CN114277092A

CN114277092A - RNA virus macro transcriptome database building method based on nanopore sequencing platform and application

Info

Publication number: CN114277092A
Application number: CN202111473132.2A
Authority: CN
Inventors: 周水莲; 潘吾思; 戴岩; 李振; 童桢开; 李诗濛; 任用
Original assignee: Jiangsu Xiansheng Diagnostic Medical Instrument Co ltd; Jiangsu Xiansheng Diagnostic Technology Co ltd; Jiangsu Xiansheng Medical Devices Co ltd
Current assignee: Jiangsu Xiansheng Diagnostic Medical Instrument Co ltd; Jiangsu Xiansheng Diagnostic Technology Co ltd; Jiangsu Xiansheng Medical Devices Co ltd
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-04-05
Anticipated expiration: 2041-12-02
Also published as: CN114277092B

Abstract

The invention belongs to the field of gene sequencing, and particularly relates to a RNA virus macro-transcriptome database building method of a nanopore sequencing platform. Aiming at the problems that a nanopore sequencing platform is low in flux, a clinical sample is high in host content, and total RNA contains a large amount of ribosomal RNA, incomplete host cell DNA removal and ribosomal RNA removal are carried out on the clinical sample through reverse enrichment and the like; aiming at the problems of low viral load of clinical samples and high diversity of RNA viral genomes, cDNA is positively enriched by adopting single primer isothermal amplification and the like. According to the invention, through forward and reverse enrichment steps, a nanopore sequencing platform RNA virus macro-transcription set-up library detection process is successfully established, rapid clinical identification of unknown RNA virus pathogens is satisfied, and the method is suitable for popularization and application.

Description

RNA virus macro transcriptome database building method based on nanopore sequencing platform and application

Technical Field

The invention belongs to the field of gene sequencing, and particularly relates to a RNA virus macro-transcriptome database building method of a nanopore sequencing platform and application thereof.

Background

Infectious diseases are a major cause of human illness and death and have been of great clinical interest. In most cases of severe infection, RNA viruses are the major pathogens, such as the global pandemic of seasonal influenza and the worldwide pandemic of the novel coronavirus (SARS-CoV-2), posing serious challenges to the world's economy and human health, making accurate and rapid identification of the pathogen a focus of attention.

The main methods for identifying viruses in the traditional clinical microbiological laboratory are virus separation culture, immunofluorescence detection, PCR molecular detection and other technologies, but the methods have limitations. The virus isolation culture and immunofluorescence detection are low in sensitivity and time-consuming; PCR molecular detection is based only on differential diagnosis by clinicians for specific pathogen types, and the detection capability of rare and novel viral pathogens is limited. And the RNA virus in a sample can be comprehensively detected without bias by the macrotranscriptome sequencing of the RNA virus, rare and novel pathogens which cannot be detected by the traditional means can be found, and more comprehensive and accurate reference is provided for clinical decision. In addition, the macro-transcriptome sequencing of the RNA virus can further research the immune response of the virus and a host, provides possibility for searching disease markers and promotes the development of accurate diagnosis and treatment of infectious diseases.

Compared with the traditional etiology identification method, the metagenome sequencing identification method has the advantages of short identification period, low requirements on the technical level of operators and identification personnel and the like. And the metagenomic sequencing overcomes the defects of the traditional etiological diagnosis, namely a series of differential diagnosis made by a clinician according to the clinical manifestations of patients, and is increasingly applied to the identification of microorganisms, particularly pathogenic microorganisms of unknown causes. However, the existing second-generation sequencing-based technology has long sequencing analysis period, long reading length and large instrument investment, which limits the feasibility of the application of the technology in clinical rapid detection. The third-generation sequencing technology PacBio is greatly improved in sequencing and reading, but the library building process is complex, the defect of long sequencing period exists as the second-generation sequencing technology, the data downloading is finished within dozens of hours, and the clinical rapid identification is difficult to meet due to the follow-up analysis time.

The nanopore sequencing technology developed in recent years can make up for the disadvantages of other sequencing platforms, has the characteristics of reading of a sequencing super-long sequence, real-time data generation and biological information analysis, and small and portable equipment, has the characteristics of improving the accuracy of pathogenic microorganism detection and shortening the report period, is suitable for hospital and field detection, conforms to the requirement scene of clinical infection diagnosis, and is expected to become the infection diagnosis technology of the next generation.

