Method, primer group and kit for sequencing whole genome of adenovirus

Abstract

The invention discloses a method, a primer group and a kit for sequencing an adenovirus whole genome, and belongs to the technical field of DNA (Deoxyribose Nucleic Acid) detection. The method for sequencing the whole genome of the adenovirus comprises the following steps: S1, carrying out a first round of amplification by adopting a first round of amplification primers with sequences as shown in SEQ ID NO.1-SEQ ID NO.93 in a table 1; and S2, carrying out a second round of amplification by adopting the second round of amplification primers with the sequences as shown in SEQ ID NO.94-SEQ ID NO.285 in the table 2. The average length of data sequenced by the method is 0.8 kb, compared with the short read length of a second-generation platform, the uniformity of adenovirus coverage can be greatly improved, meanwhile, the adenovirus type coverage is comprehensive, and the method can be widely popularized.

Description

Technical Field

[0001] The present invention belongs to the technical field of DNA detection, and in particular relates to a method, a primer set and a kit thereof for sequencing the whole genome of an adenovirus. Background Art

[0002] Adenovirus can infect a variety of mammals, including humans. Adenovirus infection can be transmitted through multiple routes such as respiratory transmission, contact transmission, fecal-oral transmission and eye transmission, causing a variety of diseases. Respiratory transmission is through airborne droplets, causing respiratory infections such as colds, laryngitis, bronchitis and pneumonia, with symptoms including fever, cough, sore throat and runny nose. Contact transmission is through contact with contaminated objects or direct contact with infected people, and fecal-oral transmission is through contaminated food or water sources, causing gastrointestinal infections, especially in infants and young children, with symptoms including diarrhea, vomiting and abdominal pain. Eye transmission is through contact with contaminated water or objects, causing conjunctivitis, manifested as red eyes, eye pain and tearing. Adenovirus can also cause other types of infections, such as cystitis, hepatitis and meningoencephalitis.

[0003] The adenovirus genome is a linear double-stranded DNA molecule with a length of approximately 26-45kb. The genome structure includes terminal repeat sequences (ITRs), which play an important role in the replication and packaging of the viral genome. The adenovirus genome is divided into early genes and late genes. Early genes are expressed in the early stages of infection and are mainly involved in viral replication and host cell regulation; late genes are expressed in the late stages of viral replication and mainly encode structural proteins. Key genes include the E1A and E1B genes in the E1 region, which regulate the cell cycle and transcription and promote viral genome replication; the E2 region encodes DNA polymerase, terminase and DNA binding protein, and participates in viral DNA replication; the E3 region is mainly involved in host immune escape and encodes a variety of proteins that regulate host immune responses; the E4 region regulates viral gene expression and DNA repair, and participates in the transcriptional regulation of the viral genome. The L region encodes viral structural proteins, including capsid proteins and other proteins related to viral assembly.

[0004] The replication and life cycle of adenovirus includes the steps of attachment and invasion, early gene expression, DNA replication, late gene expression, and viral release. The virus binds to the host cell receptor through the fibrous protein, enters the cell and enters the cell nucleus through endocytosis. After the viral genome is released into the cell nucleus, early genes begin to be transcribed, and the proteins produced regulate the host cell environment to prepare for viral replication. Subsequently, viral DNA polymerase and other related enzymes synthesize new viral genomes. Late genes are transcribed, viral structural proteins are synthesized, and the virus assembles to form progeny virus particles, which are finally released through cell lysis or exocytosis to infect new cells.

[0005] With the development of genomics technology, sequencing technology has been increasingly widely used in various aspects of infectious disease tracing, detection, typing and drug resistance assessment, and has developed rapidly in the direction of speed and economy. Traditionally, Sanger sequencing has been widely used for sequencing and verification of partial regions of the adenovirus genome due to its high accuracy, but its low throughput and high cost limit the application of whole genome sequencing. With the development of next-generation sequencing (NGS) technology, such as Illumina and MGI platforms, the efficiency and throughput of adenovirus whole genome sequencing have been greatly improved. These platforms are able to generate a large amount of sequence data in a short period of time, and through parallel sequencing, multiple samples can be analyzed simultaneously. This is of great significance for the study of adenovirus genome variation, epidemiological surveys, and the identification of new adenovirus strains.

