A method for constructing sequencing library for removing human-derived nucleic acid and application thereof

Abstract

The application discloses a sequencing library for removing human-derived nucleic acid and a construction method and application thereof, and the construction method comprises the following steps: fragmentizing nucleic acid and adding sequencing adapters, constructing a DNA or RNA library, or constructing a RNA and DNA co-library, then taking the library with the added sequencing adapters as a template, performing denaturation annealing on the library and specific human-derived hybridization probes, and performing digestion on the library by using DSN enzyme, and in the renaturation process, human-derived nucleic acid with a higher content is preferentially annealed with the human-derived hybridization probes to form double strands, which are degraded by the DSN enzyme, pathogenic microorganism with a low content is slowly renatured and remains in a single-stranded state, and cannot be digested by the DSN enzyme, and the human-derived nucleic acid is effectively removed, and the nucleic acid of the pathogenic microorganism is further enriched through subsequent PCR. The method can effectively reduce the proportion of human-derived nucleic acid in the library, and maximize the retention of nucleic acid information of the pathogenic microorganism.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic technology, in particular to a sequencing library construction method for removing human nucleic acid and application thereof. BACKGROUND

[0002] Infectious diseases are one of the main causes of global human death. The number of deaths directly caused by pathogenic microorganism infection ranks among the world's top every year, which brings a heavy burden to the public health system. Rapid and clear pathogen can greatly improve the efficiency of treatment and prevention, but traditional microorganism detection is greatly limited in clinical use due to multiple steps, complex operation and single target. Literature reports that more than 20-60% of infections cannot identify the pathogen. Metagenomics detection provides a new solution for difficult and critical or special infection conditions with its rapid identification, comprehensive coverage, no need for culture and no need for pre-set. However, due to high human background in the sample, the sensitivity of metagenomics detection is low, and the sequencing data volume is high, which requires high detection cost, causing a relatively large financial burden on patients.

[0003] Currently, in terms of reducing human nucleic acid interference, the main method is differential lysis. Based on the characteristic that the cell membrane of human is more fragile than the outer wall of microorganism, before nucleic acid extraction, a mild detergent (such as saponin) is used to lyse human cells, thereby releasing host nucleic acid, and then deoxyribonuclease I is used to degrade host DNA. Although this method can reduce the interference of human nucleic acid to some extent, it has obvious disadvantages. It cannot avoid the loss of mycoplasma, chlamydia and other microorganisms with fragile cell membranes, as well as viruses and other microorganisms without cell structure. At the same time, this method also removes free nucleic acid in the sample, resulting in the loss of possible pathogenic nucleic acid information, thereby affecting the accuracy and comprehensiveness of the detection result.

[0004] In summary, the current infectious disease detection faces many challenges, and an sequencing library construction method for removing human nucleic acid is urgently needed, which can minimize the loss of pathogenic microorganisms during the removal of human nucleic acid, and provide more powerful support for the diagnosis and treatment of infectious diseases. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a sequencing library construction method for removing human nucleic acid and application. The library construction method for removing human nucleic acid comprises library preparation: fragmenting nucleic acid and adding sequencing adapters; according to the type of nucleic acid, the library is divided into a DNA library, an RNA library, and an RNA and DNA co-library; then, the library with added sequencing adapters is used as a template, the library is denatured and annealed with specific human hybridization probes, and then is digested by a heat-stable double-stranded specific nuclease (Duplex-specific nuclease, DSN); during the renaturation process, human nucleic acid with a higher content preferentially anneals with human hybridization probes to form double-stranded DNA, which is degraded by the DSN enzyme; pathogenic microorganisms have a low content and slow renaturation, and thus remain in a single-stranded state and cannot be digested by the DSN enzyme, so that human nucleic acid is effectively removed, and pathogenic microorganism nucleic acid is further enriched through subsequent PCR. The present application also provides a preparation method of human probes, which specifically bind to widely distributed interspersed repeat sequences, rRNA sequences, and gene sequences in the human genome. The method of the present application can effectively reduce the proportion of human nucleic acid in the library and maximize the retention of nucleic acid information of pathogenic microorganisms.