In the research field of RNA virus macro transcriptome, the amount of host nucleic acid in clinical samples is extremely high, the total RNA contains a large amount of ribosomal RNA (rRNA for short), the viral load in the samples is extremely low, RNA virus Reads cannot be detected or the detected amount is very small, and the establishment of a library of RNA virus macro transcriptome becomes a challenge. In practice, an efficient and convenient macro transcriptome database building method for RNA viruses is urgently needed for accurate diagnosis and treatment of infectious diseases.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

Aiming at the problem that the RNA virus of a clinical sample is difficult to detect, the core problem or purpose to be solved by the invention is to set up a set of detection flow of the RNA virus macro-transcription component library of the nanopore sequencing platform so as to improve the sensitivity of RNA virus detection of the nanopore sequencing platform, meet the clinical requirement for rapidly detecting the sample suspected of RNA virus infection, and be suitable for popularization and application.

According to the invention, through the operation steps of reverse enrichment and forward enrichment, the detection process of establishing the RNA virus macro-transcriptome library of the nanopore sequencing platform is successfully established, the sensitivity of RNA virus macro-transcriptome detection is improved, the rapid clinical identification of unknown pathogens is met, and the method is suitable for popularization and application.

Specifically, the technical scheme adopted by the invention is as follows:

the invention firstly provides a RNA virus macro transcriptome database building method based on a nanopore sequencing platform, which comprises the following steps:

1) removing host DNA from the sample;

2) a nucleic acid extraction step;

3) a cDNA synthesis step;

4) enrichment of cDNA;

5) eliminating human rRNA;

further, the method can also comprise the following steps:

6) and (5) library construction.

Further, the step of 1) removing the host DNA from the sample is a step of incompletely removing the host DNA.

Further, in the step 1), DNase is adopted to carry out incomplete removal of host DNA on the sample;

preferably, the DNase is added in an amount of 26U-260U, so that incomplete host removal corresponding to clinical samples can be effectively realized.

Further, the 2) nucleic acid extraction step can adopt RNA virus extraction by adopting Zymo _ D7021 or Qiagen _ 57704; preferably, Zymo-D7021 is used.

Further, in the step 4) of cDNA enrichment, a single primer isothermal amplification method is adopted to actively enrich cDNA;

furthermore, the single-primer isothermal amplification method is an SPIA isothermal amplification method for actively enriching cDNA.

Preferably, the SPIA Isothermal amplification uses a NuGEN Trio RNA-seq (Single Primer Isotermal amplification) kit.

Further, 5) removing the human rRNA by a probe method.

Further, the probe method comprises a hybridization capture step and a specific nuclease treatment step.

Preferably, the hybrid capture can use NuGEN Trio RNA-seq (transcript deletion with AnyDeplete) kit.

Further, the library constructing step of 6) adopts a PCR-free library building computer kit; more preferably, SQK-LSK109 kit is used.

Further, the samples include, but are not limited to: a body fluid sample, an alveolar lavage fluid sample, a sputum sample, a cerebrospinal fluid sample.

Further, the RNA virus macro-transcriptome is a nanopore sequencing platform based RNA virus macro-transcriptome.

The invention also provides the application of any one of the methods in RNA virus macro-transcriptome sequencing.

The invention has the beneficial technical effects that:

(1) the invention has high host content and total RNA of clinical samples contains a large amount of ribosome RNA (rRNA), and incomplete host cell DNA removal and rRNA removal are carried out on the clinical samples through reverse enrichment.

(2) The invention actively enriches cDNA by adopting a single primer isothermal amplification method aiming at low viral load of clinical samples and high diversity of RNA viral genomes.

(3) The invention effectively improves the sensitivity of RNA virus macro-transcriptome detection through reverse enrichment and forward enrichment, meets the clinical identification of unknown pathogens, and is suitable for popularization and application.

(4) The invention carries out detailed parameter optimization on the whole process to obtain a set of method process which is suitable for a nanopore sequencing platform and used for RNA virus macro-transcription library building and sequencing analysis.

