A method for constructing sequencing libraries based on closed primers and dehumanized nucleic acids and its application.

Abstract

This invention discloses a method for constructing sequencing libraries based on blocking primers to remove human nucleic acids, and its application. The blocking primers include blocking primers for binding human repetitive sequences, blocking primers for binding human rRNA sequences, and blocking primers for binding human housekeeping gene sequences. These blocking primers can specifically bind to widely distributed scattered repetitive sequences, rRNA sequences, and housekeeping gene sequences in the human genome. The construction method includes the following steps: constructing a DNA library or an RNA library, or a co-constructed RNA and DNA library; using a library with added sequencing adapters as a template; and performing PCR amplification using adapter primers and blocking primers. The blocking primers can specifically bind to human nucleic acids through annealing and prevent modification by DNA polymerase extension, thus preventing the enrichment of human nucleic acid libraries through PCR amplification, while allowing normal amplification of pathogenic microorganism nucleic acids. This achieves the goal of reducing the proportion of human nucleic acids and provides guidance for pathogen detection.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology and relates to a method for constructing sequencing libraries based on closed primers and dehumanized nucleic acids, as well as its application. Background Technology

[0002] Infectious diseases are one of the leading causes of death worldwide. Rapid identification of pathogens is fundamental to effective infection control. Traditional microbial testing, due to its numerous steps and complex procedures, significantly limits its clinical application in my country. Currently, there are over 30 million meningitis patients globally each year. Due to the lack of effective testing methods for most pathogens, while the presence or absence of infection is easily diagnosed clinically, the specific type of infection cannot be determined, resulting in over 90% of patients not receiving a definitive diagnosis and accurate treatment. Metagenomics testing, with its advantages of rapid identification, unbiasedness, broad coverage, no culture required, and no pre-setting required, plays a crucial role in detecting complex, critical, or special infections. However, metagenomic sequencing still faces many challenges, particularly the high human background in samples leading to a low proportion of pathogens in the sequencing data, resulting in low data utilization and affecting detection sensitivity. Furthermore, the high data volume requirements result in high metagenomic costs, limiting its widespread clinical application.

[0003] Currently, methods for removing human nucleic acids from samples mainly include differential lysis, antimethylated DNA magnetic beads, microporous membrane filtration, and differential centrifugation. Differential lysis is the most commonly used method. Before nucleic acid extraction, a mild detergent (such as saponin) is used to lyse the host cell membrane, which is more fragile than the microbial outer wall, releasing the host nucleic acid. Then, deoxyribonuclease I is used to degrade the host DNA. However, this method cannot avoid the loss of microorganisms, especially mycoplasma, chlamydia, and viruses, which are significantly affected. Furthermore, this method can only remove host DNA, not RNA, and also loses free pathogen nucleic acids. Antimethylated DNA magnetic beads are based on the phenomenon that human DNA has a high degree of methylation while most microbial genomes lack methylated DNA, and are used to specifically remove human DNA. However, this method is costly and removes a relatively small proportion of host DNA. Microporous membrane filtration and differential centrifugation utilize the physical properties of human cells and microbial cells for separation, but the separation effect is generally poor and the loss is significant, so their application is limited. Summary of the Invention

[0004] To overcome the aforementioned technical problems, this invention proposes a method and application for constructing sequencing libraries based on human-derived nucleic acid removal using blocking primers. First, this invention provides blocking primers for human-derived nucleic acid removal, including blocking primers for binding human repetitive sequences and blocking primers for binding human rRNA sequences and human housekeeping gene sequences. The blocking primers for binding human repetitive sequences are obtained by fragmenting Human Cot-1 DNA and modifying it with ddNTPs. The sequences of the blocking primers for binding human rRNA sequences and human housekeeping gene sequences are shown in SEQ ID NO. 1-464, with a blocking group at their 3' end. The blocking primers of this invention can specifically bind to widely distributed scattered repetitive sequences, rRNA sequences, and housekeeping gene sequences in the human genome. Secondly, this invention provides a method for constructing a sequencing library dehumanized based on blocking primers. This method includes: constructing a DNA or RNA library, or a co-construction of an RNA and DNA library; using the library with added sequencing adapters as a template; and performing PCR amplification using adapter primers and blocking primers. The blocking primers specifically bind to human nucleic acids through annealing, and the 3' end of the blocking primers is modified with Spacer C3 or ddNTP to inhibit DNA polymerase extension, preventing the enrichment of human nucleic acid libraries by PCR amplification, while allowing normal amplification of pathogenic microorganism nucleic acids, thus reducing the proportion of human nucleic acids. Using this method, the proportion of human nucleic acids in the library can be effectively reduced, while maximizing the retention of pathogenic microorganism nucleic acid information, providing guidance for pathogen detection.