[0006] The application of NGS technology has greatly promoted the development of adenovirus genomics research. For example, researchers used NGS technology to systematically sequence the whole genome of adenovirus samples isolated from different regions and different populations, revealing the genetic diversity and evolution of adenoviruses. By comparing and analyzing adenovirus genome data, new gene mutations, recombination events, and genome rearrangements can be identified, which is of great reference value for understanding the pathogenic mechanism of adenovirus and vaccine development. In addition, NGS technology is also used to monitor the epidemic trends and transmission paths of adenoviruses, providing data support for public health decision-making.

[0007] Although NGS technology has shown many advantages in adenovirus whole genome sequencing, there are still some challenges. The accuracy and integrity of high-throughput sequencing data depend on multiple factors during sample preparation and data processing. In order to improve the accuracy of sequencing, researchers usually need to perform high-coverage sequencing and combine bioinformatics tools to strictly control the data quality and conduct subsequent analysis. Adenovirus has more than 80 different serotypes, and the genome sequences between different serotypes vary greatly. This diversity increases the data complexity of whole genome sequencing. When using NGS technology, different types of adenovirus need to be compared and sequenced with reference genomes separately, which requires a lot of computing resources and time. In addition, for new or rare adenovirus types, there may be a lack of applicable reference genomes, which further increases the difficulty of sequencing and analysis. The high variability of adenovirus genomes and frequent recombination events make the assembly and interpretation of NGS data complicated. NGS technology usually produces shorter reads (usually 150-300 bases). When dealing with highly variable regions and repetitive sequences in the genome, short reads may not be able to accurately parse these complex regions, resulting in assembly errors or data loss. For example, some gene regions of adenovirus have high GC content and repetitive sequences, which often show uneven sequencing coverage and difficult assembly in NGS technology. In addition, adenovirus infection is common in different populations and environments, and the infection is widespread. NGS technology has certain limitations when processing mixed samples (such as environmental samples or mixed infection samples of patients). For mixed samples, NGS technology needs to distinguish the sequences of different adenovirus strains, which requires higher sequencing depth and complex bioinformatics analysis processes. Low sequencing depth may lead to cross-contamination of sequences between virus strains, affecting the accuracy of the results. In addition, host DNA and other microbial DNA in mixed samples may also interfere with the sequencing and analysis of the adenovirus genome, further increasing the difficulty of data processing.

[0008] For the whole genome detection of adenovirus, the current common method is to use the second-generation sequencing platform to sequence the metagenomics. This platform has the problems of long sequencing time, short sequencing read length, large amounts of human genomic DNA contamination in the sequencing results, high sequencing data volume, and it is difficult to obtain sequences covering the entire genome.

[0009] With the rise of third-generation sequencing technologies (such as PacBio SMRT and Oxford Nanopore), the accuracy and efficiency of adenovirus whole genome sequencing have been further improved. Third-generation sequencing technologies can read longer DNA fragments, reduce the complexity of sequence assembly, and have unique advantages in detecting genome structural variations.

[0010] However, there are currently very few reports on the use of third-generation sequencing technology to type and trace the origin of adenovirus. Existing methods can only cover a few types, and the genomic regions covered by sequencing are limited, and the complete genome cannot be well covered. Therefore, the field continues to develop sequencing methods for typing and tracing adenovirus that cover comprehensive types, sequence wider genomic regions, and can well cover the complete genome. Summary of the invention

[0011] In order to overcome the above-mentioned shortcomings and defects of the prior art, such as limited coverage type and sequencing coverage of the genome region, and inability to cover the entire genome well, the present invention provides a method, primers and a kit for adenovirus whole genome sequencing.

[0012] The technical solution of the present invention is as follows:

[0013] A method for whole genome sequencing of adenovirus, comprising the following steps:

[0014] S1. Perform the first round of amplification using the first round of amplification primers of the sequences shown in SEQ ID NO.1 to SEQ ID NO.93 in Table 1;

[0015] S2. Use the second round amplification primers with sequences shown in SEQ ID NO.94 to SEQ ID NO.285 in Table 2 to perform the second round amplification.

[0016] The method for whole genome sequencing of adenovirus further comprises the following steps: S3. Sequencing adapter connection.

[0017] The method for whole genome sequencing of adenovirus further comprises the following steps: S4. sequencing on a machine.