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 is a schematic diagram of an experimental procedure according to the present invention;

FIG. 2 is a graph comparing the results of each thousand Reads for different viruses of the library;

FIG. 3 is a comparison graph of the detection results of the average coverage depth of sequencing of different on-machine library kits;

FIG. 4 details of virus identification and coverage map of virus identification for sample 187;

FIG. 5 sample 725 details of virus identification and a virus identification coverage map;

FIG. 6 sample A1 details of virus identification and a coverage map of virus identification;

FIG. 7 sample B1 virus identification details and a virus identification coverage map;

FIG. 8 sample C1 virus identification details and a virus identification coverage map;

FIG. 9 shows the detailed results of virus identification and the coverage map of virus identification for the cell quality control product D1;

FIG. 10 details of virus identification and a virus identification coverage map of the cell quality control product E1;

FIG. 11 shows the detailed results of virus identification and the coverage map of virus identification for the cell quality control F1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, 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.

The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The invention relates to a RNA virus macro-transcriptome database building method based on a nanopore sequencing platform, which mainly comprises the following steps:

1) removing host DNA from the sample; 2) a nucleic acid extraction step; 3) a cDNA synthesis step; 4) enrichment of cDNA; 5) eliminating human rRNA; 6) and (5) library construction.

In some embodiments, in step 1) removing the host DNA from the sample, the sample contains a large amount of host DNA and the RNA virus is in low abundance and only a very small fraction. On one hand, only a very small part of obtained sequencing data can be used for species identification due to host DNA interference, on the other hand, the extracted nucleic acid is high, the input volume of subsequent DNase digestion is also influenced, the input amount of total RNA pathogens is further influenced, the detection sensitivity of macro-transcription library construction of low-abundance RNA viruses is directly influenced, and the host DNA is required to be incompletely removed through analysis. For example, a combination of (i) a reagent DH + a reagent SS + a reagent XX (liberation and degradation of host DNA) or (ii) a combination of (ii) a reagent SS + a reagent XX (degradation of host DNA) is adopted, and (ii) a combination of (ii) a reagent SS + a reagent XX (degradation of host DNA) is preferably adopted for viruses, so that incomplete removal of host DNA is carried out; preferably, the combination (ii) is used.

In some embodiments, clinical RNA samples are limited in access and subsequently optimized library-building procedures are performed using cell-cultured virus quality controls simultaneously.

In some embodiments, 2) the nucleic acid extraction step, with low clinical viral load, qPCR assesses kit efficacy, demonstrating that Zymo-D7021 is superior to Qiagen-57704, and therefore, Zymo-D7021 may be preferred for RNA virus extraction.

In some embodiments, in the step 4) of cDNA enrichment, in the step of actively enriching cDNA by using a single-primer isothermal amplification method, the results of qPCR and on-machine detection show that SPIA > SISIPA, preferably SPIA isothermal amplification method is used for actively enriching cDNA.

In some embodiments, 5) in the step of human rRNA elimination, the flux of the nanopore sequencing platform is low, the total RNA contains a large amount of conserved ribosomal RNA (rRNA), and the rRNA is removed by a probe method, so that the Reads of RNA viruses is improved.

In some embodiments, 6) in the library construction step, the PCR FREE kit is selected and tested according to the sequencing data amount, Unmapped, virus Reads and sequencing average coverage depth, so that the PCR FREE kit has better effect, such as SQK-LSK109> SQK-PBK004, and thus SQK-LSK109 kit is preferably used for library construction.

Further, the method for identifying the macro transcriptome can be applied to the fields including but not limited to clinical research, scientific research and the like.

It will be appreciated that the inventive methodology is not limited to sample types, but is applicable in the field involving macrotranscriptome rna viruses, for example, samples may include, but are not limited to: a body fluid sample, an alveolar lavage fluid sample, a sputum sample, or a cerebrospinal fluid sample, among others.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

The following examples and experimental examples relate to apparatus comprising: biological safety cabinet, vibration metal bath, pipettor, centrifuge, breaking instrument, super clean bench, PCR instrument, magnetic frame, GridION, Qubit 4.0, refrigerator etc..

