A capture probe set, kit and method for whole genome sequencing of hepatitis a virus

Abstract

This invention provides a capture probe set for whole-genome sequencing of hepatitis A virus (HAV). The nucleotide sequences of the capture probe set are shown in SEQ ID No:1 to SEQ ID No:880. This capture probe set can be used to prepare kits for whole-genome sequencing of HAV. This invention also provides a method for whole-genome sequencing of HAV using the capture probe set. The method involves liquid-phase hybridization of a total RNA library from a sample to be tested with the capture probe set. DNA fragments hybridized with the capture probe set are isolated from the hybridization reaction solution. These DNA fragments are then used as templates for capture-and-PCR amplification. The PCR products are sequenced, and after data analysis, the full-length HAV genome sequence is obtained. The capture probe set designed in this invention can capture the HAV genome. By optimizing the probe length, it can specifically enrich ultra-low copy numbers of the viral genome from high-background host nucleic acids, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology gene sequencing detection technology, specifically relating to a capture probe set, kit, and method for whole genome sequencing of hepatitis A virus. Background Technology

[0002] Hepatitis A virus (HAV) is the main pathogen causing acute viral hepatitis in humans, primarily transmitted via the fecal-oral route. It can cause outbreaks or epidemics of acute hepatitis, making research on this virus crucial for public health. HAV belongs to the Picornaviridae family and is a single-stranded positive-sense RNA virus with a genome length of approximately 7.5 kb. Whole-genome sequencing of HAV is essential for tracing the virus's origin, analyzing its transmission chain, identifying genotypes, monitoring mutations, and developing vaccine and drug targets.

[0003] Hepatitis A virus (HAV) is genetically conserved, with only one serotype, but its genome still contains sufficient variations for differentiation. Currently, based on the genotyping criteria of less than 15% nucleotide sequence difference within the same genotype and less than 7.5% nucleotide sequence difference within the same genotype subtype, HAV is classified into six genotypes (I-VI) according to the nucleotide sequence differences in the VP1-2A linker region. Among them, genotypes I, II, and III mainly infect humans and are further divided into subtypes A and B (such as IA, IB, IIA, IIB, IIIA, and IIIB). Genotype I is the most prevalent globally, with subtype IA being more common than subtype IB, followed by subtype IIIA; while genotypes IV, V, and VI are mainly found in non-human primates. In my country, subtype IA is currently the most prevalent, with a small number of subtypes of IB. Obtaining the complete genome sequence is an important technical support for pathogen surveillance.

[0004] Currently, obtaining the complete viral genome from clinical or environmental samples (such as serum, feces, and sewage) mainly relies on the following methods: 1. Sequencing after Viral Isolation and Culture: Viral isolation and culture is the "gold standard" in virology research. However, for hepatitis A virus (HAV), its isolation and culture in conventional cell lines present significant challenges. HAV grows slowly in vitro (28 days per generation), has low replication efficiency, and typically does not produce obvious cytopathic effects; this results in lengthy virus isolation cycles (often requiring weeks or even months), low success rates, high costs, and a high dependence on the presence of intact infectious viral particles in the sample, greatly limiting its application in rapid clinical diagnosis and large-scale epidemiological surveillance. After successful virus isolation, qualitative or quantitative detection techniques are still required, such as antigen detection (commonly using enzyme-linked immunosorbent assays, immunofluorescence, etc.) and nucleic acid qualitative detection (real-time quantitative PCR, conventional PCR combined with nucleic acid electrophoresis, etc.) for identification. Further whole-genome sequencing analysis of the successfully isolated viral culture is essential for identifying viral genotypes, neutralizing antigen sites, and other molecular biological characteristics. Therefore, high-throughput sequencing technology for hepatitis A virus is still necessary after viral isolation and culture.

[0005] 2. Metagenomic sequencing (mNGS): While mNGS can be used to directly obtain viral genomes, its effectiveness in detecting HAV cannot be guaranteed in practical applications. The fundamental reason is that in clinical and environmental samples, the nucleic acids of the host and symbiotic microorganisms are absolutely dominant, resulting in low viral sequence abundance. Directly performing metagenomic sequencing leads to the following problems: Limited sensitivity: To obtain the minimum coverage depth of the viral genome, the total amount of sequencing data required is extremely high, resulting in low cost-effectiveness; Poor genome integrity: Due to insufficient effective depth, the obtained viral reads are fragmented, making it difficult to assemble a complete and reliable whole genome sequence; Data analysis and storage challenges: Analyzing viral genome sequences is difficult and costly due to the extremely high background noise from host and other genomes.