[0005] The technical solution adopted in this invention is as follows:

[0006] In a first aspect, the present invention provides a blocking primer for dehumanized nucleic acid, the blocking primer comprising a blocking primer for binding human repetitive sequences, and a blocking primer for binding human rRNA sequences and human housekeeping gene sequences; the blocking primer for binding human repetitive sequences is prepared by the following steps:

[0007] A1. Fragment the Human Cot-1 DNA and purify and recover it;

[0008] A2. Modify the purified and recovered fragmented Human Cot-1 DNA with ddNTPs and then recover it.

[0009] In one embodiment of the present invention, in step A1, the Human Cot-1 DNA is Thermo Fisher Scientific 15279.

[0010] As one embodiment of the present invention, in step A1, the fragmentation method includes enzymatic fragmentation.

[0011] Preferably, the fragmented fragment distribution is less than 100bp.

[0012] Furthermore, the fragmentation enzyme used in the enzymatic fragmentation is a nuclease. In some embodiments, the fragmentation enzyme is... dsDNA Fragmentase.

[0013] In one embodiment of the present invention, in step A1, the purification and recovery are performed using DNA purification magnetic beads.

[0014] Furthermore, the purification and recovery includes the following steps:

[0015] B1. Equilibrate DNA purification magnetic beads (to room temperature);

[0016] B2. Take the purified DNA beads after equilibration and add them to the fragmented Human Cot-1 DNA. Mix well and let the reaction stand.

[0017] B3. Transfer the reaction solution to a centrifuge tube and place it on a magnetic rack. After standing, discard the supernatant (using a pipette).

[0018] B4. Add ethanol solution, let stand, and discard the supernatant;

[0019] B5. Repeat step B4;

[0020] B6. Keep the centrifuge tubes on the magnetic rack and dry the magnetic beads;

[0021] B7. Add sterile ultrapure water to elute the magnetic beads and let them stand. After centrifugation, place them on a magnetic rack to stand and obtain the supernatant, which is the purified and recovered fragmented Human Cot-1 DNA.

[0022] In some embodiments, in step B2, the amount of DNA purification magnetic beads after equilibration is 45-60 μl, and the reaction time is 3-5 min; in step B3, the solution is allowed to stand for 1-2 min until it is clear and the magnetic beads are completely adsorbed; in step B4, the volume ratio of ethanol solution is 70-80% (v / v), the amount is 400-500 μl, and the solution is allowed to stand for 20-30 s; in step B5, step B4 is repeated once; in step B5, the magnetic beads are air-dried for 5-10 min until there is no ethanol residue; in step B7, 22-55 μl of sterile ultrapure water is added for elution, and the mixture is gently blown and stirred thoroughly with a pipette. The solution is placed at room temperature for 3-5 min, the centrifuge tube is briefly centrifuged and placed on a magnetic rack to stand until the solution is clear (about 2 min). The supernatant is carefully transferred to a new centrifuge tube, taking care not to touch the magnetic beads.

[0023] As one embodiment of the present invention, in step A2, the enzyme used for ddNTP modification includes terminal transferase.

[0024] As one embodiment of the present invention, in step A2, the ddNTP modification reaction program includes: 37℃ for 1.5h, 1 cycle; 70℃ for 10min, 1 cycle.

