Sequencing library construction method based on closed primer human nucleic acid removal and application

Abstract

The invention discloses a sequencing library construction method for removing human nucleic acid based on a closed primer and application. The closed primer comprises a closed primer for combining a human repetitive sequence and a closed primer for combining a human rRNA sequence and a human housekeeping gene sequence, and the closed primer can be specifically combined with a scattered repetitive sequence, an rRNA sequence and a housekeeping gene sequence which are widely distributed in a human genome. The construction method comprises the following steps that a DNA library or an RNA library or an RNA and DNA co-construction library is constructed, a library with a sequencing connector added is used as a template, a connector primer and a closing primer are used for PCR amplification, the closing primer can be specifically combined to human source nucleic acid through annealing, DNA polymerase extension reaction modification is prevented, and the DNA library or the RNA library or the RNA and DNA co-construction library is constructed. A human nucleic acid library cannot be enriched through PCR amplification, and pathogenic microorganism nucleic acid can be normally amplified, so that the purpose of reducing the proportion of human nucleic acid is achieved, and guidance is provided for pathogenic microorganism detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and relates to a method for constructing a sequencing library based on blocking primers to remove human nucleic acids and its application. Background Art

[0002] Infectious diseases are one of the main causes of global human death. Rapidly identifying pathogens is the basis for effectively controlling infections. Traditional microbial detection has many steps and complex operations, which greatly limits its clinical use in China. Currently, there are more than 30 million encephalitis (meningitis) patients globally every year. Due to the lack of effective detection methods for most pathogenic microorganisms, although it is easy to clinically diagnose whether there is an infection, it is impossible to clarify what type of infection it belongs to, and more than 90% of the patients have not received a clear diagnosis and accurate treatment. Metagenomics detection plays an important role in the detection of difficult, critical or special infectious diseases with its advantages of rapid identification, unbiasedness, wide coverage, no need for culturing, and no need for presetting. However, there are still many problems to be solved in current metagenomic sequencing. In particular, the high human background in samples leads to a small proportion of pathogenic microorganisms in the sequencing data, low data utilization rate, affecting the detection sensitivity, and the high requirement for the amount of sequencing data results in a high cost of the metagenome, restricting its popularization and application in clinical practice.

[0003] Currently, the main methods for removing human nucleic acids from samples include differential lysis, anti-methylated DNA magnetic beads, microfiltration membrane filtration, differential centrifugation, etc. Among them, differential lysis is the most commonly used method. In this method, before nucleic acid extraction, a mild detergent (such as saponin) is used to lyse host cells whose cell membranes are more fragile than the outer walls of microorganisms, releasing host nucleic acids, and then deoxyribonuclease I is used to degrade host DNA. However, this method cannot avoid the loss of microorganisms, especially the loss of mycoplasma, chlamydia, and viruses is relatively serious. Moreover, this method can only remove host DNA, cannot remove RNA, and will also lose free pathogenic nucleic acids; Anti-methylated DNA magnetic beads are based on the phenomenon that human DNA has a high degree of methylation while most microbial genomes lack methylated DNA to specifically remove human DNA. However, this method has a high cost and a small proportion of host removal; Microfiltration membrane filtration and differential centrifugation use the physical properties of human cells and microbial cells for separation, but the separation effect is average and the loss is large, so they are less used. Summary of the Invention