[0006] Specifically, the purpose of the present application is achieved by the following technical solutions:

[0007] <First aspect>

[0008] The technical solutions adopted by the present application are as follows:

[0009] A sequencing library construction method for removing human nucleic acid comprises the following steps:

[0010] S1, library preparation: fragmenting nucleic acid and adding sequencing adapters; according to the type of nucleic acid, the library is divided into a DNA library, an RNA library, and an RNA and DNA co-library;

[0011] S2, human nucleic acid hybridization: adding human hybridization probes to the library constructed in step 1, and performing denaturation and annealing treatment to make the human probes specifically bind to the human sequence library; the human hybridization probes specifically bind to widely distributed interspersed repeat sequences, rRNA sequences, and housekeeping gene sequences in the human genome;

[0012] S3, heat-stable double-stranded specific nuclease digestion: under the condition of keeping the annealing temperature unchanged, adding a heat-stable double-stranded specific nuclease (Duplex-specific nuclease, DSN) to digest the library in step S3, and then adding a termination solution to terminate the reaction after the digestion is completed; the DSN enzyme can specifically degrade double-stranded DNA and can maintain stable activity at a relatively high annealing temperature;

[0013] S4, amplifying the product after digestion, the product after digestion of the heat-stable double-stranded specific nuclease in step S3 is amplified by PCR with the library universal primer to enrich the library and obtain a sequencing library without human nucleic acid; the PCR amplification takes the product after digestion of the heat-stable double-stranded specific nuclease in S3 as a template, and the library universal primer is a sequencing adapter universal sequence, and the sequencing adapter and the library universal primer are selected based on different sequencing platforms.

[0014] In S2, the preparation method of the human hybridization probe comprises the following steps:

[0015] A, RNA extraction: extracting total RNA of Hela cells;

[0016] B, RNA fragmentation: mixing an appropriate amount of total RNA with fragmentation buffer under certain reaction conditions to break the RNA into fragments of about 100-150 bp;

[0017] C, reverse transcription reaction: mixing the fragmented RNA with reverse transcription reaction buffer and reverse transcriptase to perform reverse transcription reaction, synthesize cDNA, and realize the conversion from RNA to cDNA;

[0018] D, RNA digestion to prepare single-stranded cDNA: mixing the cDNA product with digestion reaction buffer and RNase H enzyme to perform reaction and recovery to prepare single-stranded cDNA;

[0019] E, preparation of human hybridization probe: mixing Human Cot-1 DNA with the single-stranded cDNA prepared in step D in equal quality to prepare a probe mixture capable of specifically binding different types of human nucleic acid sequences, and complete the preparation of the human hybridization probe.

[0020] In step B, the fragmentation buffer comprises 50-250 mM Tris-HCl (pH 8-8.5), 2.5 μM-15 μM MgCl2, and 100 mM-400 mM KCl. The fragmentation reaction program is 94℃ for 10 min, 1 cycle.

[0021] In step C, the reverse transcription reaction buffer comprises 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).

[0022] The reverse transcriptase is M-MLV Reverse Transcriptase.

[0023] The reverse transcription reaction procedure is 10 min at 25°C, 15 min at 42°C, and 15 min at 70°C, each for one cycle, to synthesize cDNA.

[0024] In step D, the digestion reaction buffer includes 200 mM Tris-HCl (pH 7.8), 400 mM KCl, 80 mM MgCl2, and 10 mM DTT.

[0025] The digestion reaction procedure is 30 min at 37°C for one cycle to prepare single-stranded cDNA, which is recovered and stored.

[0026] In step E, Human Cot-1 DNA (Thermo Fisher catalog number 15279, derived from human placental genomic DNA, with a maximum length of about 50-300 bp, rich in scattered repeat sequences); Human Cot-1 DNA can bind human DNA sequences in the DNA library, and cDNA products can bind human rRNA sequences and commonly expressed gene sequences (such as housekeeping gene sequences) in the RNA library.

[0027] The human hybridization probe includes DNA single-stranded probes with a length of 100-150 bp and supplemented 50-300 bp Human Cot-1 DNA.

[0028] In step S1, the preparation of the DNA library is performed by physical disruption, enzymatic fragmentation, or Tn5 transposase fragmentation for DNA fragmentation, followed by end repair and A addition, and then ligation of sequencing adapters.

[0029] Generally, the DNA template is mechanically disrupted or enzymatically disrupted, and then the fragmented DNA is end-repaired and A-added, and then the sequencing adapters are ligated to both ends of the DNA fragments by T4 DNA ligase, and finally the library is PCR amplified and enriched by the library universal primer matched with the sequencing adapter; or the DNA template is disrupted by transposase and added with adapters, and then the sequencing adapters are ligated to the fragments by PCR amplification to prepare a complete DNA library.

[0030] In step S1, the preparation of the RNA library is performed by thermal fragmentation of RNA in a divalent metal ion buffer, followed by reverse transcription and double-strand synthesis, and then end repair and A addition, and ligation of sequencing adapters.