[0018] The system for the first round of amplification includes: 2.5 pg to 5 ng / μl template, 500 mM KCl, 100 mM Tris HCl, 250 mM MgCl2, 10 mM dNTP, 5 U / μl Taq polymerase, 10 uM first round amplification primer and water;

[0019] Preferably, the template refers to: adenovirus nucleic acid;

[0020] Preferably, the reaction procedure of the first round of amplification includes: 95°C for 3 min; 98°C for 20 s, 60°C for 2 min, and 65°C for 3 min as one cycle, for a total of 30 cycles;

[0021] Preferably, the system for the second round of amplification includes: 0.0025-0.5 μl / μl of the first round of amplification product, 500 mM KCl, 100 mM Tris HCl, 250 mM MgCl2, 10 mM dNTP, 5 U / μl Taq polymerase, 10 uM of the second round of amplification primer and water;

[0022] Preferably, the reaction procedure of the second round of amplification includes: 95°C for 2 min; 98°C for 20 s, 60°C for 1 min, and 65°C for 2 min as one cycle, for a total of 6 cycles; 72°C for 5 min;

[0023] Preferably, the 5' end of the second round primer has phosphorylation modification.

[0024] The sequencing adapter connection comprises: performing a connection reaction after purification with magnetic beads;

[0025] Preferably, the ligation reaction system includes: 1-10 ng / μl second-round amplification product, 50 mM MgCl2, 5 mM ATP, 50 mM DTT, 250 mM Tris-HCl, 400 U / μl T4 ligase, 0.03 μl / μl sequencing adapter;

[0026] Preferably, the ligation reaction is carried out at 25°C for 10 min.

[0027] The on-machine sequencing refers to placing the ligation product on a Nanopore sequencer for on-machine reaction.

[0028] A primer set for adenovirus whole genome sequencing includes first-round amplification primers and second-round amplification primers; the first-round amplification primers have sequences shown in SEQ ID NO.1 to SEQ ID NO.93; the second-round amplification primers have sequences shown in SEQ ID NO.94 to SEQ ID NO.285.

[0029] The 5' end of the second round primer has phosphorylation modification.

[0030] A kit for adenovirus whole genome sequencing, comprising the primer set for adenovirus whole genome sequencing.

[0031] The kit for adenovirus whole genome sequencing further comprises: an amplification reagent and a ligation reagent;

[0032] Preferably, the amplification reagents include: KCl, Tris HCl, MgCl2, dNTP, Taq polymerase, water;

[0033] Preferably, the ligation reagents include: purified magnetic beads, MgCl2, ATP, DTT, Tris-HCl, T4 ligase, and sequencing adapters.

[0034] The beneficial effects of the present invention are as follows:

[0035] Based on the third generation sequencing technology, the present invention has developed a set of primers and kits for sequencing the whole genome of adenovirus. First, the sequencing technology based on PacBio and Nanopore can directly read long fragment sequences of thousands to tens of thousands of bases, significantly reducing the risk of assembly mismatch. These technologies perform particularly well in sequencing high GC content and repetitive sequence regions, and can more accurately reveal complex structural variations in the adenovirus genome. Secondly, by designing universal multiple primer sequences in the conserved regions of the whole genome of adenovirus, the whole genome sequence of adenovirus can be effectively enriched from clinical samples, which can effectively solve the typing and traceability problems of adenovirus.

[0036] The method of the present invention has the following characteristics:

[0037] 1. A method for designing universal adenovirus whole genome specific targeted primers based on various adenovirus types and an adenovirus whole genome sequencing kit developed based on this method, which can complete the detection of adenovirus whole genome in 10 hours;

[0038] 2. When the multiple primers designed based on this method are used for testing, there is no need to remove human genomic DNA during sample extraction. DNA and RNA extracted using conventional magnetic bead methods and other automated instruments can meet the requirements of subsequent library construction and sequencing;

[0039] 3. This method has extremely high sensitivity and can be applied to the whole genome sequencing of clinical samples with adenovirus Ct values ​​as high as 35;

[0040] 4. During amplification, the universal sequence is added to the amplified target band through multiple amplification of low-concentration specific primers in the first round, and then the universal Barcode primer and ordinary rapid amplification enzyme are directly added for amplification, so that the adenovirus full genome library construction can be quickly completed;

[0041] 5. The 5' end of the Barcode used is additionally phosphorylated. The rapid amplification enzyme used during amplification will additionally add A to the extension end. The resulting amplification product can be directly connected to the TA of the sequencing adapter, which greatly simplifies the library construction steps and shortens the experimental time.