The related reagent comprises: a host removal and microbiome DNA isolation Kit, a nucleic acid extraction Kit, a SPIA amplification Kit, an RNA-Seq transcript deletion with AnyDesplete Kit (Human rRNA), a Universal End prediction Module, a Universal Adapter Ligation Module, a recombinant DNA isolation Kit, a nucleic acid amplification Kit, a recombinant DNA, and a recombinant DNA, a nucleic acid, a recombinant DNA, and a recombinant DNA, a Kit, a recombinant DNA, and a recombinant DNA, a recombinant,

GXL DNA Polymerase, ONT library construction kit, AMPure XP purified magnetic bead and Qubit^TMA detection kit, an ONT sequencing chip and the like.

Example 1RNA Virus derhosting procedure optimization

Considering that the sample contains a large amount of host DNA, the abundance of RNA virus is low and only a very small part is occupied. On one hand, due to the interference of host DNA, only a very small part of obtained sequencing data can be used for species identification, on the other hand, the extracted nucleic acid is high, the input volume of subsequent DNase digestion is also influenced, the input amount of total RNA pathogen is further influenced, the detection sensitivity of macro-transcription library construction of low-abundance RNA virus is directly influenced, and therefore the incomplete removal of the host DNA is considered.

In this embodiment, host DNA is incompletely removed from clinical Influenza B virus samples (IVB) and Rhinovirus samples (Rhinovirus RhV) so as to screen a combination that can not only incompletely remove host DNA but also has a small influence on viruses.

The invention sets two combinations as follows: combining a saponin and DNase combined mode, and carrying out host DNA dissociation and degradation for experimental purposes; combination of two to only the host mode for DNase degradation.

The two combined operation flows set by the invention are as follows:

combining a flow: taking 500 mu L of sample, centrifuging for 10min at 4 ℃ of 18000 rcf; carefully transferring the supernatant to a new 2mL centrifuge tube; add 10. mu.L of 5% DH; adding 56 mu L of SS, adding 1-10 mu L of solution XX, immediately mixing uniformly, and incubating at 37 ℃ and 1000rpm for 10 min.

Combining a flow II: taking 500 mu L of sample, centrifuging for 10min at 4 ℃ of 18000 rcf; carefully transferring the supernatant to a new 2mL centrifuge tube; adding 56 mu L of SS, adding 1-10 mu L of solution XX, immediately mixing uniformly, and incubating at 37 ℃ and 1000rpm for 10 min.

The experimental design is shown in table 1:

table 1: optimized different combination forms of RNA virus decoating process

Combination of	Reagent DH + reagent SS + reagent XX	Host DNA liberation and degradation
			Combination 2	Reagent SS + reagent XX	Host DNA degradation

Remarking: the component of reagent DH is saponin, reagent SS is DNase Buffer, and reagent XX is DNase.

The results of the experiment are shown in table 1:

table 2: qPCR result after host removal of different combinations of clinical RNA virus samples

qPCR validated combined test results: RhV clinical specimens Human and viral targets were quantified to see if the effect of reagent DH removal was negligible; in the aspect of IVB clinical sample Human quantification, the influence of reagent DH removal is negligible, but the difference of the virus target is 4.48CT, the difference is large, and the risk of reagent DH on virus removal hosts is prompted. In summary, the invention preferably combines reagents SS + XX (DNase Buffer + DNase) with the reagent for viruses, i.e. incomplete removal of host DNA is performed only by DNA degradation without involving DH-based DNA dissociation step, and a preferred amount of DNase added is 26U to 260U, which enables correspondingly effective incomplete removal of host.

Example 2 virus off-host procedure the necessity for building a nanopore sequencing platform RNA virus macro-transcriptome library

In the process of sample extraction, besides a very small amount of RNA viruses are extracted, the sample also contains a large amount of human genome nucleic acids, DNA of other pathogens and ribosomal RNA (rRNA), and if the parts are not removed, a large amount of sequencing space is occupied, so that the RNA detection is influenced; however, in the process of host removal, part of RNA viruses are also removed, which affects the load and detection of RNA viruses, so how to select host removal and the removal degree in practice affects the final result.