[0031] The library construction process of the RNA library generally includes the following steps: fragmentizing the RNA template by thermal cracking, reverse transcribing and double-strand synthesizing the fragmented RNA to generate double-strand DNA, end repairing and adding A to the double-strand DNA, connecting sequencing adapters to both ends of the DNA fragments by T4 DNA ligase, and finally performing PCR amplification and enrichment on the library by using library universal primers matched with the sequencing adapters; or reverse transcribing and double-strand synthesizing the RNA template to generate double-strand DNA, breaking the double-strand DNA by transposase and adding adapters, and connecting the sequencing adapters to the fragments by PCR amplification to prepare a complete DNA library.

[0032] The RNA and DNA co-constructed library in step S1 is prepared by reverse transcribing and double-strand synthesizing the RNA, fragmentizing the RNA and DNA together by enzymatic fragmentation, end repairing and adding A, and then connecting the sequencing adapters.

[0033] The RNA and DNA co-constructed library process uses total nucleic acid containing DNA and RNA as the input template, reverse transcribes and double-strand synthesizes the nucleic acid to generate double-strand DNA, mechanically breaks or enzymatically breaks the double-strand DNA, end repairs and adds A to the fragmented DNA, connects the sequencing adapters to both ends of the DNA fragments by T4 DNA ligase, and finally performs PCR amplification and enrichment on the library by using library universal primers matched with the sequencing adapters; or breaks and adds adapters to the generated double-strand DNA by transposase, and connects the sequencing adapters to the fragments by PCR amplification to prepare a complete DNA library.

[0034] In the above library construction process, the library universal primers are sequencing adapter universal sequences, and the sequencing adapters and library universal primers are selected based on different sequencing platforms.

[0035] Preferably, the library universal primer sequence is as follows:

[0036] The upstream primer is 5'-CAAGCAGAAGACGGCAT-3', such as SEQ ID No. 1.

[0037] The downstream primer is 5'-AATGATACGGCGACCAC-3', such as SEQ ID No. 2.

[0038] In step S2, the human hybridization probe includes a DNA single-strand probe with a length of 100-150 bp and a Human Cot-1 DNA supplement of 50-300 bp.

[0039] Human nucleic acid hybridization involves adding human hybridization probes to a library (DNA library, RNA library, RNA and DNA co-constructed library), followed by denaturation and annealing. The reaction system is prepared in a 200µl PCR tube with the following composition: 5-10µl library, 1-2µl human hybridization probe, and 2-5µl hybridization buffer.

[0040] The amount of the library input is 10~500 ng;

[0041] The amount of the human hybridization probe used is 0.1~5µg.

[0042] The hybridization buffer contains 50–400 mM HEPES (pH 7.5 @ 25°C), 0.5–2 M NaCl, and 0.2–2 mM EDTA.

[0043] The hybridization reaction procedure is as follows:

[0044] Step 1: 98℃ for 3 minutes, 1 cycle;

[0045] Step 2: 68℃ for 1~7h, 1 cycle.

[0046] In step S3, a thermostable double-stranded specific nuclease (DSN enzyme) is used for digestion. DSN enzyme is added to the mixture from step S3 while maintaining the annealing temperature. The reaction is then terminated by adding a stop solution: Specifically, the DSN enzyme reaction buffer is preheated at 68°C, then 1 µL is added to the hybridization product. After mixing, the mixture is quickly returned to a 65-70°C thermostat. Then, 0.5-1.5 µL of DSN enzyme is added, mixed, and the mixture is quickly returned to 65-70°C for digestion for 7-30 min. After the reaction is complete, 3-7 µL of stop solution is added, and the reaction is terminated at 65-70°C for 5-10 min.

[0047] 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;

[0048] The amount of the thermostable double-stranded specific nuclease used is 0.2~1U.

[0049] The stop solution can be an SDS solution or an EDTA solution.

[0050] Step S4: Amplification of the digested product. The product digested by DSN enzyme in the previous step is amplified by PCR using universal library primers to enrich the library. 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 digested product from the previous step, and add water to make up to a total volume of 50-100µl.

[0051] The universal library primer pair is a primer that matches the universal sequence of the library sequencing adapter;

[0052] The amount of the universal library primers used is 5-80 pmol;

[0053] 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).

[0054] The PCR amplification procedure is as follows:

[0055] Step 1: 95℃ for 3 minutes, 1 cycle;

[0056] Step 2: 95℃ for 20s, 60℃ for 15s, 72℃ for 30s, 3~18 cycles;

[0057] Step 3: 72℃ for 3 minutes, 1 cycle.

[0058] Step S4 also includes a step of purifying the PCR amplification.

