阅读说明：本技术 一种基于宏基因组学的呼吸道咽拭子样本的建库方法和病原检测方法 (Macrogenomics-based respiratory tract pharynx swab sample database building method and pathogen detection method ) 是由 李鹏 宋宏彬 林彦锋 王凯英 李瑾慧 杨朗 李沛翰 贾雷立 于 2020-12-30 设计创作，主要内容包括：本发明提供了一种基于宏基因组学的呼吸道咽拭子样本的建库方法和病原检测方法,涉及微生物检测技术领域。所述建库方法包括(a)待测样本溶液预处理：加入溶菌酶进行消化；(b)样本总核酸的提取,在提取的过程中不添加carrier RNA；(c)cDNA合成：采用随机引物进行反转录；(d)使用磁珠对步骤(c)合成的产物进行纯化；(e)样本DNA文库的构建。本发明基于DNA-RNA共测序原理,提供了一种基于宏基因组学的适合于咽拭子样本建库与检测的方法,通过单次反应就能实现咽拭子样本宏基因组的病原检测。(The invention provides a method for establishing a library of a respiratory tract pharynx swab sample based on metagenomics and a method for detecting pathogen, and relates to the technical field of microbial detection. The library building method comprises the following steps of (a) preprocessing of a sample solution to be detected: adding lysozyme for digestion; (b) extracting total nucleic acid of the sample, wherein carrier RNA is not added in the extraction process; (c) and (3) cDNA synthesis: carrying out reverse transcription by adopting a random primer; (d) purifying the product synthesized in the step (c) by using magnetic beads; (e) and constructing a sample DNA library. The invention provides a method suitable for pharynx swab sample library establishment and detection based on metagenomics based on a DNA-RNA co-sequencing principle, and can realize pathogen detection of pharynx swab sample metagenome through a single reaction.)

1. A metagenomics-based method for constructing a database of respiratory tract pharyngeal swab samples, comprising:

(a) pretreating a sample solution to be detected: adding lysozyme for digestion;

(b) extracting total nucleic acid of the sample, wherein carrier RNA is not added in the extraction process;

(c) and (3) cDNA synthesis: carrying out reverse transcription by adopting a random primer;

(d) purifying the product synthesized in the step (c) by using magnetic beads;

(e) and constructing a sample DNA library.

2. The library-building method according to claim 1, wherein in step (a), the lysozyme is added in an amount of 1/50 based on the volume of the sample solution to be tested, and the concentration of lysozyme is 50 mg/mL.

3. The library-building method according to claim 1 or 2, wherein in step (a), after adding lysozyme, the mixture is mixed well and incubated at 37 ℃ for 15 min.

4. The library construction method according to claim 3, wherein in the step (b), during the nucleic acid elution, the nucleic acid elution is performed using a volume of 40. mu.l or less of an eluent to obtain the extracted total nucleic acids.

5. The library construction method of claim 4, wherein in step (c), the volume ratio of total extracted nucleic acids to random primers added is 25: 3.

6. the library construction method according to claim 5, wherein in the step (b), the total nucleic acid in the sample is extracted using QIAamp MinElute Virus Spin Kit (QIAGEN); the eluent is Buffer AVE.

7. The library construction method according to claim 1 or 5, wherein in the step (e), the MGIEasy enzyme digestion DNA library preparation reagent set is used for sample DNA library construction.

8. The metagenomic library obtained by the library construction method according to any one of claims 1 to 7.

9. A method for detecting pathogens in respiratory tract pharyngeal swab samples for non-disease diagnostic use based on metagenomics, comprising the steps of:

(1) the library construction method of any one of claims 1-7;

(2) performing high-throughput sequencing and performing bioinformatics analysis on sequencing results.

10. The detection method of claim 9, wherein the high-throughput sequencing is performed on a BGISEQ-500 sequencer; and performing metagenome comparison analysis on BGISEQ-500 sequencing data by using Centrifuge software, and performing visual analysis on comparison results by using Pavian.

Technical Field

The invention relates to the technical field, in particular to a method for establishing a database of a respiratory tract pharynx swab sample based on metagenomics and a method for detecting pathogeny.

