 minutes until no ethanol remains; 7) Add 22-50µl sterile ultrapure water for elution, mix thoroughly with a pipette, and place at room temperature for 5-10 minutes. Centrifuge the tube instantaneously and place it on a magnetic rack to stand. After the solution becomes clear, collect the supernatant.

10. A library constructed according to the construction method according to any one of claims 1 to 8.

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