[0059] The purification steps of the amplification product include:

[0060] 1) Equilibrate the magnetic beads to room temperature;

[0061] 2) Add 45-50µl of magnetic beads to the PCR product, mix thoroughly, and let stand for 5-10 minutes.

[0062] 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.

[0063] 4) Add 500-600µl of 75-80% (v / v) ethanol solution, let stand for 30-60 seconds, and then discard the supernatant;

[0064] 5) Repeat step 4) once;

[0065] 6) Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue;

[0066] 7) Add 20-50µl of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5-10 min, centrifuge the centrifuge tube briefly (usually 2-5 s) and place it on a magnetic rack to stand until the solution is clear (2-5 min), collect the supernatant to obtain the purified amplification product.

[0067] Compared with the prior art, the present invention has the following significant advantages:

[0068] 1. Precise Removal of Human Nucleic Acids while Preserving Pathogen Information. Accurately obtaining nucleic acid information from pathogenic microorganisms is crucial for diagnosis and treatment in the detection of infectious diseases. Existing technologies often suffer from loss of pathogenic microbial nucleic acids during the removal of human nucleic acids. For example, traditional differential lysis methods, while aiming to reduce human nucleic acid interference through specific steps, cannot accurately distinguish between human cells and some vulnerable pathogenic microorganisms. This leads to significant loss of cell membrane-sensitive microorganisms such as mycoplasma and chlamydia, as well as cell-agnostic microorganisms such as viruses, severely impacting the integrity of pathogenic nucleic acid information. This invention utilizes a unique technical approach, employing a thermostable double-stranded specific nuclease (DSN) and specific human hybridization probes, to efficiently and precisely remove human nucleic acids during library construction. During renaturation, human nucleic acids, due to their higher content, preferentially anneal with the human hybridization probe to form double strands, which are then specifically degraded by the DSN enzyme. Pathogenic microbial nucleic acids, due to their lower content and slower renaturation, remain in a single-stranded state, effectively avoiding the risk of digestion. This process not only effectively removes human nucleic acid, but more importantly, it maximizes the preservation of nucleic acid information of all pathogenic microorganisms in the sample, providing a comprehensive and accurate data foundation for subsequent detection and analysis, and greatly improving the reliability of test results and the accuracy of diagnosis.

[0069] 2. Simplified Process and Reduced Costs. Existing detection technologies typically involve complex processes with multiple cumbersome steps and stages. Taking traditional microbial detection methods as an example, each step, from sample collection, culture, isolation to identification, requires strict operation and a long time cycle, consuming significant manpower and resources and easily introducing errors during operation. While metagenomics detection technology has certain advantages in detection range and speed, it requires processing large amounts of sequencing data, placing extremely high demands on equipment performance. It necessitates not only advanced sequencing instruments but also powerful data analysis software and professional technicians for operation and interpretation, significantly increasing detection costs and limiting its application in resource-constrained areas. The method of this invention greatly simplifies the entire detection process. Human nucleic acid is directly removed during library construction, avoiding multiple complex preprocessing steps in traditional methods, reducing sample processing time and potential errors. Simultaneously, since it does not rely on expensive specialized equipment and complex data analysis systems, this invention significantly reduces detection costs while ensuring detection effectiveness. This is of great significance for improving detection efficiency and alleviating the economic burden on medical institutions and patients, especially suitable for resource-scarce areas and large-scale detection needs.

[0070] 3. Convenient operation and easy to promote. Existing technologies often require a high level of professional skill from operators in practice. For example, differential lysis methods require precise control of the concentration of detergent, reaction time, and the amount of deoxyribonuclease I used; slight errors can lead to deviations in experimental results. Metagenomics detection technology involves complex instrument operation and data analysis, requiring professional technicians to undergo extensive training to master it. The method of this invention is simple and convenient to operate, and easy to learn. Each step in the entire process has clear operating guidelines and parameter ranges, allowing even inexperienced operators to quickly get started. Moreover, the reagents and equipment used in this invention are readily available on the market, further reducing the difficulty and barrier to entry. This ease of operation enables the method of this invention to be widely used in medical institutions and research units at different levels, providing strong technical support for promoting the rapid diagnosis and control of infectious diseases, and has extremely high promotional value.