[0004] To overcome the above technical problems, the present invention provides a method for constructing a sequencing library for removing human nucleic acids based on blocking primers and its application. First, the present invention provides a blocking primer for removing human nucleic acids, including a blocking primer for binding to human repetitive sequences, a blocking primer for binding to human rRNA sequences and human housekeeping gene sequences; among them, the blocking primer for binding to human repetitive sequences is obtained by fragmenting Human Cot-1 DNA and performing ddNTP modification; the sequences of the blocking primers for binding to human rRNA sequences and human housekeeping gene sequences are shown in SEQ ID NO.1-464, and their 3' ends are provided with a blocking group. The blocking primers of the present invention can specifically bind to the interspersed repetitive sequences, rRNA sequences and housekeeping gene sequences widely distributed in the human genome. Secondly, the present invention provides a method for constructing a sequencing library for removing human nucleic acids based on blocking primers, which includes: constructing a DNA or RNA library, or co-constructing a library of RNA and DNA. Using the library with sequencing adapters added as a template, PCR amplification is performed using adapter primers and blocking primers. Among them, the blocking primers can specifically bind to human nucleic acids by annealing, and the 3' ends of the blocking primers are modified with Spacer C3 or ddNTP, etc., which can prevent the DNA polymerase extension reaction, so that the human nucleic acid library cannot be enriched by PCR amplification, while the nucleic acids of pathogenic microorganisms can be amplified normally, achieving the purpose of reducing the proportion of human nucleic acids. Using 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, providing guidance for the detection of pathogenic microorganisms.

[0005] The technical solution adopted by the present invention is as follows:

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

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

[0008] A2. Perform ddNTP modification on the purified and recovered fragmented Human Cot-1 DNA, and recover it to obtain.

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

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

[0011] Preferably, the fragments after fragmentation are less than 100 bp in distribution.

[0012] Further, the fragmentation enzyme used in the enzymatic fragmentation is an endonuclease. In some embodiments, the fragmentation enzyme is dsDNA Fragmentase.

[0013] As an embodiment of the present invention, in step A1, the purification and recovery is carried out using DNA purification magnetic beads.

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

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

[0016] B2. Take the equilibrated DNA purification magnetic beads and add them to the fragmented Human Cot-1 DNA, mix well and let stand for reaction;

[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 an ethanol solution, let stand and then discard the supernatant;

[0019] B5. Repeat step B4;

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

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

[0022] In some embodiments, in step B2, the amount of the equilibrated DNA purification magnetic beads is 45 - 60 μl, and the standing reaction time is 3 - 5 min; in step B3, let stand for 1 - 2 min until the solution is clear and the magnetic beads are completely adsorbed; in step B4, the volume ratio of the ethanol solution is 70 - 80% (v / v), and the amount used is 400 - 500 μl, let stand for 20 - 30 s; in step B5, repeat step B4 once; in step B5, open the lid and air-dry the magnetic beads for 5 - 10 min until there is no ethanol residue; in step B7, add 22 - 55 μl of sterile ultrapure water for elution, gently pipette and mix well, place at room temperature for 3 - 5 min, briefly centrifuge the centrifuge tube and place it in the magnetic rack to stand. After the solution is clear (about 2 min), carefully transfer the supernatant to a new centrifuge tube, and do not touch the magnetic beads.

[0023] As an embodiment of the present invention, in step A2, the enzymes used for the ddNTP modification include terminal transferase.

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

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

[0026] As an embodiment of the present invention, the blocking primers for binding to the human rRNA sequence and the human housekeeping gene sequence include the sequences shown in SEQ ID NO. 1-464; wherein, the 3' end of the sequence is provided with a blocking group.

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

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

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

[0030] The present invention designs blocking primers for binding to human dispersed repetitive sequences widely distributed in the human genome; designs blocking primers for binding to the human rRNA sequence and the human housekeeping gene sequence for the rRNA sequences 5.8s rRNA, 18s rRNA, 28s rRNA, and the human housekeeping genes GAPDH, β-actin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, NONO, ARHGDIA, RPL32, RPS18, HSPCB, etc.; the length of the blocking primer is 20-40 bp, and the annealing temperature of the blocking primer is 65-72°C. The blocking primers of the present invention can specifically bind to the dispersed repetitive sequences, rRNA sequences, and housekeeping gene sequences widely distributed in the human genome.

[0031] In a second aspect, the present invention provides a method for constructing a sequencing library of human-removed nucleic acids based on the blocking primers, including the following steps:

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

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

[0034] S3. Purify and recover the PCR amplification products to obtain the sequencing library of the dehumanized nucleic acid.