[0025] As one embodiment of the present invention, in step A2, the recovery method includes centrifugal adsorption column method.

[0026] As one embodiment of the present invention, the blocking primer for binding the human rRNA sequence and the human housekeeping gene sequence includes the sequence shown in SEQ ID NO.1-464; wherein the 3' end of the sequence has a blocking group.

[0027] Furthermore, the blocking group includes any one of Spacer C3 and ddNTP. The blocking group is a modifying group that prevents DNA polymerase extension reaction.

[0028] Furthermore, during primer synthesis, the blocking primer is modified with Spacer C3 at its 3' end;

[0029] Alternatively, after primer synthesis, the blocking primer is modified at its 3' end with a ddNTP, and the enzyme used for modification includes a terminal transferase.

[0030] This invention designs blocking primers for binding human repetitive sequences, targeting scattered repetitive sequences widely distributed in the human genome; and designs blocking primers for binding human rRNA sequences and human housekeeping gene sequences, targeting 5.8S rRNA, 18S rRNA, 28S rRNA, and human housekeeping genes such as GAPDH, β-actin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, NONO, ARHGDIA, RPL32, RPS18, and HSPCB. The blocking primers are 20–40 bp in length and annealed at 65–72 °C. The blocking primers of this invention can specifically bind to scattered repetitive sequences, rRNA sequences, and housekeeping gene sequences widely distributed in the human genome.

[0031] Secondly, the present invention provides a method for constructing a sequencing library of dehumanized nucleic acid based on the aforementioned closed primers, comprising the following steps:

[0032] S1. Fragment the sample nucleic acid, repair the ends of the nucleic acid fragments and add A, and ligate sequencing adapters (at both ends of the nucleic acid fragments) to obtain a library; the sample nucleic acid contains human nucleic acid;

[0033] S2. Using the library as a template, perform PCR amplification with adapter primers and blocking primers;

[0034] S3. The PCR amplification product is purified and recovered to obtain the sequencing library containing the dehumanized nucleic acid.

[0035] As one embodiment of the present invention, in step S1, the fragmentation method includes any one of mechanical fragmentation, enzymatic fragmentation, and thermal lysis.

[0036] Furthermore, the mechanical fragmentation includes ultrasonic fragmentation. In some embodiments, an ultrasonic fragmenter is used for DNA fragmentation.

[0037] Furthermore, the solution used for thermal lysis is a divalent metal ion buffer. Thermal lysis is used for RNA fragmentation.

[0038] Furthermore, the fragmentation enzyme used in the enzymatic fragmentation method is an endonuclease. Enzymatic fragmentation is used for DNA fragmentation.

[0039] As one embodiment of the present invention, step S1 further includes: using Tn5 transposase to fragment the sample nucleic acid and ligate sequencing adapters to obtain a library. That is, Tn5 transposase can complete fragmentation and sequencing adapter ligation in one step.

[0040] In one embodiment of the present invention, in step S1, the ligation is performed using T4 DNA ligase.

[0041] In one embodiment of the present invention, in step S1, the library includes any one of a DNA library, an RNA library, and a co-constructed RNA and DNA library. The library is a metagenomic or metagenomic library.

[0042] Furthermore, the method for preparing the DNA library includes: fragmenting the sample DNA, repairing the ends of the DNA fragments and adding A, and ligating sequencing adapters to obtain the DNA library;

[0043] Alternatively, Tn5 transposase can be used to fragment the sample DNA and ligate sequencing adapters to obtain a DNA library;

[0044] The method for preparing the RNA library includes: fragmenting the sample RNA, reverse transcribing the RNA fragments and synthesizing them into double-stranded DNA, then performing end repair and adding A, and ligating sequencing adapters to obtain the RNA library;

[0045] Alternatively, the sample RNA can be reverse transcribed and synthesized into double-stranded DNA, and then the double-stranded DNA can be fragmented and ligated with sequencing adapters using Tn5 transposase to obtain an RNA library.