[0042] 6. By using a long-read third-generation sequencing platform, the amplified long-fragment data only needs to be simply spliced ​​to obtain the full genome sequence of the adenovirus, which can greatly improve the stability of adenovirus type determination.

[0043] The present invention has developed a method for sequencing all adenovirus types, which can complete the whole genome sequencing of adenovirus within 10 hours and achieve full coverage assembly, tracing and typing of the virus.

[0044] The method of the present invention has a wide coverage, and the primer sequences are designed for all adenovirus types. In theory, it can adapt to all adenovirus types and can perform whole genome sequencing. However, in the more than 200 adenovirus-positive swab samples collected by the first applicant's unit in the past two years, it was found that the main common adenovirus types in my country are concentrated in 3, 7, 1, 2, 5, 21, 11, 14, and 55. Therefore, the present invention verifies the detection effect of the method and primer set of the present invention on all common adenovirus types in my country within the scope that can be obtained in clinical practice. The result data are shown in Table 7 of Experimental Example 4.

[0045] This method is applicable to various clinical sample types such as conventional swabs and bronchoalveolar lavage fluid. There is no need to remove human genomic DNA during sample extraction. DNA extracted using conventional magnetic bead methods and other automated instruments can meet the requirements of subsequent library construction and sequencing.

[0046] This method optimizes and adjusts the multiple amplification system. When the Ct value of adenovirus in the original nucleic acid sample reaches 35, whole genome sequencing can still be performed. It is an ultra-high sensitive genome sequencing method.

[0047] The present invention designs specific primers for the detection target and adds a universal sequence to the 5' end of the primer, so that the universal sequence can be added to the amplified target band while amplifying and enriching adenovirus. By directly adding universal barcode primers and ordinary rapid amplification enzymes for amplification, a library for sequencing can be obtained.

[0048] The present invention optimizes the conditions of the multiplex amplification system in multiple ways. After the optimization, the amplification primers can account for more than 80% of the adenovirus data in the sequencing data without removing the human genomic DNA, thus solving the problem of human genomic DNA contamination in the whole genome detection of adenovirus.

[0049] The present invention uses a third-generation sequencing platform with a long read length. The amplified long fragment data only needs simple splicing to directly obtain the whole genome data. The average length of the data sequenced by this method is 0.8kb, which can greatly improve the uniformity of adenovirus coverage compared to the short read length of the second-generation platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A specific experimental principle flow chart of a method for adenovirus whole genome sequencing provided for a specific embodiment of the present invention.

[0051] Figure 2 This is a distribution diagram of sequencing data of sample 1 of Experimental Example 1 of the present invention.

[0052] Figure 3 For Experimental Example 2 of the present invention, the μ 1. μ 1.2.5 μ 1.5 μ l The genome coverage distribution diagram of a round of sequencing without purification, where A to D correspond to 0.5 μ l, 1μl, 2.5μl, 5μl.

[0053] Figure 4 This is a distribution diagram of the average coverage of adenovirus in Experimental Example 3 of the present invention under different conditions (Ct values ​​are 35, 32, 29, and 26, respectively), where A to D correspond to Ct values ​​of 35, 32, 29, and 26, respectively. DETAILED DESCRIPTION

[0054] The details of the present invention are further described in detail below in conjunction with the accompanying drawings, specific experimental examples and experimental examples, but the protection scope of the present invention is not limited thereto.

[0055] Sources of biological materials

[0056] All adenovirus-positive swab samples used in Experimental Examples 1 to 4 were from Peking Union Medical College Hospital, Chinese Academy of Medical Sciences.

[0057] The first group of embodiments, the sequencing method of the present invention

[0058] This group of embodiments provides a method for sequencing the whole genome of an adenovirus. All embodiments in this group have the following common features: the method for sequencing the whole genome of an adenovirus comprises the following steps:

[0059] S1. Perform the first round of amplification using the first round of amplification primers of the sequences shown in SEQ ID NO.1 to SEQ ID NO.93 in Table 1;

[0060] S2. Use the second round amplification primers with sequences shown in SEQ ID NO.94 to SEQ ID NO.285 in Table 2 to perform the second round amplification.