[0006] Therefore, although metagenomic sequencing is a powerful exploratory tool, its non-targeted nature makes it significantly less efficient, costly, and prone to inconsistent data output quality in the specific task of obtaining the whole genome of known low-abundance viruses (such as HAV).

[0007] 3. Traditional Multiplex PCR-based methods: Traditional PCR-based methods are also the mainstream technology for detecting and obtaining viral sequence fragments. They typically involve designing specific primers to target and amplify specific regions (such as conserved regions) of the target viral genome, followed by Sanger sequencing or high-throughput sequencing to obtain the sequence information of that fragment. However, when this technology is applied to obtain the complete genome sequence of hepatitis A virus (HAV), the following inherent and insurmountable limitations are exposed: Primer design is challenging and coverage is difficult to guarantee: Whole-genome sequencing requires splicing a large number of overlapping amplicones, which relies on designing multiplex PCR primer sets that cover the entire genome. Primer design heavily depends on known reference sequences. For regions with high genomic variability or new mutations, amplification loss can easily occur due to decreased primer binding efficiency or failure, resulting in missing genome sequences and preventing true genome-wide coverage.

[0008] Amplification bias: Different primer pairs have varying amplification efficiencies, resulting in severely uneven sequencing depths across different genomic regions. Regions with high GC content, complex secondary structures, or sequence variations may experience significantly reduced amplification efficiency, or even fail, thus affecting the accuracy and completeness of downstream analyses.

[0009] Strict requirements for sample quality: PCR amplification requires a certain level of integrity of the template nucleic acid. For viral RNA samples that are common in clinical settings or the environment and have already degraded, long-fragment amplicon may not be generated effectively, leading to difficulties in genome splicing, especially affecting the acquisition of easily degraded regions such as the 5' and 3' ends.

[0010] Primer optimization is difficult: Multiplex PCR systems covering the entire genome often require the design of dozens or even hundreds of primer pairs, and complex multiplex reaction optimization is required (such as avoiding primer dimers and avoiding non-specific amplification). This increases the cycle and cost of designing experiments, making it difficult to achieve standardized and high-throughput operations.

[0011] Therefore, the strategy based on multiplex PCR amplification and splicing has problems such as incomplete coverage, poor uniformity, incompatibility with degraded samples and sequence variations, and system complexity when applied to HAV whole genome sequencing, which restricts its application in large-scale epidemiological surveillance and precise source tracing research. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a capture probe set, kit and method for whole genome sequencing of hepatitis A virus (HAV) in response to the shortcomings of the prior art. The capture probe set can capture the HAV virus genome, including highly variable regions and conserved regions, and the optimized probe length can specifically enrich the viral genome with ultra-low copy number from high background host nucleic acid. It is suitable for clinical samples and environmental samples with low viral load and has broad application prospects.

[0013] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a capture probe set for whole genome sequencing of hepatitis A virus, wherein the nucleotide sequence of the capture probe set is shown as SEQ ID No:1 to SEQ ID No:880.

[0014] Preferably, each of the capture probes is labeled with biotin.

[0015] This invention provides a kit for preparing a whole genome sequence of hepatitis A virus using the above-mentioned capture probe set. The kit includes RNA library construction reagents, library hybridization reaction reagents, the capture probe set, and streptavidin-labeled magnetic beads.

[0016] Preferably, the RNA library construction reagents include RNA fragmentation reagents, reverse transcription reagents, cDNA two-strand synthesis reagents, adapter ligation reagents, purification reagents, PCR pre-reaction reagents, and capture-after-PCR amplification reaction reagents.

[0017] Preferably, the library hybridization reaction reagents include TargetSeq One® Hyb Buffer v2, HybHuman Block, TargetSeq® Blocking Oligo, and RNase Block.