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

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

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

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

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

[0040] As an embodiment of the present invention, in step S1, T4 DNA ligase is used for the ligation.

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

[0042] Further, the preparation method of the DNA library includes: fragmenting the sample DNA, performing end repair and A-tailing on the DNA fragment, and ligating a sequencing adapter to obtain a DNA library;

[0043] Or, fragmenting the sample DNA with Tn5 transposase and ligating a sequencing adapter to obtain a DNA library;

[0044] The preparation method of the RNA library includes: fragmenting the sample RNA, performing reverse transcription and second-strand synthesis on the RNA fragment to generate double-stranded DNA, then performing end repair and A-tailing, and ligating a sequencing adapter to obtain an RNA library;

[0045] Or, performing reverse transcription and second-strand synthesis on the sample RNA to generate double-stranded DNA, and then fragmenting the double-stranded DNA with Tn5 transposase and ligating a sequencing adapter to obtain an RNA library;

[0046] The preparation method of the co - constructed library of RNA and DNA includes: reverse transcribing the sample RNA and synthesizing the second strand to generate double - stranded DNA, fragmenting it together with the sample DNA, repairing the ends of the DNA fragments and adding A, and then ligating sequencing adapters to obtain the co - constructed library of RNA and DNA;

[0047] Or, reverse transcribing the sample RNA and synthesizing the second strand to generate double - stranded DNA, then using Tn5 transposase to fragment the double - stranded DNA and the sample DNA and ligating sequencing adapters to obtain the co - constructed library of RNA and DNA.

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

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

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

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

[0052] As an embodiment of the present invention, in step S2, the molar ratio of the blocking primers to the adapter primers is 1 - 10:1.

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

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

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

[0056] Step 1: 95°C for 3 min, 1 cycle;

[0057] Step 2: 95°C for 20 s, 60°C for 15 s, 72°C for 30 s, 3 - 15 cycles;

[0058] Step 3: 72°C for 3 min, 1 cycle.

[0059] As an embodiment of the present invention, the purification and recovery are carried out using DNA purification magnetic beads.

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

[0061] In a third aspect, the present invention provides a sequencing library obtained by the described construction method.

[0062] In a fourth aspect, the present invention provides an application of the described sequencing library in the preparation of a product 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 the interspersed repetitive sequences, rRNA sequences, and housekeeping gene sequences widely distributed in the human genome, and prevent the modification of the DNA polymerase extension reaction, so that the human nucleic acid library cannot be enriched by PCR amplification, while the nucleic acids of pathogenic microorganisms can be amplified normally, achieving the purpose of reducing the proportion of human nucleic acids.

[0065] 2. The present invention uses the blocking primers in the construction of the sequencing library. The method can remove human nucleic acids during the library construction process without losing the nucleic acids of pathogenic microorganisms, maximizing the retention of all pathogenic microorganism nucleic acid information in the sample. The process is simple, low-cost, and easy to operate, and has high reference value and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objectives, and advantages of the present invention will become more apparent:

[0067] Figure 1 is the flow chart for constructing the sequencing library of the present invention;

[0068] Figure 2 is the library fragment distribution in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0069] The present invention will be described in detail below with reference to the drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, providing detailed implementation manners and specific operation processes, which will help those skilled in the art to further understand the present invention. It should be noted that the protection scope of the present invention is not limited to the following embodiments. Several adjustments and improvements made on the premise of the concept of the present invention all belong to the protection scope 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, and its maximum length distribution is approximately 50 to 300 bp. Due to the widespread distribution of interspersed repetitive sequences (IRS) such as SINE (short interspersed nuclear elements, such as Alu sequences) and LINE (long interspersed nuclear elements, such as L1 sequences) on the human genome, under conditions rich in these repetitive sequences, Human Cot-1 DNA can be prepared from human placenta DNA through shearing, denaturation, and reannealing. Further fragment Human Cot-1 DNA (purchased from Thermo Fisher Scientific in this example, catalog number 15279) so that its fragment distribution is less than 100 bp, and then modify ddNTP at the ends with terminal deoxynucleotidyl 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 and use dsDNA Fragmentase (catalog number M0348L) for fragmentation, and prepare the reaction system as shown in Table 1 below:

[0074] Table 1

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

[0076] The fragmentation reaction procedure is as follows:

[0077] Step 1: 37°C for 10 min, 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. Take 60 μl of DNA purification magnetic beads and add them to the fragmented product from the previous step. After thorough mixing, let it stand for 5 min;

[0081] C. Place the centrifuge tube on the magnetic rack. After standing for 1 - 2 min until the solution is clear and the magnetic beads are completely adsorbed, carefully discard the supernatant with a pipette;

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

[0083] E. Repeat the previous step once;

[0084] F. Keep the centrifuge tube always on the magnetic rack, open the lid and air-dry the magnetic beads for 5 - 10 min until there is no ethanol residue;

[0085] G. Add 55 μl of sterilized ultrapure water for elution, gently pipette and mix well, let it stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on the magnetic stand to stand still. After the solution becomes clear (about 2 min), carefully transfer 52.5 μl of the supernatant to a new centrifuge tube, taking care not to touch the magnetic beads.

[0086] (3) ddNTP modification

[0087] Thaw the ddNTP stock solution on ice, shake and mix well. Dilute it with water to a 1 mM working solution in a new EP tube. Using the terminal transferase kit (product number M0315L) from NEB, prepare the reaction system as shown in Table 2 below:

[0088] Table 2

[0089]

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

[0091] Step 1: 37 °C for 1.5 h, 1 cycle;

[0092] Step 2: 70 °C for 10 min, 1 cycle;

[0093] (4) Recovery of modified product: Recover the modified product using the centrifugal adsorption column method. The recovered product can be used as a blocking primer for binding to human repetitive sequences.

[0094] A. Place the adsorption column in the collection tube, then add 500 μl of equilibration buffer, centrifuge at 12000 rpm for 1 min, then pour out the liquid in the collection tube and place the adsorption column back into the collection tube for standby;

[0095] B. Add 375 μl of binding buffer to 75 μl of the product and mix well;

[0096] C. Transfer the mixture from the previous step to the adsorption column, let it stand at room temperature for 5 min, then centrifuge at 12000 rpm for 30 s, pour out the liquid in the collection tube and place the adsorption column back into the collection tube;

[0097] D. Add 600 μl of wash buffer to the adsorption column, centrifuge at 12000 rpm for 30 s, pour out the liquid in the collection tube and place 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 wash buffer as possible, then let it stand at room temperature to dry for 3 min;

[0100] G. Take out the adsorption column and place it in a clean centrifuge tube. Add 20 μl of eluent to the middle position of the membrane, let it stand at room temperature for 5 min, and then centrifuge at 12,000 rpm for 2 min to collect the DNA solution.

[0101] (5) Product quantification: Use Qseq100 to detect the size of the product, use qubit 2.0 to detect the concentration, and then calculate the molar concentration according to the fragment size for standby.

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

[0103] (1) Blocking primer design: Design primers for human housekeeping genes such as 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 is 20 - 40 bp, and the primer annealing temperature is 65 - 72 °C. The primer sequences are shown in Table 3 (SEQ ID NO.1 - 464).

[0104] (2) Blocking primer synthesis: The blocking primer can be blocked by modifying Spacer C3 at the 3' end during synthesis (as shown in Table 3), or a conventional primer can be synthesized, and then ddNTP can be added to the 3' end of the primer according to the procedure of 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 a Library Constructed with DNA and RNA

[0122] This example refers to the construction flow chart as shown in Figure 1 Preparation of reference samples: ZymoBIOMICS TM Microbial Community Standard (Zymo, catalog number D6300) and synthetic influenza A virus pseudovirus (H1N1 nucleic acid reference, Jingliang Biotech, catalog number GW-IAF110) were added to a human Hela cell line at 2×10 5 cells / mL at different concentration gradients, with PBS buffer as the matrix solution, as shown in Table 4 below:

[0123] Table 4

[0124] Serial number Zymo standard (Cells / mL) Influenza A 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: A DNA and RNA co-extraction kit was used to co-extract nucleic acids from the reference samples.