Background

The pathogens causing respiratory tract infection are various, including viruses, bacteria, fungi and the like, wherein most pathogens use DNA as a carrier of genetic information, but some viruses (such as influenza virus, coronavirus and the like) use RNA as a carrier of genetic information. The metagenome sequencing of a respiratory tract clinical sample becomes an important method for detecting respiratory tract infection pathogens at present, and can detect not only a plurality of pathogens existing in the sample, but also some new or rare pathogens which are difficult to identify by a conventional method by directly detecting all microbial nucleic acids in the sample. Because of some differences in sequencing methods for DNA or RNA, the conventional sequencing and library building method for detecting metagenome pathogens selects DNA library building mainly aiming at bacterial detection, RNA library building mainly aiming at virus detection or DNA and RNA library building twice simultaneously according to the judgment of the clinician on the infection pathogens experience. However, in practice, many cases may not be able to determine empirically whether the pathogen causing the infection is a bacterium, virus or other pathogen, and if a wrong library construction method is selected, the pathogen may be missed. On the other hand, the throat swab is a conventional noninvasive respiratory tract sample and is commonly used for respiratory tract pathogen detection, but the content of nucleic acid in the sample is generally low, if 2-time library establishment is carried out on DNA and RNA at the same time, the detection time and cost are increased, and the content of nucleic acid is generally difficult to meet the requirement of 2-time library establishment. Therefore, it is necessary to develop a method for detecting metagenomic pathogens in throat swab samples by a single reaction.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for detecting pathogens in a respiratory tract pharynx swab sample based on metagenomics. The invention provides a method suitable for pharynx swab sample library establishment and detection based on metagenomics based on a DNA-RNA co-sequencing principle, and can realize pathogen detection of pharynx swab sample metagenome through a single reaction.

The technical scheme provided by the invention is as follows:

in one aspect, the invention provides a method for banking a respiratory tract pharyngeal swab sample based on metagenomics, the method comprising:

(a) pretreating a sample solution to be detected: adding lysozyme for digestion;

(b) extracting total nucleic acid of the sample, wherein carrier RNA is not added in the extraction process;

(c) and (3) cDNA synthesis: carrying out reverse transcription by adopting a random primer;

(d) purifying the product synthesized in the step (c) by using magnetic beads;

(e) and constructing a sample DNA library.

In the invention, the method considers that pathogens which are possibly existed in the throat swab sample and are difficult to crack, such as gram positive bacteria and the like, need to be considered in the treatment of the throat swab sample solution, so the method can improve the nucleic acid extraction efficiency by adding the step of lysozyme digestion before the nucleic acid extraction.

Collecting a pharynx swab sample, and preparing a sample solution to be detected (pharynx swab sample liquefaction); adding lysozyme into the sample solution to be detected for digestion.

In one embodiment, in step (a), the lysozyme is added in an amount of 1/50 based on the volume of the sample solution to be tested, and the concentration of the lysozyme is 50 mg/mL.

In one embodiment, the method is that in step (a), after adding lysozyme, the mixture is mixed well and incubated at 37 ℃ for 15 min.

Carrier RNA is a mixture of RNA fragments between 200 and 3000nt, dissolved in an RNA protectant. In the process of purifying trace nucleic acid by using a column, such as viral RNA purification, viral DNA purification, trace DNA extraction, etc., the binding and elution efficiency of trace nucleic acid on the purification column is reduced, which results in failure to recover sufficient PCR template, and ultimately results in detection failure. The Carrier RNA is added into a column purification nucleic acid purification system, so that the recovery efficiency of trace nucleic acid can be improved by more than 10 times, and meanwhile, due to the existence of the Carrier RNA, trace RNA templates can be particularly protected, and the attack probability of RNase to the trace RNA templates is reduced. However, in the experimental process of the present invention, it was found that carrier RNA affects the efficiency of the subsequent library construction step, and at the same time, carrier RNA is not easily removed in the subsequent step, affecting the nucleic acid quantification result, so carrier RNA is not added in the sample processing process.

In one embodiment, the method in step (b), during nucleic acid elution, nucleic acid elution is performed using a volume of 40. mu.l or less, preferably 20. mu.l of eluent to obtain the total nucleic acids extracted.

In the technical scheme of the invention, a smaller elution volume is adopted, so that the final nucleic acid can have higher concentration, and the utilization efficiency of the nucleic acid in the cDNA synthesis step is improved.