[0018] This invention also provides a method for performing whole-genome sequencing of hepatitis A virus using the above-mentioned capture probe set, the method being as follows: S1. Construct a total RNA library from the samples to be tested; S2. Hybridize the total RNA library of the sample to be tested constructed in S1 with the capture probe set in liquid phase to obtain the hybridization reaction solution; S3. Separate the DNA fragment that hybridized with the capture probe group from the hybridization reaction solution in S2 to obtain the DNA fragment; S4. Using the DNA fragment obtained in S3 as a template, perform capture-and-PCR amplification to obtain the PCR product. S5. The PCR products obtained in S4 were subjected to high-throughput sequencing. After data analysis, the full-length genome sequence of the hepatitis A virus was obtained.

[0019] Compared with the prior art, the present invention has the following advantages: 1. The capture probe set of the present invention has high efficiency and broad spectrum: the capture probe is designed based on all whole genome reference sequences in NCBI, which can efficiently capture the main known HAV genotypes (type I, including IA and IB genotype subtypes) in China, covering all regions of the genome, including highly variable and conserved regions, and is not afraid of any mutations in the sample, effectively avoiding the decrease in genome coverage caused by sequence variation.

[0020] 2. The capture probe set of the present invention has high sensitivity and specificity: the optimized probe length, coverage and hybridization system can specifically enrich the viral genome with ultra-low copy number from high background host nucleic acid, and is particularly suitable for clinical samples and environmental samples with low viral load.

[0021] 3. The capture hybridization library constructed using the capture probe in this invention has strong compatibility: it is applicable to various mainstream high-throughput sequencing platforms such as Illumina and MGI, as well as third-generation sequencing platforms, and the operation process is standardized.

[0022] 4. The capture probe of this invention has a wide range of applications: it can be used in multiple fields such as clinical diagnosis, epidemic tracing, virus mutation monitoring, and basic research, providing key technical support for the precise prevention and in-depth research of hepatitis A.

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 This is a design diagram of the capture probe group in Embodiment 1 of the present invention.

[0025] Figure 2 This is a flowchart of the whole genome sequencing process of hepatitis A virus in Embodiment 2 of the present invention.

[0026] Figure 3 This is a schematic diagram of HAV1 sequencing coverage and depth in Embodiment 3 of the present invention.

[0027] Figure 4 This is a schematic diagram of HAV2 sequencing coverage and depth in Embodiment 3 of the present invention.

[0028] Figure 5 This is a schematic diagram of HAV3 sequencing coverage and depth in Embodiment 3 of the present invention.

[0029] Figure 6 This is a schematic diagram of HAV4 sequencing coverage and depth in Example 3 of the present invention. Detailed Implementation

[0030] Example 1 This embodiment is a capture probe set design scheme for whole genome sequencing of hepatitis A virus.

[0031] Design schemes for capture probe sets targeting the entire hepatitis A virus (HAV) genome, such as... Figure 1 As shown, the design of the capture probe set is based on all 142 complete HAV genome reference sequences marked "complete" downloaded from the NCBI database. Through sequence alignment and design optimization, probes in hypervariable regions of the genome are retained, and probes in conserved regions are merged to remove redundancy, generating a set of probes covering the entire HAV genome. The probe length is preferably 100 nucleotides to ensure the continuity and uniformity of coverage. The nucleotide sequences of the capture probe set are shown in SEQ ID No:1 to SEQ ID No:880, and all are labeled with biotin.

[0032] Example 2 This embodiment describes a method for using the capture probe set designed in Example 1 for whole-genome sequencing of hepatitis A virus. The specific procedure of this method is as follows: Figure 2 As shown, the method is as follows: (I) Construction of total RNA library: (1) RNA was extracted from the sample using the QIAamp Viral RNA Mini kit (catalog number 52906, Qiagen GmbH, Germany). The RNA concentration was quantified using the Qubit RNA HS assay kit and the Qubit Fluorometer (LifeTechnologies, USA).

[0033] (2) RNA fragmentation: Remove the RNA sample from the -80℃ freezer in advance, and remove the Fast Frag Buffer from the -20℃ freezer in advance. Place them on an ice box to thaw. After thawing, briefly vortex to mix and then centrifuge briefly. Place them on an ice box for later use. The fragmentation reaction system is: 13 μL of RNA sample and 4 μL of Fast Frag Buffer. After adding the sample, use a pipette to mix and centrifuge briefly. Set the PCR instrument parameters as follows: 105℃ hot cap temperature, 94℃ for 7 min, and 4℃ incubation. Place the reaction solution on the PCR instrument and run the program. When the temperature of the PCR instrument drops to 4℃, remove the PCR tube and centrifuge briefly to obtain fragmented RNA. Proceed to step (3) immediately.