The purpose of this embodiment is: according to the optimized virus host-removing flow reagent combination II in the embodiment 1, whether the host-removing flow is necessary for constructing an ONT platform RNA virus macro-transcription component library or not is tested aiming at a clinical sample.

Table 3: effect of non-decovered and decovered clinical samples on viral load

Table 4: clinical sample uncarved and uncarved nanopore sequencing platform library building and computer result

The result shows that clinical IVB samples are respectively tested for non-host removal and host removal process library building, and from the sequencing off-line result in 1 hour, the target pathogen is not detected in the non-host removal, the IVB can be well detected in the host removal, and the sequence number is 442.

In summary, in the RNA virus macro-transcript building library based on the nanopore sequencing platform, although the host removing process reduces the virus load, from the point of the computer result, the host removing process is very necessary for building the RNA virus macro-transcript building library of the nanopore sequencing platform.

Example 3 viral nucleic acid extraction reagent screening

The metagenome clinical sample has low virus load, and in practice, the existing RNA virus nucleic acid extraction kit is not suitable for RNA virus nucleic acid extraction of the metagenome, and a high-quality RNA nucleic acid template is required for obtaining a high-quality sequencing result. The RNA virus nucleic acid extraction kit of different manufacturers has great difference in extraction effect, extraction time and the like, so that the effective detection of pathogens in the subsequent RNA virus sequencing stage is influenced. The difference of the effect of extracting the pathogene from the RNA virus nucleic acid is dozens of times to thousands of times, which not only influences the sensitivity of the RNA virus macro-transcriptome process, but also can cause the omission of the RNA pathogene to socioeconomic and immeasurable and even life-threatening loss of patients. Therefore, the present example explores and tests two RNA virus nucleic acid extraction kits, and adopts qPCR to evaluate the extraction effect of the kit.

The experimental scheme is as follows: alveolar lavage fluid sample Spike in RNA virus

1mL of alveolar lavage fluid sample is put into a 1.5mL centrifuge tube, 100 μ L of Enterovirus 71 (Enterovirus 71, EV71) and human Parainfluenza 3 (Parailnza virus type 3, PIV3) quality control products (copy number is 1 ^ 10^5copies/mL) are added, nucleic acid extraction of Zymo _ D7021 and Qiagen _57704 is carried out on average by dividing into two parts, and the kit is commercially available.

Table 5: qPCR result for extracting nucleic acid from different kit viruses

As shown in Table 5, Zymo-D7021 is obviously superior to Qiagen-57704 in the quantitative result of the virus target, and the difference of the extraction effects of the viruses is significant, and the difference of the quantitative result evaluation is 3-10 CTs, and the difference of the extraction efficiency of the RNA viruses is about 10-1000 times.

In the extraction time, the nucleic acid extraction time of Zymo-D7021 is about 0.5 hour, the nucleic acid extraction time of Qiagen _57704 is about 1.5 hours, and Zymo-D7021 can better meet the requirement of quick detection of clinical infection samples.

In summary, Zymo-D7021 is preferred for RNA virus extraction in the present invention, because of the combination of the extraction effect and the extraction time.

Example 4 cDNA enrichment necessity for building a nanopore sequencing platform RNA Virus Macro-transcriptome library

The cDNA has less virus content, and the direct library construction and sequencing may not be effective, and this example contrasts and tests the influence of direct library construction and machine installation of cDNA and machine installation after cDNA enrichment on the virus sequencing result.

Table 6: influence of cDNA enrichment on RNA virus macrotranscriptome sequencing result of construction of nanopore sequencing platform

The experimental results are shown in Table 6, and compared with the three treatment conditions, the number of sequences detected by SPIA enrichment of Rhinovirus A and the relative abundance of cDNA in the method 3 are obviously better than those detected by methods 1 and 2 without cDNA enrichment. Thus, in RNA virus macrotranscriptome sequencing, cDNA enrichment has a significant gain effect on RNA virus enrichment.

Example 5 rRNA knock-out necessities for building a nanopore sequencing platform RNA viral transcriptome library

The transcriptome comprises a collection of different types of RNA molecules, coding RNA and non-coding RNA. Ribosomal RNA (rrna), which accounts for about 80% of the total RNA, is the largest class of RNA, but usually such RNA is large in molecular weight and is not metabolically active, and if these RNA fractions are not removed, it may occupy a large amount of sequencing space, which may affect RNA detection.