In one embodiment, the cDNA obtained by reverse transcription is amplified with random primers to obtain a loading sufficient for the next high throughput sequencing. In step (c), the volume ratio of the total extracted nucleic acids to the random primers added is 25: 3.

the random primer component is 6 bases long random polynucleotide 5'd (N6)3' [ N ═ A, C, G, T ], and the random primer used in the present invention is the self-contained component of NEBNext Ultra II RNA First Strand Synthesis Module reagent.

The ratio of the initial nucleic acid to the random primer probably influences the utilization rate of the nucleic acid, and the invention optimizes the ratio of the initial nucleic acid to the random primer and improves the utilization rate of the nucleic acid.

In one embodiment, in the step (b), the extraction of the total nucleic acid of the sample is performed using a QIAamp MinElute Virus Spin Kit from QIAGEN; the eluent is Buffer AVE.

In one embodiment, the purification in step (d) is performed using AMPure XP magnetic beads (Agencourt AMPure XP nucleic acid purification kit), wherein the Agencourt AMPure XP magnetic bead reagents and the product synthesized in step (c) have a volume of 1: 1.8.

in one embodiment, in the step (e), the sample DNA library is constructed using the MGIEasy enzyme digestion DNA library preparation kit.

In another aspect, the invention provides a metagenomic library obtained by the above library construction method.

In yet another aspect, the present invention provides a method for detecting the etiology of a respiratory tract pharyngeal swab sample for non-disease diagnostic use based on metagenomics, said method comprising the steps of:

(1) the aforementioned library building method;

(2) performing high-throughput sequencing and performing bioinformatics analysis on sequencing results.

In one embodiment, the high throughput sequencing is performed on a BGISEQ-500 sequencer; and performing metagenome comparison analysis on BGISEQ-500 sequencing data by using Centrifuge software, and performing visual analysis on comparison results by using Pavian.

Has the advantages that:

the invention is based on the DNA-RNA co-sequencing principle, can realize pathogen detection of throat swab sample metagenome through single reaction, and can realize simultaneous detection of DNA and RNA pathogens.

The database construction method and the detection method provided by the invention directly perform cDNA synthesis on total nucleic acid extracted from throat swabs (without removing host DNA), convert all RNA in a sample into cDNA for database construction and detection, and save detection time and cost.

The method optimizes the sample pretreatment process and the cDNA synthesis process, and has high nucleic acid extraction efficiency and accurate result. And high-throughput sequencing and analysis are carried out by subsequently combining a BGISEQ-500 platform, and the detection result has higher consistency with the result of a fluorescence quantitative PCR method.

The method can realize the simultaneous detection of different types of pathogens in the throat swab and has good application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the fluorescence quantitative PCR detection result of mixed pharyngeal swab of healthy human according to the embodiment of the present invention (wherein 1: Staphylococcus aureus, 2: Klebsiella pneumoniae, 3: influenza A virus, 4: human adenovirus, 5: Aspergillus fumigatus, 6: human);

FIG. 2 is a scattergram of single pathogen simulation sample dilution factor and Ct value provided by the embodiment of the present invention;

FIG. 3 is a comparison of the sequencing depth of a Staphylococcus aureus single pathogen sample provided by an embodiment of the invention with reference genome ATCC 27217;

FIG. 4 is a graph showing the results of electrophoresis of the effect of the absence or addition of Carrier RNA on nucleic acid extraction and pooling in comparative example 1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

1. Clinical sample nucleic acid extraction

Extracting total nucleic acid of the sample by using a QIAamp MinElute Virus Spin Kit of QIAGEN, and adjusting the experimental scheme to a certain extent according to the instruction, wherein the specific operation steps are as follows:

an initial amount of 500. mu.L of clinical specimens such as pharyngeal swab/sputum/alveolar lavage fluid was equilibrated to room temperature and mixed well before use.

Adding 10 μ L lysozyme with concentration of 50mg/mL, mixing well, and incubating at 37 deg.C for 15 min. The lysozyme was obtained from Sigma-Aldrich lysozyme in dry powder, cat # L6876-1G, and the lysozyme in dry powder was dissolved in 10mM Tris-HCl, pH 8.0 to a final concentration of 50 mg/ml.

Adding 62.5 mu L of QIAGEN protease and 500 mu L of Buffer AL, and uniformly mixing for 15s by oscillation to ensure that the final liquid is uniform; incubate at 56 ℃ for 15min and briefly centrifuge.

Adding 625 μ L of anhydrous ethanol, shaking and mixing for 15s, standing at room temperature for 5min, and centrifuging for a short time.