[0034] (3) Reverse transcription: Remove the Fast First Strand Buffer from the kit from the -20℃ freezer beforehand, place it on an ice box to thaw, briefly vortex to mix and then centrifuge briefly, and place it on an ice box for later use; remove the Fast First Strand Enzyme from the kit from the -20℃ freezer, invert to mix and then centrifuge briefly, and place it on an ice box for later use; prepare the reaction system on the ice box: 17μL fragmented RNA, 6μL Fast First Strand Buffer, and 2μL Fast First Strand Enzyme; after preparation, use a pipette to mix (avoid vigorous shaking), centrifuge briefly, and set the PCR instrument parameters as follows: 105℃ hot cap temperature, 25℃ for 10min, 42℃ for 15min, 70℃ for 15min, and 4℃ incubation; place the reaction solution on the PCR instrument, run the program, and when the PCR instrument temperature drops to 4℃, remove the PCR tube, centrifuge briefly to obtain cDNA, and immediately proceed to step (4).

[0035] (4) cDNA double strand synthesis, addition of "A" to the 3' end: Remove the Fast Second Strand Buffer with dUTP from the -20℃ freezer beforehand, place it on an ice box to thaw, briefly vortex to mix and then centrifuge briefly, and place it on an ice box for later use; remove the Fast Second Strand Enzyme from the -20℃ freezer, invert to mix and then centrifuge briefly, and place it on an ice box for later use; prepare the reaction system on the ice box: 25μL cDNA, 30μL Fast Second Strand Buffer with dUTP, and 5μL Fast Second Strand Enzyme. After preparation, use a pipette to mix (avoid vigorous shaking), centrifuge briefly, and set the PCR instrument parameters as follows: 105℃ hot cap temperature, 16℃ for 30min, 72℃ for 15min, and 4℃ incubation; place the reaction solution on the PCR instrument, run the program, and when the PCR instrument temperature drops to 4℃, remove the PCR tube, centrifuge briefly, and immediately proceed to step (5).

[0036] (5) Connector connection: Remove the Adapter from the -20℃ freezer beforehand, place it on an ice box to thaw, briefly vortex to mix and then centrifuge briefly, and place it on an ice box for later use; dilute the Adapter (15μM) to 1.5μM in advance according to the amount of RNA added for library construction; remove the Fast Ligation Buffer from the kit from the -20℃ freezer beforehand, place it on an ice box to thaw, briefly vortex to mix and then centrifuge briefly, and place it on an ice box for later use; remove the Fast Ligase Mix from the -20℃ freezer, invert to mix and then centrifuge briefly, and place it on an ice box for later use; prepare the reaction system on the ice box: 60μL of sample, 30μL of Fast Ligation Buffer, 5μL of Fast Ligase Mix, and 5μL of Adapter after the reaction is completed in step (4), use a pipette to mix (avoid vigorous shaking), centrifuge briefly, and set the PCR instrument parameters (close the hot lid) to: 20℃ 15 min, keep warm at 4℃; place the PCR tube on the PCR instrument, run the program, when the temperature of the PCR instrument drops to 4℃, take out the PCR tube, centrifuge briefly, and immediately proceed to step (6).