TABLE 7 influence of rRNA knock-out effect on the construction of nanopore sequencing platform RNA virus macro-transcriptome sequencing results

The experimental results show in the table, the detected sequence number and relative abundance of Rhinovirus A after rRNA elimination of cDNA in the method 2 are obviously superior to those of the method 1 without rRNA elimination operation flow under two treatment conditions; in addition, it is clear from the above example 4 that the effect of rRNA elimination alone is not ideal, but the "rRNA elimination + SPIA enrichment" effect is optimal, and the principle is not clear at all. In conclusion, after SPIA enrichment, rRNA elimination is performed on cDNA, so that the effective data volume is remarkably improved, and the rRNA elimination is very key for building a nanopore sequencing platform RNA virus macro-transcription component library.

Example 6 on-Board library reagent selection

Considering that the Nanopore kit is divided into PCR and PCR-free, and whether performing PCR again in the selection of the kit in the machine will further amplify the sequencing result, this example compares the effect of constructing library kit on QK-LSK109(PCR-free) and SQK-PBK004(LP with PCR).

As can be seen from FIGS. 2 and 3, in the selection of the on-line kit, SQK-LSK109(PCR-free) > SQK-PBK004(LP with PCR) is selected from the aspects of sequencing data amount, virus Reads detection and sequencing average coverage depth, so that the SQK-LSK109 kit is preferably used for library on-line in the invention.

Example 7 establishment of the basic Process of the invention

The basic process of the present invention was finally determined by the search of examples 1-6 (the following is only an optimal solution and is not intended to limit the scope of the patent claims).

The basic flow of the experiment is shown in FIG. 1, and the concrete sample macro-transcription library construction and detection method is as follows:

first, sample pretreatment (removal of host DNA):

1. taking 500 mu L of body fluid sample, and centrifuging for 10min at 4 ℃ of 18000 rcf;

2. carefully transferring the supernatant to a new 2mL centrifuge tube;

3. adding 56 mu L of SS, adding 1-10 mu L of solution XX, immediately mixing uniformly, and incubating at 37 ℃ and 1000rpm for 10 min.

And II, nucleic acid extraction, namely extracting RNA virus by adopting Zymo-D7021 according to the kit instruction.

Third and 1/2 chain cDNA Synthesis: the conventional cDNA synthesis process is adopted.

Fourth, cDNA enrichment (SPIA Pre-amplification)

1. In a 0.2mL PCR tube, the following system was prepared: specifically, Trio RNA-Seq (Single Primer Isothermal Amplification kit, purchased from NuGEN) was used.

2. The PCR tube was placed on a PCR instrument for the following reactions:

temperature (. degree.C.)	Time (min)
		4	1
47	90
		80	20
4	Hold

Purification after SPIA Pre-amplification

(1) Adding 33 mu L of nucleic-free water into the PCR tube after the previous step, adding 60 mu L of AMPure XP beads suspension for purification, uniformly mixing, incubating at room temperature for 5-10 minutes, centrifuging for a short time, standing on a magnetic frame, and removing supernatant;

(2) adding 200 μ L of freshly prepared 80% ethanol, and repeating for 2 times;

(3) after the short-time centrifugation, resetting the centrifugal tube on a magnetic frame, removing residual liquid by using a 10-mu-L pipettor, and airing for 2min at room temperature;

(4) adding 12 mu L of DNA Resuspension Buffer, resuspending the magnetic beads, incubating at room temperature for 2min, standing on a magnetic frame, sucking the supernatant, putting into the next step for reaction, and taking the remaining 1 mu L in the previous step for the concentration determination of the Qubit.

Five, human rRNA elimination treatment

1. And (3) probe binding: specifically, Trio RNA-seq (transcript deletion with AnyDesplete) kit was used, and purchased from NuGEN.