[0126] (3) Library construction with DNA and RNA

[0127] The co-extracted nucleic acids contain DNA and RNA. Using a co-library construction scheme, 3 libraries were constructed in parallel for each reference sample.

[0128] A. Nucleic acid denaturation

[0129] Prepare the reaction system according to Table 5 below and gently pipette 10 times to mix well:

[0130] Table 5

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

[0132] The denaturation reaction procedure is as follows:

[0133] Step 1: 70°C for 5 min, 1 cycle;

[0134] Step 2: Immediately ice-bath for 3 min;

[0135] B. Reverse transcription reaction

[0136] Take out the components required for the reverse transcription reaction from -30 to -15 °C, thaw on ice, invert the tube up and down to mix well, and briefly centrifuge to collect at the bottom of the tube. Prepare the first-strand cDNA synthesis reaction system according to Table 6 below:

[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, 2 mM dNTPs; the reverse transcriptase is M-MLV Reverse Transcriptase (100 U / μL).

[0141] The reverse transcription reaction procedure is as follows:

[0142] Step 1: 25 °C for 5 min, 1 cycle;

[0143] Step 2: 42 °C for 15 min, 1 cycle;

[0144] Step 3: 85 °C for 5 min, 1 cycle;

[0145] C. Second-strand synthesis

[0146] Take out the components required for the double-stranded cDNA synthesis from -30 to -15 °C, thaw on ice, invert the tube up and down to mix well, and briefly centrifuge to collect at the bottom of the tube. Prepare the second-strand cDNA synthesis reaction system according to Table 7 below:

[0147] Table 7

[0148] Reagent components Volume (μL) Previous cDNA product 25 Second-strand synthesis reaction buffer 5 Second-strand synthesis enzyme 5 Total volume 35

[0149] The second-strand synthesis reaction buffer in Table 7 includes 350 mM Tris-HCl (pH 7.5 at 25 °C), 50 mM MgCl 2 , 5 mM DTT; the second-strand synthesis enzyme contains RNase H (0.1 U / μL), DNA Polymerase I (3 U / μL).

[0150] The second-strand synthesis reaction procedure is as follows:

[0151] Step 1: 16 °C for 30 min, 1 cycle;

[0152] D. Fragmentation and end repair plus A

[0153] Take out the components required for fragmentation, end repair, and A-tailing from -30 to -15 °C, thaw on ice, invert the tube to mix well, briefly centrifuge to collect at the bottom of the tube, and prepare the reaction system according to Table 8 below:

[0154] Table 8

[0155]

[0156]

[0157] The fragmentation, end repair, and A-tailing mixed enzyme and buffer in Table 8 were purchased from Yeasen's Rapid Fragmentation / End Repair / A-Tailing Module (Catalog No. 12619ES).

[0158] The fragmentation, end repair, and A-tailing reaction procedure is as follows:

[0159] Step 1: 37 °C for 20 min, 1 cycle;

[0160] Step 2: 65 °C for 30 min, 1 cycle;

[0161] E. Adapter Ligation

[0162] Take out the components required for adapter ligation from -30 to -15 °C, thaw on ice, invert the tube to mix well, briefly centrifuge to collect at the bottom of the tube, and prepare the reaction system according to Table 9 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 buffer in Table 9 includes 400 mM Tris-HCl, 40 mM MgCl 2 , 4 mM DTT, 4 mM ATP, and 30% PEG 6000; DNA Adapter X for Illumina was purchased from Illumina's TruSeq DNA UDIndexes (24 indexes, 96 samples).