[0046] The method for preparing the RNA and DNA co-constructed library includes: reverse transcription of sample RNA and two-strand synthesis to generate double-stranded DNA, fragmentation of sample DNA together, end repair and A addition of DNA fragments, and ligation of sequencing adapters to obtain the RNA and DNA co-constructed library.

[0047] Alternatively, the sample RNA can be reverse transcribed and synthesized into double-stranded DNA. Then, Tn5 transposase can be used to fragment the double-stranded DNA and sample DNA and ligate sequencing adapters to obtain an RNA-DNA co-constructed library.

[0048] Furthermore, the sample RNA is denatured sample RNA.

[0049] As one embodiment of the present invention, step S1 further includes: performing PCR amplification on the library using adapter primers (to increase the concentration).

[0050] The adapter primers in this invention are universal library primers that match the sequencing adapters.

[0051] In one embodiment of the present invention, in step S2, the concentration of the adapter primer is 1-20 pmol; the concentration of the blocking primer is 2-100 pmol.

[0052] In one embodiment of the present invention, in step S2, the molar ratio of the blocking primer to the linker primer is 1 to 10:1.

[0053] In one embodiment of the present invention, in step S2, a 50 μl reaction system is prepared in a 200 μl PCR tube: 25 μl nucleic acid amplification reaction solution, 4 μl blocking primer set, 2 μl universal library primer pair, and 19 μL library. The amount of library added is 0.1–100 ng.

[0054] Furthermore, the nucleic acid amplification reaction solution includes 2×PCR reaction buffer, dNTPs, and DNA polymerase.

[0055] In one embodiment of the present invention, the PCR amplification procedure in step S2 is as follows:

[0056] Step 1: 95℃ for 3 minutes, 1 cycle;

[0057] Step 2: 95℃ for 20s, 60℃ for 15s, 72℃ for 30s, repeat 3 to 15 times;

[0058] Step 3: 72℃ for 3 minutes, 1 cycle.

[0059] In one embodiment of the present invention, the purification and recovery are performed using DNA purification magnetic beads.

[0060] The sequencing library in this invention can selectively inhibit the enrichment of human nucleic acid libraries, thereby increasing the proportion of pathogenic microorganism libraries.

[0061] Thirdly, the present invention provides a sequencing library obtained by the construction method described above.

[0062] Fourthly, the present invention provides an application of the sequencing library in the preparation of products for detecting pathogenic microorganisms.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] 1. The present invention provides blocking primers that can specifically bind to scattered repetitive sequences, rRNA sequences and housekeeping gene sequences widely distributed in the human genome, and prevent the modification of DNA polymerase extension reaction, so that human nucleic acid libraries cannot be enriched by PCR amplification, while pathogenic microbial nucleic acids can be amplified normally, thereby achieving the purpose of reducing the proportion of human nucleic acids.

[0065] 2. This invention uses blocking primers to construct sequencing libraries. The method can remove human nucleic acid without losing pathogenic microbial nucleic acid during library construction, maximizing the retention of all pathogenic microbial nucleic acid information in the sample. The process is simple, low-cost, and easy to operate, and has high reference value, making it suitable for widespread application. Attached Figure Description

[0066] 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:

[0067] Figure 1 This is a flowchart of the sequencing library construction process for this invention;

[0068] Figure 2 This refers to the distribution of library fragments in Example 3. Detailed Implementation

[0069] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0070] Example 1: Preparation of blocking primers based on Human Cot-1 DNA

[0071] Human Cot-1 DNA is derived from genomic DNA extracted from human placenta, with a maximum length ranging from approximately 50 to 300 bp. Since the human genome is rich in interstitial repetitive sequences (IRS) such as SINEs (small interstitial repetitive sequences, e.g., Alu sequences) and LINEs (large interstitial repetitive sequences, e.g., L1 sequences), Human Cot-1 DNA can be prepared from human placental DNA through shearing, denaturation, and re-annealing under conditions rich in these repetitive sequences. The Human Cot-1 DNA (purchased from Thermo Fisher Scientific, catalog number 15279 in this example) was further fragmented to a fragment size of less than 100 bp. Then, ddNTPs were modified at the ends using terminal transferase (TdT) to prepare blocking primers. The experimental procedure is as follows:

[0072] (1) Fragmentation of Human Cot-1 DNA

[0073] Take 3 μg of Human Cot-1 DNA for use dsDNA fragmentation was performed using dsDNA Fragmentase (catalog number M0348L), and the reaction system was prepared as shown in Table 1 below:

[0074] Table 1

[0075] reagent components Volume (μL) Human Cot-1 DNA (1 μg / μL) 3 Sterile Water 11 10×Fragmentase Reaction Buffer v2 2 <![CDATA[200mM MgCl2]]> 2 dsDNA Fragmentase 2 total 20

[0076] The fragmentation reaction procedure is as follows:

[0077] Step 1: 37℃ for 10 minutes, 1 cycle;

[0078] (2) Purification and recovery of fragmented Human Cot-1 DNA

[0079] A. Equilibrate the DNA purification magnetic beads to room temperature;

[0080] B. Add 60 μl of DNA purification magnetic beads to the fragmentation product from the previous step, mix thoroughly, and let stand for 5 min.

[0081] 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.

[0082] D. Add 500 μl of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant;

[0083] E. Repeat the previous step once;

[0084] 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.

[0085] G. Add 55 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 52.5 μl of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0086] (3) ddNTP modification

[0087] Thaw the ddNTP stock solution on ice, vortex to mix, and dilute with water to prepare a 1 mM working solution in a new EP tube. Use the NEB terminal transferase kit (catalog number M0315L) to prepare the reaction system as shown in Table 2 below:

[0088] Table 2

[0089]

[0090] The reaction procedure for ddNTP modification is as follows:

[0091] Step 1: 37℃ for 1.5 hours, 1 cycle;

[0092] Step 2: 70℃ for 10 minutes, 1 cycle;

[0093] (4) Recovery of modified products: The modified products are recovered by centrifugal adsorption column method. The recovered products can be used as blocking primers for binding human repetitive sequences.

[0094] A. Place the adsorption column into the collection tube, then add 500 μl of equilibration solution, centrifuge at 12000 rpm for 1 min, then discard the liquid in the collection tube, and put the adsorption column back into the collection tube for later use.

[0095] B. Add 375 μl of binding solution to 75 μl of product and mix thoroughly;

[0096] 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.

[0097] D. Add 600 μl of washing solution to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the liquid in the collection tube, and put the adsorption column back into the collection tube.

[0098] E. Repeat the previous step once;

[0099] F. Centrifuge at 12000 rpm for 2 min to remove as much of the washing solution as possible, then let it stand at room temperature to dry for 3 min;

[0100] G. Remove the adsorption column and place it in a clean centrifuge tube. Add 20 μl of elution buffer to the middle of the membrane, let it stand at room temperature for 5 min, and then centrifuge at 12000 rpm for 2 min to collect the DNA liquid.

[0101] (5) Product quantification: Qseq100 was used to detect the size of the product, and qubit 2.0 was used to detect the concentration. Then, the molar concentration was calculated based on the fragment size for later use.

[0102] Example 2: Design and synthesis of blocking primers

[0103] (1) Design of blocking primers: Primers were designed for human housekeeping genes 5.8S rRNA, 18S rRNA, 28S rRNA, GAPDH, β-actin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, NONO, ARHGDIA, RPL32, RPS18, HSPCB, etc. The primer length was 20-40 bp, and the primer annealing temperature was 65-72℃. The primer sequences are shown in Table 3 (SEQ ID NO.1-464).

[0104] (2) Synthesis of blocking primers: The blocking primers can be blocked by modifying the 3' end with Spacer C3 during synthesis (as shown in Table 3), or conventional primers can be synthesized and then ddNTPs can be added to the 3' end of the primers according to the procedure in Example 1.