[0071] 4. Because the method of this invention can completely preserve 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 multiple fields. In clinical diagnosis, it can quickly and accurately detect pathogenic microorganisms, providing important basis for doctors to formulate personalized treatment plans, helping to improve treatment effects and shorten the patient's recovery period. In the field of public health, it can be used for large-scale disease surveillance and control, timely detection of potential pathogens, and implementation of effective prevention and control measures to prevent the 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, promoting the continuous in-depth development of research in related fields. Attached Figure Description

[0072] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0073] Figure 1 This is a schematic diagram of the process of the present invention;

[0074] Figure 2 This is a distribution diagram of the document fragments. Detailed Implementation

[0075] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0076] Figure 1 This is a schematic diagram of the process of the present invention.

[0077] Example 1: Human Total RNA Extraction

[0078] Take 10 HeLa cells 7 Total RNA was extracted from cells using the Tiangen Biotech Total RNA Extraction Kit (DP451).

[0079] A. Centrifuge HeLa cells at 300×g for 5 min, discard the supernatant, and retain the cell pellet. Centrifuge at low speed to precipitate the cells from the culture medium, which facilitates subsequent processing and removes impurities such as serum and culture medium components that may interfere with RNA extraction.

[0080] B. Add 600µl of lysis buffer RLA and 10µl of proteinase K to the cell pellet and vortex to mix. The role of lysis buffer RLA is to disrupt the cell membrane and nuclear membrane structure and release intracellular RNA. 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 and ensure complete lysis reaction.

[0081] C. Centrifuge at 12000 rpm for 5 min, transfer the supernatant to a new centrifuge tube, add 600 µl of 70 v / v ethanol, mix well, and transfer all solutions, including the precipitate, to the RNA adsorption column CR4; high-speed centrifugation precipitates cell debris and insoluble matter, and the supernatant is used to ensure the purity of the extracted RNA. 70% ethanol is used to precipitate RNA, reduce its solubility, and facilitate subsequent capture in the adsorption column.

[0082] D. Centrifuge at 12000 rpm for 30 seconds, discard the filtrate, and return the adsorption column to the collection tube; this operation allows the RNA-containing solution to pass through the adsorption column, the RNA binds to the medium inside the adsorption column, and the filtrate containing unbound impurities is discarded.

[0083] E. Add 80µl of DNase I enzyme working solution to the center of the adsorption column and let it stand at room temperature for 15 min; DNase I enzyme specifically degrades DNA, removing any possible genomic DNA contamination in the sample and ensuring that the extracted RNA is free of DNA impurities;

[0084] F. Add 350µl of protein removal 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 protein removal solution removes residual proteins and enzymes from the lysis buffer, and the filtrate containing protein impurities is removed by centrifugation to further purify the RNA.

[0085] G. Add 500µl of wash buffer RW to the center of the adsorption column, let it stand at room temperature for 2 min, then centrifuge at 12000 rpm for 30 s, discard the filtrate, and put the adsorption column back into the collection tube; the wash buffer removes residual salt ions, buffer components and a small amount of unbound impurities from the adsorption column, and further purifies the RNA.

[0086] H. Repeat step G;

[0087] 1. Centrifuge at 12000 rpm for 30 seconds, discard the filtrate, and allow to air dry at room temperature for 2 minutes. Remove residual rinsing solution from the adsorption column by high-speed centrifugation to prevent it from affecting subsequent RNA elution and quality. Air dry at room temperature to remove moisture, avoid diluting the eluted RNA or introducing impurities, and ensure RNA purity and stability.

[0088] J. Place the adsorption column into a new centrifuge tube, add 30µl of enzyme-free water to the center of the adsorption column, let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to obtain the RNA solution.

[0089] Example 2 Preparation of human hybridization probe

[0090] (1) RNA fragmentation

[0091] Prepare the reaction system according to Table 1, and break the RNA into fragments of approximately 100-150 bp:

[0092] Table 1

[0093]

[0094] Fragmentation reaction program: 94℃ for 10 min, 1 cycle;

[0095] (2) Reverse transcription reaction

[0096] Prepare the reaction system according to Table 2, and perform reverse transcription of total RNA to synthesize cDNA:

[0097] Table 2

[0098]

[0099] The reverse transcription procedure is as follows:

[0100] Step 1: 25℃ for 10 minutes, 1 cycle;

[0101] Step 2: 42℃ for 15 minutes, 1 cycle;

[0102] Step 3: 70℃ for 15 minutes, 1 cycle;

[0103] (3) RNA digestion reaction

[0104] Prepare the reaction system according to Table 3, digest RNA with RNase H, and prepare single-stranded cDNA:

[0105] Table 3

[0106]

[0107] The digestion reaction procedure is as follows:

[0108] Step 1: 37℃ for 30 minutes, 1 cycle;

[0109] (4) Recovery of single-stranded cDNA products (using Tiangen Biotech's common DNA product purification kit (catalog number DP204)): cDNA was recovered using the centrifugal adsorption column method.