[0061] According to the records and teachings of the present invention, those skilled in the art can use the above steps S1 and S2, combined with common experimental steps in the field of molecular biology, such as the Molecular Cloning Experiment Guide, known sequencing machine operations, etc., to achieve the whole genome sequencing, typing and tracing of respiratory syncytial virus. Any use of the above steps to sequence, type and trace the whole genome of respiratory syncytial virus falls within the scope of protection of the present invention.

[0062] In a further embodiment, the method for whole genome sequencing of adenovirus further comprises the following steps: S3. Sequencing adapter connection.

[0063] In a further embodiment, the method for whole genome sequencing of adenovirus further comprises the following steps: S4. On-machine sequencing.

[0064] In a specific embodiment, the system of the first round of amplification includes: 2.5pg ~ 5ng / μ l Template, 500mM KCl, 100mM Tris HCl, 250mM MgCl2, 10mM dNTP, 5U / μ l Taq polymerase, 10uM first round amplification primer and water;

[0065] Preferably, the template refers to: adenovirus nucleic acid;

[0066] Preferably, the reaction procedure of the first round of amplification includes: 95°C for 3 min; 98°C for 20 s, 60°C for 2 min, and 65°C for 3 min as one cycle, for a total of 30 cycles;

[0067] Preferably, the second round of amplification system includes: 0.0025-0.5 μ l / μ lFirst round amplification product, 500mM KCl, 100mM Tris HCl, 250mM MgCl2, 10mM dNTP, 5U / μl Taq polymerase, 10uM second round amplification primer and water;

[0068] Preferably, the reaction procedure of the second round of amplification includes: 95°C for 2 min; 98°C for 20 s, 60°C for 1 min, and 65°C for 2 min as one cycle, for a total of 6 cycles; 72°C for 5 min;

[0069] Preferably, the 5' end of the second round primer has phosphorylation modification.

[0070] In a specific embodiment, the sequencing adapter connection includes: performing a connection reaction after magnetic bead purification;

[0071] Preferably, the ligation reaction system includes: 1-10 ng / μl second-round amplification product, 50 mM MgCl2, 5 mM ATP, 50 mM DTT, 250 mM Tris-HCl, 400 U / μl T4 ligase, 0.03 μl / μl sequencing adapter;

[0072] Preferably, the ligation reaction is carried out at 25°C for 10 min.

[0073] In a more specific embodiment, the on-machine sequencing refers to: placing the ligation product on a Nanopore sequencer for on-machine reaction.

[0074] Second embodiment, primer set of the present invention

[0075] This group of embodiments provides a primer set for adenovirus whole genome sequencing. All embodiments in this group have the following common features: the primer set for adenovirus whole genome sequencing includes a first round amplification primer and a second round amplification primer; the first round amplification primer has a sequence shown in SEQ ID NO.1 to SEQ ID NO.93; the second round amplification primer has a sequence shown in SEQ ID NO.94 to SEQ ID NO.285.

[0076] According to the records and teachings of the present invention, those skilled in the art can use primers with sequences shown in SEQ ID NO.1 to SEQ ID NO.285, combined with common experimental steps in the field of molecular biology, such as the Molecular Cloning Experiment Guide, known sequencing machine operations, etc., to achieve the whole genome sequencing, typing and tracing of respiratory syncytial virus. Any act of synthesizing, amplifying, reacting, preparing and producing primers with sequences shown in SEQ ID NO.1 to SEQ ID NO.285 falls within the scope of protection of the present invention.

[0077] In a specific embodiment, the 5' end of the second round primer has a phosphorylation modification.

[0078] The third group of embodiments, the kit of the present invention

[0079] This group of embodiments provides a kit for adenovirus whole genome sequencing. All embodiments in this group have the following common features: the kit for adenovirus whole genome sequencing includes a primer set for adenovirus whole genome sequencing as described in any one of the second group of embodiments.

[0080] In a further embodiment, the kit for adenovirus whole genome sequencing further comprises: an amplification reagent, a ligation reagent;

[0081] Preferably, the amplification reagents include: KCl, Tris HCl, MgCl2, dNTP, Taq polymerase, water;

[0082] Preferably, the ligation reagents include: purified magnetic beads, MgCl2, ATP, DTT, Tris-HCl, T4 ligase, and sequencing adapters.