The sample was transferred to an adsorption column, centrifuged at 8000rpm for 1min and the waste liquid discarded.

The collection tube was replaced, 500. mu.L of Buffer AW1 was added, and centrifugation was carried out at 8000rpm for 1 min.

The collection tube was replaced, 500. mu.L of Buffer AW2 was added, and centrifugation was carried out at 8000rpm for 1 min.

The collection tube was replaced, 500. mu.L of absolute ethanol was added, and the mixture was centrifuged at 8000rpm for 1 min.

The collection tube was replaced and idled at 14000rpm for 3 min.

The column was placed in a new 1.5mL EP tube, uncapped and dried at room temperature for 5 min.

Add 40. mu.L Buffer AVE to the center of the adsorption column membrane, let stand at room temperature for 5min, and centrifuge at 14000rpm for 1 min.

Sucking the eluate out with a pipette, adding into the center of the adsorption column membrane again, standing at room temperature for 5min, centrifuging at 14000rpm for 1min to obtain a liquid, which is total nucleic acid of the sample, and immediately performing the next experiment or storing at-20 deg.C.

cDNA Synthesis and magnetic bead purification

2.1 reverse transcription Single-Strand Synthesis (NEBNext Ultra II RNA First Strand Synthesis Module)

mu.L of the total nucleic acid extracted + 1.5. mu.L of random primers (primers from NEBNext Ultra II RNA First Strand Synthesis Module), pipetting, mixing, centrifuging briefly, reacting at 65 ℃ for 5min, covering with a hot lid at 105 ℃, and immediately placing on ice after the reaction.

Configuring a first chain synthesis reaction system:

after mixing on ice with a pipette, the sample was placed into a PCR instrument to start the reaction.

Reaction conditions are as follows:

2.2 Second Strand Synthesis (NEBNext Ultra II Non-directed RNA Second Strand Synthesis Module)

Configuring a second-chain synthesis reaction system:

after mixing on ice with a pipette, the reaction was carried out for 1h at 16 ℃ without hot-capping (lid temperature <40 ℃).

2.3 magnetic bead purification

144 μ L (1.8 ×) of Agencour AMPure XP magnetic beads were added to 80 μ L of the reaction product, pipetted and mixed well, and incubated at room temperature for 5 min.

Centrifuging for a short time, placing the centrifuge tube on a magnetic frame, standing for 2-5min until the liquid is clear, carefully sucking and discarding the supernatant.

The tube was kept on a magnetic stand, 200. mu.L of freshly prepared 80% ethanol was added, allowed to stand for 30s, and the supernatant was carefully discarded with suction.

Adding 200 mu L of freshly prepared 80% ethanol again, standing for 30s, carefully sucking and discarding the supernatant, sucking the liquid at the bottom of the tube as much as possible by using a small-range pipettor, and drying at room temperature for about 5min until the surfaces of the magnetic beads have no reflection and no cracking.

The centrifuge tube was removed from the magnetic frame, 20. mu.L of nucleic acid-free water was added to elute the DNA, and the mixture was gently pipetted and mixed, followed by incubation at room temperature for 5 min.

And (3) performing short-time centrifugation, placing the centrifugal tube on a magnetic frame, standing for 2-5min until the liquid is clear, and sucking out 18 mu L of supernatant to obtain the DNA.

The dsDNA concentration of the purified nucleic acids was quantified using the Qubit 3.0.

Construction and sequencing of BGI sequencing platform DNA library

The method comprises the steps of constructing a sample DNA library by adopting a MGIEasy enzyme digestion DNA library preparation reagent set (an experimental process refers to a reagent specification), sequencing on a BGISEQ-500 sequencer by adopting a PE100 sequencing strategy, and predicting and distributing 100M reads data volume for each sample.

4. Analysis of sequencing results

And performing metagenome comparison analysis on BGISEQ-500 sequencing data by using Centricugage software, setting the shortest length of comparison hit to be 60bp, and setting a comparison database to be an nt database carried by the Centricugage. The alignment was visually analyzed using Pavian.

Examples

Specific pathogens with different concentrations are artificially added into a healthy human pharyngeal swab, metagenome pathogen detection and fluorescence quantitative PCR (polymerase chain reaction) pathogen detection based on the method are respectively carried out, and the result shows that the detection result of the method has higher consistency with the result of the fluorescence quantitative PCR method, and the detected pathogen sequence number has higher correlation with the pathogen concentration in a sample and the Ct value of the fluorescence quantitative PCR.