[0105] Table 3

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] Example 3: Removal of human nucleic acids from DNA and RNA co-constructed libraries

[0122] This embodiment refers to, as follows: Figure 1 The construction flowchart is shown. Reference sample preparation: using ZymoBIOMICS. TM Microbial community standards (Zymo, catalog number D6300) and synthetic H1N1 pseudovirus (H1N1 nucleic acid reference, Jingliang Biotechnology, catalog number GW-IAF110) were added to 2×10⁻⁶ cells / mL at different concentration gradients. 5 In the human HeLa cell line with cells / mL, the matrix medium was PBS buffer, as shown in Table 4 below:

[0123] Table 4

[0124] Serial Number Zymo standards (Cells / mL) H1N1 pseudovirus (Copies / mL) Human cells (Cells / mL) 1 1.00E+05 1.00E+06 2.00E+05 2 1.00E+04 1.00E+05 2.00E+05 3 1.00E+03 1.00E+04 2.00E+05

[0125] (2) Nucleic acid extraction: The reference sample was co-extracted using a DNA and RNA co-extraction kit.

[0126] (3) DNA and RNA co-construction library

[0127] 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.

[0128] A. Nucleic acid denaturation

[0129] Prepare the reaction system according to Table 5, and mix thoroughly by gently pipetting 10 times:

[0130] Table 5

[0131] reagent components Volume (μL) Total nucleic acids (RNA & DNA) 16 Random haxamers (50μM) 1 Oligo dT (10 μM) 1 Total volume 18

[0132] The denaturation reaction procedure is as follows:

[0133] Step 1: 70℃ for 5 minutes, 1 cycle;

[0134] Step 2: Immediately give the ice bath for 3 minutes;

[0135] B. Reverse transcription reaction

[0136] Remove the components required for the reverse transcription reaction from -30 to -15°C, thaw on ice, mix thoroughly by inverting the tube, and collect to the bottom of the tube by brief centrifugation. Prepare the first-strand cDNA synthesis reaction system according to Table 6:

[0137] Table 6

[0138]

[0139]

[0140] The reverse transcription reaction buffer in Table 6 includes 200 mM Tris-HCl (pH 8.3), 300 mM KCl, 12 mM MgCl2, 40 mM DTT, 10 mM Random Hexamers, and 2 mM dNTPs; the reverse transcriptase is M-MLV Reverse Transcriptase (100 U / uL).

[0141] The reverse transcription procedure is as follows:

[0142] Step 1: 25℃ for 5 minutes, 1 cycle;

[0143] Step 2: 42℃ for 15 minutes, 1 cycle;

[0144] Step 3: 85℃ for 5 minutes, 1 cycle;

[0145] C. Two-chain synthesis

[0146] Remove the components required for double-stranded cDNA synthesis from -30 to -15°C, thaw on ice, mix thoroughly by inverting the tube, and collect the mixture to the bottom of the tube by brief centrifugation. Prepare the second-stranded cDNA synthesis reaction system according to Table 7 below:

[0147] Table 7

[0148] reagent components Volume (μL) Previous cDNA product 25 Dichain synthesis reaction buffer 5 Two-chain synthase 5 Total volume 35

[0149] The reaction buffers for the two-strand synthesis in Table 7 include 350 mM Tris-HCl (pH 7.5 at 25 °C), 50 mM MgCl2, and 5 mM DTT; the two-strand synthases contain RNase H (0.1 U / uL) and DNA Polymerase I (3 U / uL).

[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] Take out the components required for fragmentation and end-repair A from -30 to -15°C, thaw on ice, mix by inverting, collect to the bottom of the tube by brief centrifugation, and prepare the reaction system according to Table 8:

[0154] Table 8

[0155]

[0156]

[0157] The fragmentation and end repair plus A-tail mixing enzyme and buffer in Table 8 were purchased from Yisheng Biotechnology's rapid fragmentation / end repair / A-tail addition module (catalog number 12619ES).