[0037] (6) Purification after ligation: Prepare an 80% ethanol-water solution in advance using anhydrous ethanol and nuclease-free water, and keep it at room temperature (use freshly prepared 80% ethanol-water solution for magnetic bead purification if possible). The magnetic beads used for purification are IGT™ Pure Beads. Take the purified magnetic beads out of the 4°C freezer in advance, mix them, and equilibrate at room temperature for 30 minutes. Vortex the purified magnetic beads that have been equilibrated to room temperature and keep them ready for use. Add 0.45 times the volume (45 μL) of purified magnetic beads to 100 μL of reaction solution after step (5), vortex to mix, and let stand at room temperature for 5 minutes. Centrifuge briefly, place the PCR tube on a magnetic rack for 3 minutes, and wait for the solution to become clear. Keep the PCR tube on the magnetic rack, carefully discard the supernatant, add 200 μL of 80% ethanol-water solution to the PCR tube, and let stand for 30 seconds. Keep the PCR tube on the magnetic rack, discard the supernatant, and add another 200 μL of ethanol-water solution to the PCR tube. 80% ethanol aqueous solution, let stand for 30s, discard the supernatant; cap the tube, centrifuge briefly to remove residual ethanol to the bottom of the tube, place it on a magnetic rack, carefully use a 10μL pipette to remove the residual ethanol at the bottom, being careful not to pick up the magnetic beads; keep the PCR tube on the magnetic rack, let stand at room temperature for 4min, let the magnetic beads dry, and allow the residual ethanol to evaporate completely; add 22μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, vortex to mix, let stand at room temperature for 2min; centrifuge briefly, place the PCR tube on the magnetic rack for 2min, and wait for the solution to become clear; use a pipette to draw 20μL of supernatant, transfer it to a new PCR tube, label it, and proceed to step (7).

[0038] (7) Pre-PCR reaction: Remove the PCR Master Mix with UDG from the -20℃ freezer beforehand, place it on an ice box to thaw, mix by inversion and centrifuge briefly, and place it on an ice box for later use; remove the UDI Primer from the -20℃ freezer beforehand, place it on an ice box to thaw, mix by vortex briefly and centrifuge briefly, and place it on an ice box for later use; prepare the PCR reaction solution on the ice box: 20μL of the sample from step (6) after the reaction, 25μL of PCR Master Mix with UDG, and 5μL of UDI Primer. Record the index number used. After preparation, use a pipette to mix (avoid vigorous shaking), centrifuge briefly, and set the PCR program as follows: 105℃ hot cap temperature; 98℃ 1min; 98℃ 10s, 60℃ 30s, 72℃ 30s, 11 cycles; 72℃ 5 min; keep warm at 4℃; place the PCR tube on the PCR instrument, run the program, and when the temperature of the PCR instrument drops to 4℃, take out the PCR tube, centrifuge briefly, and immediately proceed to step (8).

[0039] (8) Purification after PCR amplification: Prepare an 80% ethanol-water solution in advance using anhydrous ethanol and nuclease-free water, and keep it at room temperature (use freshly prepared 80% ethanol-water solution for magnetic bead purification if possible). The magnetic beads used for purification are IGT™ Pure Beads. Take the purified magnetic beads out of the 4°C freezer in advance, mix them, and equilibrate at room temperature for 30 min. Vortex the purified magnetic beads that have been equilibrated to room temperature and set them aside. Add 0.9 times the volume of purified magnetic beads (45 μL) to the 50 μL reaction solution after step (7), vortex to mix, and let stand at room temperature for 5 min. Centrifuge briefly, place the PCR tube on a magnetic rack for 3 min, and wait for the solution to become clear. Keep the PCR tube on the magnetic rack, carefully discard the supernatant, add 200 μL of 80% ethanol-water solution to the PCR tube, and let stand for 30 s. Keep the PCR tube on the magnetic rack, discard the supernatant, and add another 200 μL of ethanol-water solution to the PCR tube. Add 80% ethanol aqueous solution, let stand for 30 seconds, discard the supernatant; cap the tube, centrifuge briefly to remove residual ethanol to the bottom, place the PCR tube on a magnetic rack, carefully use a 10 μL pipette to remove the residual ethanol at the bottom, being careful not to aspirate the magnetic beads, keep the PCR tube on the magnetic rack, let stand at room temperature for 5 minutes to allow the magnetic beads to dry completely and the residual ethanol to evaporate; add 30 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, vortex to mix, let stand at room temperature for 2 minutes; centrifuge briefly, place the PCR tube on the magnetic rack for 2 minutes until the solution is clear; use a pipette to transfer 28 μL of supernatant to a new PCR tube and label it; take 1 μL of the library and use the Qubit dsDNA HS AssayKit reagent to determine the library concentration on a Qubit 4.0 Fluorometer, and record the library concentration; take 1 μL of the sample and use a fragment analyzer to determine the fragment length.