[0110] A. Place the adsorption column into the collection tube, then add 500µl of equilibration solution BL, centrifuge at 12000rpm for 1min, then discard the liquid in the collection tube, and put the adsorption column back into the collection tube for later use.

[0111] B. Add 150 µl of binding solution PB to 30 µl of product and mix thoroughly.

[0112] C. Transfer the mixture from the previous step to the adsorption column, let it stand at room temperature for 5 minutes, then centrifuge at 12000 rpm for 30 seconds, discard the liquid in the collection tube, and put the adsorption column back into the collection tube.

[0113] D. Add 600µl of washing buffer PW to the adsorption column, centrifuge at 12000rpm for 30s, discard the liquid in the collection tube, and put the adsorption column back into the collection tube.

[0114] E. Repeat step D;

[0115] F. Centrifuge at 12000 rpm for 2 min to remove the rinsing solution, then let it stand at room temperature to dry for 3 min;

[0116] G. Remove the adsorption column and place it in a clean centrifuge tube. Add 20 µl of elution buffer EB to the center of the silica matrix adsorption membrane, let it stand at room temperature for 5 min, and then centrifuge at 12000 rpm for 2 min to collect the DNA liquid.

[0117] (5) Product quantification: The concentration of ssDNA was detected using qubit 2.0 and diluted to 100 ng / uL for later use.

[0118] Purity testing: The absorbance (OD value) of the product at 260nm and 280nm was measured using NanoDrop. The OD260 / OD280 ratio should be between 1.7 and 1.9.

[0119] Fragment detection: Fragment detection of the product was performed using an Agilent 2100 instrument, and the fragments were distributed between 100 and 150 bp.

[0120] (6) Preparation of human hybridization probes

[0121] Human Cot-1 DNA (Thermo Fisher Scientific catalog number 15279) is derived from genomic DNA extracted from human placenta. Its maximum length ranges from approximately 50 to 300 bp, and it is rich in interstitial repeats (IRS) such as SINE (small interstitial repeats, such as Alu sequences) and LINE (large interstitial repeats, such as L1 sequences), which are widely distributed in the human genome.

[0122] Human Cot-1 DNA and the cDNA product prepared in step (5) are mixed in equal mass 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 commonly expressed gene sequences (such as housekeeping gene sequences) in the RNA library.

[0123] Fragment detection: Fragment detection of the product was performed using an Agilent 2100 instrument, and the fragments were distributed between 50 and 300 bp.

[0124] Example 3: Removal of human nucleic acid

[0125] (1) Preparation of reference materials: ZymoBIOMICS™ microbial community standards (Zymo, catalog number D6300) and H1N1 influenza pseudovirus (H1N1 nucleic acid reference material, Jingliang Biotechnology, catalog number GW-IAF110) were added to human HeLa cell lines of 2×10⁵ cells / mL at different concentration gradients. The matrix solution was PBS buffer, as shown in Table 4 below:

[0126] Table 4

[0127]

[0128] (1) Total nucleic acid extraction: The reference sample was co-extracted using a DNA and RNA co-extraction kit (Novozymes, magnetic bead method for pathogen DNA / RNA co-extraction kit, catalog number RM601) to obtain total nucleic acid (RNA & DNA).

[0129] (2) DNA and RNA co-construction library

[0130] The extracted nucleic acids contained DNA and RNA. A co-construction library scheme was adopted, and three libraries were constructed in parallel for each reference sample.

[0131] A. Nucleic acid denaturation

[0132] Prepare the reaction system according to Table 5, and mix thoroughly by gently pipetting 10 times:

[0133] Table 5

[0134]

[0135] The denaturation reaction procedure is as follows:

[0136] Step 1: 70℃ for 5 minutes, 1 cycle;

[0137] Step 2: Immediately in an ice bath for 3 minutes; obtain denatured total nucleic acids (RNA & DNA).

[0138] B. Reverse transcription reaction

[0139] Prepare the first-strand cDNA synthesis reaction system according to Table 6:

[0140] Table 6

[0141]

[0142] The reverse transcription procedure is as follows:

[0143] Step 1: 25℃ for 5 minutes, 1 cycle;

[0144] Step 2: 42℃ for 15 minutes, 1 cycle;

[0145] Step 3: 85℃ for 5 minutes, 1 cycle, to obtain the first-strand cDNA.

[0146] C. Double-stranded DNA synthesis

[0147] Prepare the double-stranded cDNA synthesis reaction system according to the table below:

[0148] Table 7

[0149]

[0150] The procedure for the two-chain synthesis reaction is as follows:

[0151] Step 1: 16℃ for 30 minutes, 1 cycle.