[0083] A specific embodiment of the present invention has developed a set of primers for large-fragment amplification universal sequences suitable for all adenovirus types, which are used to specifically amplify and enrich adenovirus whole genome nucleic acid fragments from clinical samples such as swabs and alveolar lavage fluid. The assembly and typing of the adenovirus whole genome can be completed within 10 hours. Compared with conventional NGS detection technology, it can effectively realize the recombination of adenovirus, cover the entire adenovirus genome, and maintain good amplification uniformity.

[0084] This method does not require the removal of human genomic DNA during extraction. The DNA extracted using conventional magnetic bead method and other automated instruments can meet the requirements of subsequent library construction and sequencing. It is particularly suitable for sample types with high human genome content such as swabs and alveolar lavage fluid. The detection can be completed when the total amount of extracted DNA is above 50pg. Compared with the conventional adenovirus sequencing process, it can significantly improve the success rate of library construction for trace samples.

[0085] The present invention can achieve simultaneous coverage of the entire adenovirus genome by designing specific primers for the detection target and adding a universal sequence to the 5' end of the primer. By testing samples with different Ct values, it can be surprisingly found that the present method can achieve good whole-genome coverage for clinical samples with a Ct value as high as 35.

[0086] The present invention optimizes the conditions of the multiple amplification system for multiple rounds. After the optimization of the amplification primers, the proportion of adenovirus in the sequencing data can reach more than 90% without removing the human genomic DNA, which solves the problem of human genomic DNA contamination in the whole genome detection of adenovirus. The present invention uses a long-read third-generation sequencing platform, and the amplified long fragment data is simply spliced ​​to directly obtain the whole genome sequence of the adenovirus, which can be used for subsequent adenovirus typing and traceability analysis.

[0087] The average length of the data sequenced by this method is 0.8k, which is more than 5 times longer than the PE150 commonly used in the second-generation mNGS platform, and can greatly improve the accuracy and stability of pathogen identification. At the same time, it also maintains a highly consistent size when selecting the size of the target fragment, and improves the sensitivity of adenovirus whole genome sequencing by controlling the length of the amplified fragment while taking into account the fragment length.

[0088] The detailed implementation process of this method is as follows Figure 1 As shown, the specific operations are as follows:

[0089] 1. Primer synthesis of adenovirus genome

[0090] Based on the targeted amplification fragment length of 800 bp, the adenovirus genome includes 46 regions in total. The designed universal sequences and primer sequences are shown in Table 1 below:

[0091] Table 1

[0092]

[0093]

[0094]

[0095] The number after ADV corresponds to the region of adenovirus amplification. The F-terminal primer and R-terminal primer corresponding to the number are a pair of specific target primers, which are used to specifically amplify and enrich the corresponding target region. In specific experiments, one or more combinations can be selected for multiple amplification according to the purpose of the experiment. Primers can be synthesized in primer synthesis companies, such as Shanghai Bioengineering.

[0096] 2. Universal tag sequence primer design

[0097] The universal tag sequence consists of three parts: the tag sequence at the 5' end to identify the starting sequence, the 24bp tag sequence, and the universal primer sequence at the 3' end. The 5' end of the sequence is additionally phosphorylated, and the 96 tag sequences maintain a large difference, which is convenient for splitting and identification.

[0098] 3.Universal tag sequence primer synthesis

[0099] The tag sequences designed based on the above primer design principles are shown in Table 2 below:

[0100] Table 2

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Among them, the primer set with the corresponding number after PMGIB is a set of tag sequence primers, which are used to add tag sequences to the specific amplification enrichment target region. In specific experiments, one or more combinations can be selected for multiple amplification according to the purpose of the experiment. Primers can be synthesized in primer synthesis companies, such as Shanghai Bioengineering.

[0108] 4. Nucleic acid extraction from clinical samples

[0109] The principle of this method is based on multiple targeted enrichment, which can specifically increase the proportion of adenovirus in sequencing data. Therefore, it is not necessary to remove human genomic DNA during extraction. It can be adapted to the method of extracting DNA and RNA simultaneously. It is recommended to use the magnetic bead method of the Mindex automated extractor for nucleic acid extraction. The extraction method refers to the kit instructions and instrument instructions, and the extraction time is about 0.5h.

[0110] 5. First round of multiplex PCR amplification with adenovirus-specific primers

[0111] The specific primers for the first round of amplification include two parts. One is the universal sequence located at the 5' end. The introduction of this sequence is because in the second round of amplification, the universal sequence is used as the primer binding site, so that all samples can be amplified using the same set of primers; the second part is the adenovirus-specific primer located at the 3' end. This primer specifically binds to a specific region of the adenovirus and can specifically enrich this part of the target region to be sequenced.