[0040] (ii) High-performance liquid phase hybridization (requires two rounds of liquid phase capture probe hybridization capture experiments): (1) Preparations for the hybridization capture experiment: Remove the Hyb Human Block and RNase Block from the -20°C freezer, place them on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the matching TargetSeq® Blocking Oligo from the -20°C freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the capture probe set designed in Example 1 from the -80°C freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the total RNA library to be used for hybridization capture from the -20°C freezer, place it on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before storing on an ice box. Remove the TargetSeq One® Hyb Buffer v2, thaw it at room temperature, briefly vortex to mix, and then centrifuge briefly (if there is precipitation, the TargetSeq One® Hyb Buffer v2 needs to be heated in a 37°C water bath until the reagent is completely dissolved before use).

[0041] (2) Hybridization of total RNA library with capture probe set: When hybridizing a single library, add 750 ng of the library to a PCR tube and label it (when hybridizing multiple libraries, add 500 ng of each library); place the PCR tube in a vacuum centrifuge, open the PCR tube cap, and concentrate until dry; after the library is concentrated, prepare the hybridization reaction solution: TargetSeq One® Hyb Buffer v2 13 μL, Hyb Human Block 5 μL, TargetSeq® Blocking Oligo 2 μL, RNase Block 5 μL, Nuclease-Free Water 3 μL, and capture probe set 2 μL. After preparation, add the hybridization reaction solution to the dry library, vortex for 30 s to ensure that the dried DNA at the bottom of the tube dissolves, and centrifuge briefly; set the PCR instrument parameters as follows: 85℃ hot cap temperature, 80℃ for 5 min, and 50℃ for 12 h; place the hybridization reaction solution on the PCR instrument, run the program, and perform step (3) 30 min before the program ends.

[0042] (3) Preparations before the capture experiment: Remove TargetSeq® Cap Beads from the 4°C freezer beforehand, mix thoroughly, and equilibrate at room temperature for 30 min. Take out Wash Buffer 1 (if precipitation occurs, heat Wash Buffer 1 in a 37°C water bath until the precipitate is completely dissolved before use). Take out TargetSeq One® Wash Buffer 2 v2 and preheat it in a 50°C water bath. Add 50 µL of TargetSeq® Cap Beads to a new PCR tube, place it on a magnetic rack for 1 min, and discard the supernatant after the solution becomes clear. Remove the PCR tube from the magnetic rack, add 180 µL of Binding Buffer, and mix thoroughly to resuspend the magnetic beads. After brief centrifugation, place the PCR tube on a magnetic rack for 1 min, and discard the supernatant after the solution becomes clear. Repeat the Binding Buffer resuspension, centrifugation, and supernatant discarding process twice, washing the magnetic beads three times in total with Binding Buffer. Remove the PCR tube from the magnetic rack, add 180 µL of Binding Buffer... Mix the buffer thoroughly and proceed to step (4) immediately.

[0043] (4) Target region DNA capture: Keep the hybridization product from step (2) on the PCR instrument. Add 180 μL of TargetSeq® CapBeads prepared in step (3) to the hybridization product and mix well by aspiration. Cap the tube and remove it from the PCR instrument. Place it on a vertical spin mixer at 8 rpm and incubate at room temperature for 30 min. Remove the PCR tube, centrifuge briefly, and place it on a magnetic rack for 2 min. After the solution becomes clear, discard the supernatant. Remove the PCR tube from the magnetic rack, add 150 µL of Wash Buffer 1 to the PCR tube, and gently aspirate to resuspend the magnetic beads. Replace the cap and place the tube on a vertical spin mixer at room temperature for 15 min at 8 rpm. Remove the PCR tube, centrifuge briefly, and place it on a magnetic rack for 2 min. After the solution becomes clear, discard the supernatant. Remove the PCR tube from the magnetic rack and add 150 µL of TargetSeq One® Wash Buffer 2 preheated to 50°C. v2, gently pipette and mix, briefly centrifuge, place on a metal bath, incubate at 50℃ for 10 min; remove the PCR tube, briefly centrifuge, place on a magnetic rack for 2 min, and discard the supernatant after the solution becomes clear; repeat the steps of adding TargetSeq One® Wash Buffer 2 v2 for incubation, centrifugation, and discarding the supernatant twice, for a total of three washes of the magnetic beads with TargetSeq One® Wash Buffer 2 v2 at 50℃; keep the PCR tube on the magnetic rack, add 200µL of 80% ethanol aqueous solution to the PCR tube, let stand for 30 s, then completely discard the ethanol solution, and air dry the magnetic beads at room temperature to allow the residual ethanol to evaporate completely; add 24µL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic rack, briefly vortex to mix and resuspend the magnetic beads, and perform the capture-after-PCR amplification reaction.