[0158] The fragmentation and terminal repair plus A reaction procedure is as follows:

[0159] Step 1: 37℃ for 20 minutes, 1 cycle;

[0160] Step 2: 65℃ for 30 minutes, 1 cycle;

[0161] E. Connector Connection

[0162] Remove the components required for connector connection from -30 to -15°C, thaw on ice, mix by inverting, briefly centrifuge to collect to the bottom of the tube, and prepare reaction system 9 according to the table below:

[0163] Table 9

[0164] reagent components Volume (μL) Previous product 50 Ligation reaction buffer 25 T4 DNA ligase 5 DNA Adapter X for Illumina(10μM) 5 Enzyme-free water 15 Total volume 100

[0165] The ligation reaction buffers in Table 9 consist of 400 mM Tris-HCl, 40 mM MgCl2, 4 mM DTT, 4 mM ATP, and 30% PEG 6000; the DNA Adapter X for Illumina was purchased from Inmena Corporation's TruSeq DNA UDIndexes (24 indexes, 96 samples).

[0166] The connector connection reaction procedure is as follows:

[0167] Step 1: 20℃ for 15 minutes, 1 cycle;

[0168] F. Purification of ligation products

[0169] F1. DNA purification magnetic beads equilibrate to room temperature;

[0170] F2. Add 60 μl of DNA purification magnetic beads to the library enrichment PCR product from the previous step, mix thoroughly, and let stand for 5 min.

[0171] 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.

[0172] F4. Add 500 μl of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant.

[0173] F5. Repeat the previous step once;

[0174] 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.

[0175] F7. 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.

[0176] (4) Blocking primer amplification

[0177] The blocking primers with added ddNTPs prepared in Examples 1 and 2 (i.e., blocking primers for binding human repetitive sequences and blocking primers for binding human rRNA sequences and human housekeeping gene sequences) were mixed with universal library primers (i.e., adapter primers) to perform PCR amplification on the adapter ligation products (i.e., DNA-RNA co-constructed libraries), and the reaction system was prepared according to Table 10:

[0178] Table 10

[0179] reagent components Volume (μL) The previous step involved connecting the purified product to the adapter. 19 PCR reaction premix 25 Illumina library primers (10 μM) 2 Blocking primer (10 μM) 4 Total volume 35

[0180] The blocking primer amplification reaction procedure is as follows:

[0181] Step 1: 95℃ for 5 minutes, 1 cycle;

[0182] Step 2: 95℃ for 30s, 65℃ for 10s, 60℃ for 10s, 72℃ for 30s, 7 cycles;

[0183] Step 3: 72℃ for 5 minutes, 1 cycle;

[0184] (5) Purification of amplification products

[0185] A. Equilibrate the DNA purification magnetic beads to room temperature;

[0186] B. Add 45 μl of DNA purification magnetic beads to the library enrichment PCR product from the previous step, mix thoroughly, and let stand for 5 min.

[0187] 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.

[0188] D. Add 500 μl of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant;

[0189] E. Repeat the previous step once;

[0190] 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.

[0191] 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.

[0192] H. This yields a co-constructed library of DNA and RNA with human nucleic acids removed.

[0193] (6) Document quality control:

[0194] 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.

[0195] (7) Sequencing

[0196] 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.

[0197] (8) Data Analysis

[0198] The analysis steps include filtering adapter primer sequences, host proportion statistics and host removal, species annotation and abundance statistics, etc.

[0199] 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 this method were used as the experimental group (S1_HD, S2_HD, S3_HD). The proportions of human sequences in the control group were 97.64%, 98.78%, and 98.47%, respectively, while the proportions in the experimental group were 79.11%, 83.26%, and 85.19%, respectively. After human removal using this method, the proportion of human sequences decreased by 13%–18%, as shown in Table 11 below.

[0200] Table 11

[0201] Library Name S1_control S1_HD S2_control S2_HD S3_control S3_HD Total number of reads 18230331 22727728 22174587 20332809 19962546 19706657 Human source reads 17800533 17979798 21902950 16929051 19657677 16788048 Human-derived sequence ratio 97.64％ 79.11％ 98.78％ 83.26％ 98.47％ 85.19％

[0202] The RPM values ​​(RPM value = number of specific reads / number of valid reads per million) of species detection were statistically analyzed. 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 ​​of the experimental group were increased by about 1.5 to 14 times, as shown in Table 12 below:

[0203] Table 12

[0204]

[0205]

[0206] 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.