[0166] The adapter ligation reaction procedure is as follows:

[0167] Step 1: 20 °C for 15 min, 1 cycle;

[0168] F. Purification of Adapter Ligation Products

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

[0170] F2. Take 60 μl of DNA purification magnetic beads and add them to the library enrichment PCR product from the previous step. After thorough mixing, let it stand for 5 min;

[0171] F3. Place the centrifuge tube on the magnetic stand. After standing for 1 - 2 minutes until the solution is clear and the magnetic beads are completely adsorbed, carefully discard the supernatant with a pipette.

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

[0173] F5. Repeat the previous step once.

[0174] F6. Keep the centrifuge tube in the magnetic stand all the time. Open the lid and air-dry the magnetic beads for 5 - 10 minutes until there is no ethanol residue.

[0175] F7. Add 22 μl of sterilized ultrapure water for elution, gently pipette and mix well. Place at room temperature for 5 minutes. Briefly centrifuge the centrifuge tube and place it in the magnetic stand to stand. After the solution is clear (about 2 minutes), carefully transfer 20 μl of the supernatant to a new centrifuge tube, taking care not to touch the magnetic beads.

[0176] (4) Blocking primer amplification

[0177] Mix the blocking primers (i.e., the blocking primers for binding to human repetitive sequences, and the blocking primers for binding to human rRNA sequences and human housekeeping gene sequences) added with ddNTP prepared in Example 1 and Example 2, and the library universal primer (i.e., the adapter primer), and perform PCR amplification on the adapter ligation product (i.e., the co-library of DNA and RNA). Prepare the reaction system according to Table 10 below:

[0178] Table 10

[0179] Reagent components Volume (μL) Previous adapter-ligated and purified product 19 PCR reaction premix 25 Illumina library primer (10μM) 2 Blocking primer (10μM) 4 Total volume 35

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

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

[0182] Step 2: 95°C for 30 seconds, 65°C for 10 seconds, 60°C for 10 seconds, 72°C for 30 seconds, 7 cycles;

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

[0184] (5) Purification of amplification products

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

[0186] B. Take 45 μl of DNA purification magnetic beads and add them to the library enrichment PCR product from the previous step. After mixing well, let it stand for reaction for 5 minutes.

[0187] C. Place the centrifuge tube on the magnetic stand and let it stand for 1 - 2 min 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 it stand for 30 s, and then discard the supernatant.

[0189] E. Repeat the previous step once.

[0190] F. Keep the centrifuge tube in the magnetic stand all the time. Open the lid and air-dry the magnetic beads for 5 - 10 min until there is no ethanol residue.

[0191] G. Add 22 μl of sterilized ultrapure water for elution, gently pipette and mix well, let it stand at room temperature for 5 min, centrifuge the centrifuge tube briefly and place it in the magnetic stand. After the solution becomes clear (about 2 min), carefully transfer 20 μl of the supernatant to a new centrifuge tube, taking care not to touch the magnetic beads.

[0192] H. Thus, a DNA and RNA co-library with human nucleic acids removed is obtained.

[0193] (6) Library quality inspection:

[0194] Use Qseq100 to detect the size of the product and qubit 2.0 to perform library quality inspection on the concentration. The library fragment distribution is as Figure 2 shown.

[0195] (7) Sequencing on the machine

[0196] Standardize all the obtained products, mix them in equal amounts, and perform parallel sequencing on the mixed library. The sequencing platform is Illumina NextSeq550Dx and the sequencing type is SE75.

[0197] (8) Data analysis

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

[0199] Results: After constructing DNA and RNA co-libraries for 3 reference samples respectively, those without human source removal and directly sequenced on the machine were used as the control group (S1_control, S2_control, S3_control), and those with human source removal using this method were used as the experimental group (S1_HD, S2_HD, S3_HD). Among them, the proportions of human source sequences in the control group were 97.64%, 98.78%, and 98.47% respectively, and the proportions of human source sequences in the experimental group were 79.11%, 83.26%, and 85.19% respectively. After removing human sources using this method, the proportion of human source 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 Number of human reads 17800533 17979798 21902950 16929051 19657677 16788048 Proportion of human sequences 97.64％ 79.11％ 98.78％ 83.26％ 98.47％ 85.19％

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

[0203] Table 12

[0204]

[0205]

[0206] Conclusion: Using this method to construct a host-depleted library, the library construction process is simple, which can effectively reduce the proportion of host nucleic acids and improve the microbial detection rate.