[0044] (III) Post-capture PCR amplification: (1) Take the Post PCR Master Mix and Post PCR Primer out of the -20℃ freezer in advance, place them on an ice box to thaw, and then temporarily store them on an ice box. Briefly vortex the Post PCR Master Mix and Post PCR Primer and centrifuge them briefly. Prepare the PCR reaction solution: 24μL of magnetic bead suspension, 1μL of Post PCR Primer, and 25μL of Post PCR Master Mix. After preparation, use a pipette to mix them thoroughly. After mixing, quickly transfer them to the PCR instrument and set the PCR instrument program as follows: 105℃ hot lid temperature; 95℃ for 1min; 98℃ for 20s, 60℃ for 30s, 72℃ for 30s, 15 cycles; 72℃ for 5min; 4℃ incubation. Place the PCR reaction solution on the PCR instrument and run the program. After the program is completed, proceed to the next step of magnetic bead purification.

[0045] (2) Purification after amplification: Remove the purified magnetic beads, mix well, and equilibrate at room temperature for 30 min. Add 1.1 times the volume of magnetic beads (55 μL) to the PCR product from step (1), vortex to mix, and let stand at room temperature for 5 min; centrifuge briefly, and place the PCR tube on a magnetic rack for 3 min until the solution becomes clear; keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol aqueous solution to the PCR tube, and let stand for 30 s; keep the PCR tube on the magnetic rack, discard the supernatant, add another 200 μL of 80% ethanol aqueous solution to the PCR tube, let stand for 30 s, and then completely discard the supernatant; keep the PCR tube on the magnetic rack, let stand at room temperature for 3 min, and air dry the magnetic beads to allow the residual ethanol to evaporate completely; add 25 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, vortex to mix, and let stand at room temperature for 2 min; briefly centrifuge, place the PCR tube on the magnetic rack for 2 min, and wait for the solution to clarify; use a pipette to aspirate 23 μL of supernatant and transfer it to a new PCR tube to obtain the capture library (if not used immediately, the capture library needs to be stored in a -20℃ freezer, which can be stored for one month); take 1 μL of the capture library and use the Qubit dsDNA HS Assay Kit reagent to determine the library concentration on a Qubit 4.0 Fluorometer (LifeTechnologies, USA), and record the library concentration; take 1 μL of the library and use a fragment analyzer for fragment quality control, the fragment size is basically the same as the total RNA library size before capture.

[0046] (iv) Sequencing on the instrument: One μL of the captured library was taken and quantified using the Qubit ds DNA HS Assay Kit, and the library concentration was recorded. Another 1 μL of the sample was taken and fragment length was determined using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit). The library length was between 250 bp and 400 bp. The probe hybridization captured library was sequenced using the NovaSeq 6000 (Illumina) sequencing platform to obtain the raw sequencing data.

[0047] The raw sequencing data may contain a small number of reads containing adapter information, low-quality bases, etc. To ensure the quality of information analysis, the raw reads need to be initially filtered to obtain clean reads. Subsequent analyses are based on clean reads. The data filtering mainly involves the following: using an 8bp sliding window to cut off sequences with an average base quality value of less than 20; removing the adapter sequence at the end of the sequence; if the first or last base of the sequence is less than 20, the base will be cut off directly; generally, if the remaining sequence length is less than 40 (paired ends) after the removal is completed, the sequence pair is discarded.

[0048] Removing host sequences: Since the sequencing data may contain residual host genome sequences, we first use Bowtie2 sequencing data to align with the host reference genome to obtain the sequences that could not be aligned (unmapR1 & unmapR2). These unmapped sequences are the viral sequence information that we may actually need.

[0049] Viral genome alignment: The unmapped sequences were aligned to the viral genome using bwa software (with the designed reference species sequence information as the alignment template). The reads aligned to the genome were assembled into contig sequences using MEGAHIT software. The contig sequences were then aligned to the host genome using blastn software. The host sequence was removed again to obtain the non-human contig file. The non-human contig sequence was then aligned to the viral genome sequence to obtain the possible viral typing and sorting results-virus.txt.