[0112] The adenovirus primers were uniformly diluted to 10uM, mixed in equal volumes, and used as adenovirus-specific primers for the first round of amplification to participate in the amplification reaction.

[0113] The general formulation system for the first round of amplification reaction is shown in Table 3 below:

[0114] Table 3

[0115]

[0116] After mixing the prepared reaction system, centrifuge it instantaneously and perform amplification reaction on a PCR instrument:

[0117]

[0118] The first round of amplification takes about 180 minutes.

[0119] 6. Second round of PCR amplification reaction with tag sequence

[0120] The first-round amplification product was purified using 0.55X magnetic beads, and the purified product could be directly used for the second-round amplification.

[0121] Or directly use the first round PCR reaction product as a template and take 0.5-5 μ l Add the prepared reaction reagents as follows to perform the second round of tag sequence amplification. The preparation system is shown in Table 4 below:

[0122] Table 4

[0123]

[0124] After mixing the prepared reaction system, centrifuge it instantaneously and perform amplification reaction on a PCR instrument:

[0125]

[0126] The second round of amplification takes about 30 minutes.

[0127] The labeled primers used in this process are phosphorylated at the 5' end. The labeled primer sequences F and R form a pair. Each sample is amplified using a pair of labeled primers. Different samples use different labeled primers. During sequencing, different samples can be split directly based on the labeled primers.

[0128] 7. Connect the sequencing adapter

[0129] The second round of amplification products were purified using conventional 1X purification magnetic beads. For the specific process, please refer to the instruction manual of the purification magnetic beads. The purification magnetic beads are the purification magnetic beads of Mindray Biotechnology (device name: Nucleic Acid Extraction Kit (Magnetic Bead Method); specification model: 96 people / box). After purification, the Qubit concentration was measured, and the samples prepared for sequencing in the same batch were mixed in equal amounts. The total amount after mixing was 50-500ng, and the subsequent connection reaction could be carried out.

[0130] The connection system is formulated as shown in Table 5 below:

[0131] Table 5

[0132]

[0133]

[0134] After mixing the prepared reaction system, centrifuge it instantly, and perform the ligation reaction at 25°C for 10 minutes. After the reaction is completed, connect the sequencing adapter. The ligation product is reacted on the machine according to the official sequencing process of Nanopore. The ligation and reaction takes about 0.5 hours.

[0135] 8. Sequencing and Data Analysis

[0136] The data generated by sequencing is synchronously analyzed on the Mindex analysis software or other software. The full genome sequence of adenovirus can be detected when the sequencing time reaches 2 to 8 hours. For the specific analysis process, please refer to the software manual. Other software can be: ARTIC software or MetaSPAdes.

[0137] Experimental Example 1: Sequencing of the whole genome of adenovirus using magnetic bead purification

[0138] Three adenovirus-positive swab samples were collected. After one round of amplification, the amplification products were purified using 0.55X magnetic beads. Finally, the library was constructed and sequenced according to the standard operating procedures. All three samples were assembled into complete adenovirus genome sequences. The distribution diagram of a specific sample is shown below: Figure 2 shown.

[0139] Experimental Example 2: Directly adding a round of amplification products to perform tag sequence amplification and adenovirus whole genome sequencing

[0140] Two adenovirus-positive swab samples were collected. After one round of amplification, 5μl, 2.5μl, 1μl, and 0.5μl of the amplified product were directly taken to the second round of amplification system for amplification without using magnetic beads for purification. Finally, the library was constructed and sequenced according to the standard operating procedures. The adenovirus full genome sequence was obtained for all three samples under different conditions. The average coverage of adenovirus under different conditions of 0.5μl, 1μl, 2.5μl, and 5μl is as follows Figure 3 As shown in A-3D.

[0141] Experimental Example 3: Adenovirus Whole Genome Sequencing under Different Ct Values

[0142] Two adenovirus-positive swab samples were collected and tested for adenovirus Ct values ​​using qPCR. The samples were gradient diluted to Ct values ​​of 26, 29, 32, and 35, and then adenovirus whole genome sequencing was performed. The adenovirus whole genome sequences were obtained for all three samples under different conditions. The average coverage of adenovirus under different conditions (Ct values ​​were 35, 32, 29, and 26, respectively) is shown in the figure below. Figure 4 As shown in A~4D.