[0152] D. Fragmentation and end-point repair plus A

[0153] Table 8: Preparation of the reaction system:

[0154] Table 8

[0155]

[0156] The fragmentation and terminal repair plus A reaction procedure is as follows:

[0157] Step 1: 37℃ for 20 minutes, 1 cycle;

[0158] Step 2: 65℃ for 30 minutes, 1 cycle;

[0159] E. Connector Connection

[0160] Prepare the reaction system according to Table 9:

[0161] Table 9

[0162]

[0163] The connector connection reaction procedure is as follows:

[0164] Step 1: 20℃ for 15 minutes, 1 cycle.

[0165] F. Purification of ligation products

[0166] F1. DNA purification magnetic beads equilibrate to room temperature;

[0167] F2. Add 60µl of DNA purification magnetic beads to the adapter ligation product from step E, mix thoroughly, and let stand for 5 minutes.

[0168] 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.

[0169] F4. Add 500µl of freshly prepared 80% (v / v) ethanol solution, let stand for 30s, and then discard the supernatant.

[0170] F5, repeat F4;

[0171] F6. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0172] F7. Add 22 µl of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, centrifuge the centrifuge tube for 3 seconds and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µl of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0173] (3) Human probe hybridization

[0174] Prepare the reaction system according to Table 10:

[0175] Table 10

[0176]

[0177] The hybridization reaction procedure is as follows:

[0178] Step 1: 98℃ for 3 minutes, 1 cycle;

[0179] Step 2: 68℃ for 1 hour, 1 cycle;

[0180] Step 3: Hold at 68°C for 1 cycle.

[0181] (4) DSN enzyme digestion

[0182] Preheat the DSN enzyme reaction buffer at 68°C, then add 1µL to the hybridization product in step (3), mix well, and quickly return it to the 68°C thermostat. Add 1µL of DSN enzyme, mix well, and quickly return it to 68°C for digestion for 10 min. After the reaction is complete, add 5µL of stop solution and stop the reaction at 68°C for 5 min.

[0183] (5) Post-digestion library amplification

[0184] Prepare the reaction system according to Table 11:

[0185] Table 11

[0186]

[0187] The PCR amplification procedure is as follows:

[0188] Step 1: 95℃ for 3 minutes, 1 cycle;

[0189] Step 2: 95℃ for 20s, 60℃ for 15s, 72℃ for 30s, 18 cycles;

[0190] Step 3: 72℃ for 3 minutes, 1 cycle.

[0191] (6) Purification of amplification products

[0192] A. Equilibrate the DNA purification magnetic beads to room temperature;

[0193] B. Take 45µl of DNA purification magnetic beads and add them to the product of step (5). Mix thoroughly and let stand for 5 minutes.

[0194] C. 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.

[0195] D. Add 500µl of freshly prepared 80% (v / v) ethanol solution, let stand for 30s, and then discard the supernatant;

[0196] E. Repeat step D;

[0197] F. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0198] G. Add 22 µl of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µl of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0199] (7) Document quality control:

[0200] Qseq100 was used to detect the size of the products, and qubit 2.0 was used for library quality control to determine the concentration. The distribution of the library fragments is as follows: Figure 2 As shown.

[0201] (8) Sequencing

[0202] All products were standardized and mixed in equal amounts. The resulting libraries were then sequenced in parallel using the Illumina NextSeq550Dx sequencing platform and the SE75 sequencing type.

[0203] (9) Data Analysis

[0204] Low-quality sequences and adapter primer sequences were filtered using Fastp software. The proportion of reads of hg19 in the human reference genome was counted and removed using bowtie2. Species annotation and abundance statistics were performed using Kraken2.

[0205] Analysis steps A through E include filtering adapter primer sequences, host proportion statistics and host removal, species annotation and abundance statistics.

[0206] Results: After constructing DNA and RNA libraries from the three reference samples, those without human removal and directly sequenced were used as the control group (S1_control, S2_control, S3_control), while those with human removal using the method of this invention were used as the experimental group (S1_HD, S2_HD, S3_HD). The proportions of human sequences in the control group were 98.51%, 99.04%, and 99.38%, respectively, while the proportions in the experimental group were 76.69%, 82.96%, and 85.54%, respectively. After removing human sequences using this method, the proportion of human sequences decreased by 13%–21%, as shown in Table 12.

[0207] Table 12

[0208]

[0209] The RPM values ​​of the species were statistically analyzed.