On the basis, a plurality of different types of pathogens are added into the throat swab of a healthy person at the same time, and the method can also detect the pathogens at the same time, so that the method is proved to be capable of realizing the simultaneous detection of different types of pathogens in the throat swab, and is expected to be further popularized and applied to pathogen metagenome detection of a throat swab sample.

The specific steps and results of the experiment are as follows:

metagenomic sequencing assays were performed by adding mock samples of known concentrations of pathogen to pharyngeal swabs. After 7 healthy human pharyngeal swab samples are collected and mixed, 5 different respiratory pathogens of virus (human adenovirus type 55 and influenza A virus H1N 1), bacteria (staphylococcus aureus CICC 21600, Klebsiella pneumoniae KBJ001 (clinical isolates stored in the laboratory) and fungi (Aspergillus fumigatus Af293) with known concentrations and gradient dilution are added, and respiratory single pathogen and multi-pathogen simulation samples are respectively established.

The nucleic acid extraction and library-building sequencing of the method are carried out on the simulation sample, the pathogen is detected by adopting a real-time fluorescence quantitative PCR method on the sample, the detection effect of the DNA/RNA co-sequencing method on different types of pathogens is evaluated, and the influence of different concentrations and different species on the detection result of the metagenome sequencing pathogen is analyzed.

The result shows that the target pathogen sequence number detected by the method of the invention and the original pathogen concentration in the sample and the Ct value detected by the fluorescence quantitative PCR all present a certain linear relation aiming at the single pathogen sample; aiming at the multi-pathogen samples, the method detects 5 target pathogen sequences in 5 multi-pathogen samples with different dilution gradients, proves that the method can realize the simultaneous detection of various types of pathogens in the samples, and also provides a new method for monitoring acute respiratory tract infection pathogens.

1. Fluorescence quantitative PCR detection of mixed sample throat swab of healthy person

After the pharyngeal swabs of healthy people are mixed, six species including staphylococcus aureus, klebsiella pneumoniae, influenza a virus, human adenovirus, aspergillus fumigatus and human are detected by using fluorescence quantitative PCR, and the fluorescence quantitative PCR result is shown in figure 1.

The red curve is a positive control amplification curve, and the green curve is a sample amplification curve. As can be seen from the figure, in the healthy human mixed sample throat swab, no amplification occurs in Staphylococcus aureus, Klebsiella pneumoniae, influenza A virus, human adenovirus and Aspergillus fumigatus, but an amplification curve occurs in host (human) genes in the sample, which indicates that the sample contains host nucleic acid, but the fluorescence quantitative PCR detection results of 5 pathogens of Staphylococcus aureus, Klebsiella pneumoniae, influenza A virus, human adenovirus and Aspergillus fumigatus are negative.

2. Establishment of single pathogen simulation sample and fluorescent quantitative PCR detection

Staphylococcus aureus, streptococcus pneumoniae, aspergillus fumigatus, influenza A virus and human adenovirus 5 simulated pathogens are added into a throat swab of a healthy person after being diluted in a gradient manner, and each pathogen is provided with 7 gradients, so that 35 samples are counted. The samples were subjected to nucleic acid extraction and fluorescent quantitative PCR detection of the corresponding pathogens, and the results are shown in Table 1. The minimum detection limit of Aspergillus fumigatus is 10³Dilution multiple, the lowest detection limit of staphylococcus aureus and klebsiella pneumoniae is 10⁴Dilution multiple, minimum detection limit of influenza A virus is 10⁵Dilution factor, human adenovirus can be detected under 7 dilution factors. And (3) drawing a scatter diagram of the pathogen dilution multiple and the Ct value of the simulated sample, wherein a certain linear relation is formed between the pathogen dilution multiple and the Ct value (figure 2).

TABLE 1 fluorescence quantitative PCR detection Ct value of pathogen in single pathogen simulation sample

3. Establishment of multi-pathogen simulation sample and fluorescent quantitative PCR detection

5 multi-pathogen samples were established by simultaneously adding 5 different dilutions of the simulated pathogen to healthy human pharyngeal swabs with reference to Ct values for single pathogen simulated samples (Table 2). The fluorescence quantitative PCR detection is carried out on the content of each pathogen in the multi-pathogen sample, and the result is shown in Table 3. 5 pathogens were detected simultaneously in samples 1, 2, 3 and 4, and the Ct values increased with increasing dilution factor. No 5 pathogens were detected in sample 5, considering the pathogen nucleic acid content in the sample below the limit of fluorescent quantitative PCR detection.