[0207] 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 blocking primer for dehumanized nucleic acids, characterized in that, The blocking primer comprises a blocking primer for binding to a human repetitive sequence, and a blocking primer for binding to a human rRNA sequence and a human housekeeping gene sequence; the blocking primer for binding to the human repetitive sequence is prepared by the following steps: A1, fragmenting Human Cot-1 DNA, and purifying and recovering; A2, ddNTP-modifying the purified and recovered fragmented Human Cot-1 DNA, and recovering to obtain; The sequence of the blocking primer for binding to the human rRNA sequence and the human housekeeping gene sequence is shown in SEQ ID NO. 1-464; wherein, the 3' end of the sequence is provided with a blocking group; the blocking group comprises any one of Spacer C3 and ddNTP.

2. The blocking primer of claim 1, wherein, In step A1, the Human Cot-1 DNA is from Thermo, item number 15279; the fragmenting method comprises enzyme fragmentation, and the fragmenting enzyme used in the enzyme fragmentation is an endonuclease.

3. The blocking primer of claim 1, wherein, In step A2, the enzyme used in the ddNTP modification comprises a terminal transferase.

4. A method of constructing a sequencing library of a non-human nucleic acid based on the blocking primer of any one of claims 1-3, characterized in that, The method comprises the following steps: S1, fragmenting sample nucleic acid, end-repairing and A-tailing the nucleic acid fragments, and connecting sequencing adapters to obtain a library; the sample nucleic acid contains human nucleic acid; The library comprises any one of a DNA library, an RNA library, and an RNA and DNA co-constructed library; S2, using the library as a template, performing PCR amplification with an adapter primer and a blocking primer; S3, purifying and recovering the PCR amplification product to obtain the sequencing library for removing human nucleic acid.

5. The method of claim 4, wherein, In step S1, at least one of the following technical features is further included: The fragmenting method comprises any one of mechanical fragmentation, enzyme fragmentation, and thermal cleavage; the mechanical fragmentation comprises ultrasonic fragmentation, the thermal cleavage uses a divalent metal ion buffer solution, and the enzyme fragmentation uses an endonuclease; The connection uses T4 DNA ligase.

6. The method of claim 4, wherein, In step S1, the preparation method of the DNA library comprises: fragmenting sample DNA, end-repairing and A-tailing the DNA fragments, and connecting sequencing adapters to obtain a DNA library; Or, fragmenting sample DNA using Tn5 transposase and connecting sequencing adapters to obtain a DNA library; The preparation method of the RNA library comprises: fragmenting sample RNA, performing reverse transcription on the RNA fragments to generate double-stranded DNA, and then performing end-repairing and A-tailing on the double-stranded DNA, and connecting sequencing adapters to obtain an RNA library; Or, performing reverse transcription on sample RNA to generate double-stranded DNA, fragmenting the double-stranded DNA using Tn5 transposase, and connecting sequencing adapters to obtain an RNA library; The preparation method of the RNA and DNA co-constructed library comprises: performing reverse transcription on sample RNA to generate double-stranded DNA, fragmenting the double-stranded DNA together with sample DNA, end-repairing and A-tailing the DNA fragments, and connecting sequencing adapters to obtain an RNA and DNA co-constructed library; Or, the sample RNA is subjected to reverse transcription and double-strand synthesis to generate double-strand DNA, and then the double-strand DNA is fragmented by Tn5 transposase and connected with sequencing adapters to obtain a co-constructed library of RNA and DNA.

7. The method of claim 4, wherein, In step S2, the concentration of the adapter primer is 1-20 pmol; the concentration of the blocking primer is 2-100 pmol; and the molar ratio of the blocking primer to the adapter primer is 1-10:

1.

8. A sequencing library constructed by the method of any one of claims 4-7.

9. Use of the sequencing library of claim 8 in the preparation of a product for detecting pathogenic microorganisms.

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