[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 above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A blocking primer for removing human nucleic acid, characterized in that: The blocking primers include blocking primers for binding to human repetitive sequences, and blocking primers for binding to human rRNA sequences and human housekeeping gene sequences; the blocking primers for binding to human repetitive sequences are prepared by the following steps: A1, fragmenting Human Cot-1 DNA, and purifying and recovering it; A2. The purified and recovered fragmented Human Cot-1 DNA is modified with ddNTP and recovered.

2. The blocking primer according to claim 1, characterized in that In step A1, the Human Cot-1 DNA is Thermo Fisher 15279; the fragmentation method includes enzymatic shearing, and the fragmentation enzyme used in the enzymatic shearing is an endonuclease.

3. The blocking primer according to claim 1, characterized in that In step A2, the enzyme used for the ddNTP modification includes terminal transferase.

4. The blocking primer according to claim 1, characterized in that The blocking primer for binding to 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 carries a blocking group; the blocking group includes any one of Spacer C3 and ddNTP.

5. A method for constructing a sequencing library of humanized nucleic acid based on the blocking primers according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Fragmenting the sample nucleic acid, performing end-repair and A addition on the nucleic acid fragments, and connecting sequencing adapters to obtain a library; the sample nucleic acid contains human nucleic acid; S2, using the library as a template, PCR amplification with adapter primers and blocking primers; S3. Purify and recover the PCR amplification product to obtain the sequencing library without human nucleic acid.

6. The method according to claim 5, characterized in that In step S1, at least one of the following technical features is also included: The fragmentation method includes any one of mechanical disruption, enzymatic disruption, and thermal lysis; the mechanical disruption includes ultrasonic disruption, the thermal lysis solution is a divalent metal ion buffer, and the enzymatic disruption fragmentation enzyme is an endonuclease; The ligation was performed using T4 DNA ligase.

7. The method according to claim 5, characterized in that In step S1, the method for preparing the DNA library includes: fragmenting the sample DNA, performing end repair and A addition on the DNA fragments, and connecting sequencing adapters to obtain a DNA library; Alternatively, the sample DNA is fragmented using Tn5 transposase and connected to sequencing adapters to obtain a DNA library; The RNA library preparation method comprises: fragmenting the sample RNA, reverse transcribing the RNA fragments and synthesizing the two strands to generate double-stranded DNA, then performing end repair and A addition, and connecting sequencing adapters to obtain the RNA library; Alternatively, the sample RNA is reverse transcribed and double-stranded to generate double-stranded DNA, and then the double-stranded DNA is fragmented using Tn5 transposase and connected to sequencing adapters to obtain an RNA library; The preparation method of the RNA and DNA co-constructed library comprises: reverse transcribing the sample RNA and synthesizing the two strands to generate double-stranded DNA, fragmenting the sample RNA together with the sample DNA, performing end repair and A addition on the DNA fragments, and connecting sequencing adapters to obtain the RNA and DNA co-constructed library; Alternatively, the sample RNA is reverse transcribed and double-stranded synthesized to generate double-stranded DNA, and then the double-stranded DNA and sample DNA are fragmented using Tn5 transposase and connected to sequencing adapters to obtain an RNA and DNA co-constructed library.

8. The method according to claim 5, characterized in that In step S2, the concentration of the linker 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 linker primer is 1-10:

1.

9. A sequencing library constructed according to the method of any one of claims 5 to 8.

10. Use of the sequencing library according to claim 9 in preparing a product for detecting pathogenic microorganisms.

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