[0210] Where: RPM value = number of specific reads / number of valid reads (per million) The number of valid reads after filtering low-quality sequences and removing adapter primer sequences is obtained by using Fastp software. Kraken2 performs species annotation and abundance analysis and outputs the output file. The optimal count for the corresponding species is the number of specific reads.

[0211] All species were detected in all three reference samples in 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 were approximately 2 to 20 times higher, as shown in Table 13.

[0212] Table 13

[0213]

[0214] Conclusion: The method described above is effective in constructing host-free libraries. The library construction process is simple and can effectively reduce the proportion of host nucleic acids and improve the detection rate of microorganisms.

[0215] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can 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 with human nucleic acid removed, characterized in that, Includes the following steps: S1. Library preparation: Nucleic acid fragmentation and sequencing adapter addition are performed, and the libraries are classified according to the type of nucleic acid, including DNA libraries, RNA libraries, and RNA and DNA co-constructed libraries. S2, Human nucleic acid hybridization: The human hybridization probe is added to the library constructed in step S1 and denaturation annealing is performed to make the human probe specifically bind to the human sequence library; S3. Digestion with thermostable double-stranded specific nuclease: Under the condition of keeping the annealing temperature constant, add thermostable double-stranded specific nuclease for digestion. After digestion is completed, add a stop solution to terminate the reaction; the stop solution is SDS solution or EDTA solution. S4. Amplification of digested products: The product digested by the thermostable double-stranded nuclease in step S3 was amplified by PCR using universal library primers to enrich the library and 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 universal primers for the library are universal sequences of sequencing adapters. The matching sequencing adapters and universal primers for the library are selected based on different sequencing platforms. In S2, the preparation method of the human hybridization probe includes the following steps: A. RNA extraction: Extract total RNA from HeLa cells; B. RNA fragmentation: Mix an appropriate amount of total RNA with fragmentation buffer, and under certain reaction conditions, break the RNA into fragments of about 100-150bp. C. Reverse transcription reaction: Using fragmented RNA as a substrate, it is mixed with reverse transcription reaction buffer and reverse transcriptase to carry out reverse transcription reaction, synthesize cDNA, and realize the conversion from RNA to cDNA; D. RNA digestion to prepare single-stranded cDNA: The cDNA product is mixed with digestion reaction buffer and RNase H enzyme, reacted, and recovered to prepare single-stranded cDNA; E. Preparation of human hybridization probes: Human Cot-1 DNA and single-stranded cDNA prepared in step D are mixed in equal mass to prepare a probe mixture that can specifically bind to different types of human nucleic acid sequences, thus completing the preparation of human hybridization probes; In step S1, the DNA library is prepared by physical fragmentation, fragmentation enzyme fragmentation, or Tn5 transposase fragmentation to fragment the DNA, followed by end repair and A addition, and then ligation of sequencing adapters. The preparation of the RNA library in step S1 involves thermally lysing fragmented RNA in a divalent metal ion buffer, followed by reverse transcription and two-strand synthesis, then end repair and A addition, and finally ligation of sequencing adapters. The preparation of the RNA and DNA co-constructed library in step S1 involves reverse transcription and two-strand synthesis of RNA, followed by fragmentation and enzymatic breaking of DNA, end repair and A addition, and then ligation of sequencing adapters.

2. The method for constructing a sequencing library with human nucleic acid removed according to claim 1, characterized in that, In step S2, the amount of the library constructed in step S1 is 10~500 ng, and the amount of the human hybridization probe is 0.1~5 µg.

3. The method for constructing a sequencing library with human nucleic acid removed according to claim 1, characterized in that, In step S3, the amount of the thermostable double-stranded specific nuclease used is 0.2~1 U.

4. The method for constructing a sequencing library with human nucleic acid removed according to claim 1, characterized in that, Step S4 also includes a step of purifying the amplification product.

5. The method for constructing a sequencing library with human nucleic acid removed according to claim 4, characterized in that, The purification steps of the amplification product include: 1) Equilibrate the magnetic beads to room temperature; 2) Add 45-50µl of magnetic beads to the PCR product, mix well, and let stand for 5-10 minutes. 3) Let stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed, then discard the supernatant; 4) Add 500µl of ethanol solution, let stand for 30-60 seconds, and discard the supernatant; 5) Repeat step 4) once; 6) Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to dry the magnetic beads for 5-10 minutes until there is no ethanol residue; 7) Add 22-50µl of sterile ultrapure water to elute, mix thoroughly by pipetting, let stand at room temperature for 5-10 minutes, centrifuge the centrifuge tube briefly and place it on a magnetic rack to stand until the solution is clear, then collect the supernatant.

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