[0143]

[0144] Experimental Example 4: Whole genome sequencing of different types of adenovirus samples

[0145] Nine adenovirus-positive swab samples of different types were collected. After using qPCR to detect the adenovirus Ct value, the samples were sequenced for the adenovirus genome. The adenovirus genome sequences were obtained for all nine samples of different types. The average coverage of adenovirus under different conditions is shown in Table 7 below:

[0146] Table 7

[0147] Sample name Classification Sequencing coverage depth ADV01 Type 3 343 ADV02 Type 21 382 ADV03 Type 5 273 ADV04 Type 2 371 ADV05 Type 1 267 ADV06 Type 7 315 ADV07 Type 11 421 ADV08 Type 14 368 ADV09 Type 55 435 .

Claims

1. A method for whole genome sequencing of adenovirus, characterized in that: The steps include: S1. Perform the first round of amplification using the first round of amplification primers of the sequences shown in SEQ ID NO.1 to SEQ ID NO.93 in Table 1; S2. Use the second round amplification primers with sequences shown in SEQ ID NO.94 to SEQ ID NO.285 in Table 2 to perform the second round amplification.

2. A method for adenovirus whole genome sequencing according to claim 1, characterized in that: The following steps are also included: S3. Sequencing adapter connection.

3. A method for whole genome sequencing of adenovirus according to claim 1, characterized in that: The following steps are also included: S4. Sequencing on a machine.

4. A method for adenovirus whole genome sequencing according to claim 1, characterized in that: The system for the first round of amplification includes: 2.5 pg to 5 ng / μl template, 500 mM KCl, 100 mM Tris HCl, 250 mM MgCl2, 10 mM dNTP, 5 U / μl Taq polymerase, 10 uM first round amplification primer and water; And / or, the template refers to: adenovirus nucleic acid; And / or, the reaction procedure of the first round of amplification includes: 95°C for 3 min; 98°C for 20 s, 60°C for 2 min, and 65°C for 3 min as one cycle, for a total of 30 cycles; and / or, the system for the second round of amplification includes: 0.0025-0.5 μl / μl of the first round of amplification product, 500 mM KCl, 100 mM Tris HCl, 250 mM MgCl2, 10 mM dNTP, 5 U / μl Taq polymerase, 10 uM of the second round of amplification primer and water; And / or, the reaction procedure of the second round of amplification includes: 95°C for 2 min; 98°C for 20 s, 60°C for 1 min, and 65°C for 2 min as one cycle, for a total of 6 cycles; 72°C for 5 min; And / or, the 5' end of the second round primer has phosphorylation modification.

5. A method for adenovirus whole genome sequencing according to claim 2, characterized in that: The sequencing adapter connection comprises: performing a connection reaction after purification with magnetic beads; And / or, the ligation reaction system includes: 1-10 ng / μl second-round amplification product, 50 mM MgCl2, 5 mM ATP, 50 mM DTT, 250 mM Tris-HCl, 400 U / μl T4 ligase, 0.03 μl / μl sequencing adapter; And / or, the connection reaction is carried out at 25° C. for 10 min.

6. A method for adenovirus whole genome sequencing according to claim 3, characterized in that: The on-machine sequencing refers to placing the ligation product on a Nanopore sequencer for on-machine reaction.

7. A primer set for adenovirus whole genome sequencing, characterized in that: It comprises a first round amplification primer and a second round amplification primer; the first round amplification primer has a sequence shown by SEQ ID NO.1 to SEQ ID NO.93; the second round amplification primer has a sequence shown by SEQ ID NO.94 to SEQ ID NO.

285.

8. A primer set for adenovirus whole genome sequencing according to claim 7, characterized in that: The 5' end of the second round primer has phosphorylation modification.

9. A kit for adenovirus whole genome sequencing, characterized in that: A primer set for adenovirus whole genome sequencing as described in claim 7 or 8.

10. A kit for adenovirus whole genome sequencing according to claim 9, characterized in that: Also included: amplification reagents, ligation reagents; and / or, the amplification reagents include: KCl, Tris HCl, MgCl2, dNTP, Taq polymerase, water; And / or, the ligation reagents include: purified magnetic beads, MgCl2, ATP, DTT, Tris-HCl, T4 ligase, and sequencing adapters.

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