(1) In a 0.2mL PCR tube, the following system was prepared:

components	Volume (μ L)
		Anydeplete Buffer Mix	5
Anydeplete Probe Mix	9
		Anydeplete Enzyme Mix	1
DNA Resuspension Sample	10
		Total	25

(2) The PCR tube was placed on a PCR instrument for the following reactions:

2. targeted segment removal (specific nuclease treatment removal)

(1) The following system was added to the PCR tube at the end of the previous step:

components	Volume (μ L)
		Anydeplete Buffer Mix	5
Anydeplete Enzyme Mix II	4
		DNA Resuspension Buffer	16
Samples from the end of the previous step	25
		Total	50

(2) The PCR tube was placed on a PCR instrument for the following reactions:

temperature (. degree.C.)	Time (min)
		60	30
95	5
		4	Hold

3. And (5) purifying.

Sixthly, constructing a library, namely constructing the library by adopting an SQK-LSK109 kit according to the kit instruction.

And seventhly, sequencing analysis, namely computer sequencing and letter generation analysis according to a standard nanopore sequencing computer flow.

Example 8 clinical sample testing

Based on the established RNA virus macro-transcription component library building processing flow, 5 clinical RNA virus samples and 3 cell quality control products which are verified by qPCR are subjected to machine test according to the whole RNA virus macro-transcription component library building flow based on the nanopore sequencing platform, and the test results are shown in the following table and figures 4-11.

According to the invention, 5 parts of clinical samples verified by qPCR and 3 parts of cell culture quality control products are subjected to full-process machine test according to RNA virus macro-transcription component library based on a nanopore sequencing platform, RNA virus infection pathogeny (detected RNA viruses comprise respiratory syncytial virus, rhinovirus, human parainfluenza virus, influenza virus, enterovirus, flavivirus and the like) can be rapidly detected, and the detection process is matched with the qPCR verification result, the RNA virus macro-transcription component library detection process of the nanopore sequencing platform is successfully established, 1 hour of sequencing data shows that the sequence number, relative abundance and coverage of the viruses are good, the clinical requirement on rapid detection of suspected RNA virus infection samples is met, and the method is suitable for popularization and application.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. A method for constructing a library of RNA virus macro-transcriptomes, said method comprising the steps of:

1) removing host DNA from the sample;

2) a nucleic acid extraction step;

3) a cDNA synthesis step;

4) enrichment of cDNA;

5) eliminating human rRNA;

preferably, the method further comprises the following steps:

6) and (5) constructing a library.

2. The RNA virus macro-transcriptome library construction method of claim 1, wherein said 1) sample removal host DNA step is an incomplete host DNA removal step.

3. The RNA virus macro-transcriptome library construction method of claim 2, wherein in the step 1), the sample is subjected to incomplete removal of host DNA by DNase;

preferably, the adding amount of the DNase is 26U-260U, so that the effective incomplete host removal of the corresponding clinical samples is realized.

4. The RNA virus macro-transcriptome library construction method of any one of claims 1 to 3, wherein said 4) cDNA enrichment step actively enriches cDNA using single primer isothermal amplification;

preferably, the single-primer isothermal amplification method is an SPIA isothermal amplification method.

5. The RNA virus macro-transcriptome library construction method of any one of claims 1 to 4, wherein 5) the human rRNA is removed by probe method.

Preferably, the probe method comprises a hybrid capture step and a specific nuclease treatment step.

6. The RNA virus macrotranscriptome library construction method of any one of claims 1 to 5, wherein in said 2) nucleic acid extraction step, RNA virus extraction is performed using Zymo _ D7021 or Qiagen _ 57704; preferably, Zymo-D7021 is used.

7. The method for constructing a library of RNA viruses macro transcriptome of any of claims 1 to 6, wherein in the step of 6) library construction, a PCR-free library construction kit is used.

8. Use of the histobanking method of any one of claims 1 to 7 for sequencing the macrotranscriptome of an RNA virus.

9. The RNA virus macro-transcriptome library construction method of any one of claims 1 to 8, wherein said samples include but are not limited to: body fluid samples, alveolar lavage fluid samples, sputum samples, and cerebrospinal fluid samples.

10. The RNA virus macro-transcriptome library construction method of any one of claims 1 to 9, wherein said RNA virus macro-transcriptome is a nanopore sequencing platform based RNA virus macro-transcriptome.