TABLE 2 pathogen concentration in Multi-pathogen simulation samples

TABLE 3 fluorescence quantitative PCR detection Ct value of pathogen in multi-pathogen simulation sample

4. Simulation of sample sequencing results

Metagenomic sequencing according to the method of the invention was carried out on 30 selected mock samples (table 4). The average sequence number of 30 simulation samples is 42,455,255, and the average host sequence percentage is 68.27%. The sequence numbers of 5 pathogens including staphylococcus aureus, klebsiella pneumoniae, aspergillus fumigatus, influenza a virus and human adenovirus in the sample were counted, and the results are shown in table 5. In the single pathogen simulation sample, the number of pathogen sequences decreased with increasing dilution factor. In the multi-pathogen samples, 5 samples simultaneously detect the sequences of 5 pre-added pathogens.

TABLE 432 example sequencing sample information

TABLE 5 number of sequences detected for different pathogens in samples

To evaluate the relationship between the pathogen dilution concentration and the pathogen genome sequencing depth, taking a staphylococcus aureus single pathogen sample as an example, the staphylococcus aureus sequence in the sample is compared with a staphylococcus aureus reference genome ATCC 27217 to obtain the sequencing depth of 5 samples to the staphylococcus aureus genome, and the sequencing depth values are plotted as a broken line graph (fig. 3). As can be seen from the figure, the sequencing depths of all the sites of the samples SA-0 and SA-1 are greater than 1, while the average value of the sequencing depths of the samples SA-2, SA-3 and SA-4 is less than 1, which indicates that when the concentration of the pathogen in the sample is high, the complete sequence of the genome of the pathogen can be detected by the metagenome sequencing method, but as the concentration of the pathogen is reduced, the sequencing depth of the pathogen is reduced, only partial genome sequence fragments can be detected, and complete genome information cannot be obtained. In addition, we observed that the sequencing depth and the dilution factor of the pathogen do not vary linearly, and when the concentration of the pathogen in the sample is high, the sequencing depth significantly decreases with the decrease of the concentration of the pathogen, but when the concentration of the pathogen in the sample is low, the sequencing depth does not significantly vary with the concentration of the pathogen, and when a higher sequencing depth is required, the amount of sequencing data is further increased.

Comparative example 1

Carrier RNA validation experiment:

to verify the effect of adding Carrier RNA on nucleic acid extraction and banking, the following five samples were designed:

sample 1: pure water and carrier RNA are added to carry out nucleic acid extraction and library building;

sample 2: clinical samples and no carrier RNA are added, and nucleic acid extraction and library establishment are carried out;

sample 3: extracting nucleic acid from a clinical sample by adding carrier RNA, adding RNase digestion after extraction, and then establishing a library;

sample 4: clinical samples + carrier, nucleic acid extraction and library establishment are carried out, and the end repairing step is stopped;

sample 5: clinical samples + carrier, nucleic acid extraction and library establishment are carried out, and the step of adaptor connection is stopped;

and (3) reserving a part of samples at the following four time nodes (all links in the library are established according to the time sequence) for agarose electrophoresis quality inspection, wherein the left one is a 2000bp DNA marker:

1: after the physical ultrasound carries out segment breaking

2: magnetic bead fragments are screened

3: after PCR purification

4: original nucleic acid

The results are shown in FIG. 4: the original nucleic acid can be seen to have larger fragments, most of the fragments after physical interruption are concentrated in a range of 1000bp from 100-. No. 1 is pure water + adds Carrier RNA, and No. 3 clinical sample + adds Carrier RNA carries out nucleic acid extraction, adds one step of RNase digestion after the extraction, explains that the main reason causing unusual large fragment is Carrier RNA, and RNase digestion also can not remove Carrier RNA. The sample No. 4 and the sample No. 5 are the same sample of different time nodes, which shows that the sample added with Carrier RNA has abnormal large fragments, and the problem occurs in the link of end repair. The electrophoresis image of sample No. 2 after PCR purification shows that the sample without Carrier RNA can be successfully used for library construction. Based on the results, the scheme omits the link of adding Carrier RNA in the nucleic acid extraction process.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.