[0050] Obtain the full-length genome sequence of hepatitis A virus: The result-virus-top1 genotyping sequence, which ranks first in the possible viral genotyping ranking, is used as the reference genome. Clean reads are aligned to this reference genome using bwa software to obtain the aligned BAM file. Mutation analysis is performed using samtools software to obtain the variant sites. The variant sites are then corrected using iVar software to obtain the final full-length genome sequence of hepatitis A virus.

[0051] Example 3 This embodiment verifies the capture sequencing of the whole genome of hepatitis A virus using the capture probe set designed in Example 1 and the sequencing method in Example 2.

[0052] (a) Validation of whole genome capture and sequencing of hepatitis A virus in the sample: (1) Experimental materials: Samples: Four clinically confirmed HAV RNA-positive human serum samples (HAV1, HAV2, HAV3, HAV4) were obtained from the Pathogen Surveillance Sample Bank of the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.

[0053] Instruments and reagents: QIAamp Viral RNA extraction kit, Illumina NovaSeq 6000 sequencing platform, capture probe kit of this invention and matching reagents.

[0054] (2) Experimental method: The experimental method in this embodiment is the same as that in embodiment 2. (3) Results and Analysis: As shown in Table 1, Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown, Figure 3 For HAV1 sequencing coverage and depth. Figure 4 For HAV2 sequencing coverage and depth; Figure 5 For HAV3 sequencing coverage and depth. Figure 6 For HAV4 sequencing coverage and depth; capture coverage: after capturing, sequencing and analyzing the whole genome of 4 cases of hepatitis A virus using the kit of the present invention according to the above steps, the whole genome coverage can be >99.79%; average sequencing depth: after capturing, sequencing and analyzing the whole genome of 4 cases of hepatitis A virus using the kit of the present invention according to the above steps, the average coverage depth of whole genome sequencing can be >600x; coverage uniformity: after capturing, sequencing and analyzing the whole genome of 4 cases of hepatitis A virus using the kit of the present invention according to the above steps, the coverage at a depth of 30x can be >99%.

[0055] Table 1. Validation of whole-genome sequencing of hepatitis A virus The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A capture probe set for whole genome sequencing of a Hepatitis A virus, characterized in that, The nucleotide sequences of the capture probe set are shown in SEQ ID No:1 to SEQ ID No:

880.

2. The capture probe set for whole genome sequencing of hepatitis A virus according to claim 1, wherein, Each of the capture probes is labeled with biotin.

3. A kit for preparing hepatitis A virus whole genome sequencing using a capture probe set as described in claim 1 or 2, characterized in that, The kit includes RNA library construction reagents, library hybridization reaction reagents, the capture probe set, and streptavidin-labeled magnetic beads.

4. The kit for whole genome sequencing of hepatitis A virus according to claim 3, characterized in that, The RNA library construction reagents include RNA fragmentation reagents, reverse transcription reagents, cDNA two-strand synthesis reagents, adapter ligation reagents, purification reagents, PCR pre-reaction reagents, and capture-after-PCR amplification reaction reagents.

5. The kit for whole genome sequencing of hepatitis A virus according to claim 3, characterized in that, The library hybridization reaction reagents include TargetSeq One® Hyb Buffer v2, Hyb Human Block, TargetSeq® Blocking Oligo, and RNase Block.

6. A method for performing whole-genome sequencing of hepatitis A virus using a capture probe set as described in claim 1 or 2, characterized in that, The method is as follows: S1. Construct a total RNA library from the samples to be tested; S2. Hybridize the total RNA library of the sample to be tested constructed in S1 with the capture probe set in liquid phase to obtain the hybridization reaction solution; S3. Separate the DNA fragment that hybridized with the capture probe group from the hybridization reaction solution in S2 to obtain the DNA fragment; S4. Using the DNA fragment obtained in S3 as a template, perform capture-and-PCR amplification to obtain the PCR product. S5. The PCR products obtained in S4 were subjected to high-throughput sequencing. After data analysis, the full-length genome sequence of the hepatitis A virus was obtained.

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