A capture probe combination, kit and use thereof in whole genome sequencing of hepatitis E virus

Abstract

This invention relates to the field of gene detection technology, and more particularly to a capture probe combination, a kit, and its application in hepatitis E virus whole-genome sequencing. The probe combination comprises probes with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.951. The applications include: (1) detecting hepatitis E virus, or preparing products for detecting hepatitis E virus; (2) whole-genome sequencing of hepatitis E virus, or preparing products for whole-genome sequencing of hepatitis E virus. This invention provides a set of probes for whole-genome sequencing of hepatitis E virus, which can specifically enrich ultra-low copy number hepatitis E virus genomes from high background host nucleic acids, and thus has significant application value in hepatitis E virus detection in various scenarios such as clinical diagnosis, epidemic tracing, virus mutation monitoring, food safety testing, and basic research.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a capture probe combination, a kit, and its application in hepatitis E virus whole genome sequencing. Background Technology

[0002] Hepatitis E virus (HEV) is a major 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. HEV belongs to the Hepatitis Eviridae family and is a single-stranded positive-sense RNA virus with a genome length of approximately 7.2 kb. Whole-genome sequencing of HEV is of great significance for viral tracing, transmission chain analysis, genotyping, mutation monitoring, and vaccine and drug target research.

[0003] The genome of hepatitis E virus (HEV) exhibits significant genetic diversity. The HEV that infects humans is primarily of the species *Parajani* within the genus *Pasla hepatitis Evirus*. Currently, at least eight genotypes (1-8) have been identified. Genotypes 1 and 2 primarily infect humans, exhibiting strict species specificity and associated with large-scale waterborne outbreaks. Genotypes 3 and 4 are zoonotic, capable of cross-species transmission between animals such as pigs and humans, often leading to sporadic cases and foodborne infections. Other genotypes (5-8) are mainly found in animals. Different genotypes show significant differences in geographical distribution, host range, pathogenicity, and clinical characteristics, and their genomic information varies considerably and has low conservation. In specific regions, the hepatitis E virus carriage rate is high in animal hosts such as pigs, often transmitting to humans via foodborne transmission. Therefore, genotype monitoring, drug resistance site and neutralizing antigen site monitoring are crucial for epidemic early warning, therapeutic drug screening, and predicting vaccine efficacy. Thus, broad-spectrum, unbiased genomic testing is of significant value for epidemiological surveillance and source tracing.

[0004] Currently, obtaining the complete viral genome from clinical or environmental samples mainly relies on the following methods. First, there is sequencing after viral isolation and culture. While viral isolation and culture is the "gold standard" in virology research, HEV lacks stable and efficient passaged cell models, resulting in a lengthy isolation process, low success rate, high cost, and a high dependence on the presence of intact infectious viral particles in the sample. Even after successful viral isolation, antigen detection or nucleic acid qualitative detection is still required for identification, and whole-genome sequencing analysis of successfully isolated viral cultures remains essential. Therefore, viral isolation is challenging, and even after isolation and culture, efficient sequencing technology is still necessary.

[0005] Secondly, metagenomic sequencing (mNGS) is used to directly obtain viral genomes, but its effectiveness in detecting HEVs cannot be guaranteed in practical applications. The fundamental reason is that host and symbiotic microorganism nucleic acids dominate in clinical and environmental samples, resulting in low viral sequence abundance. Direct metagenomic sequencing leads to limited sensitivity, requiring an extremely large amount of data to obtain even the minimum coverage depth; simultaneously, genome integrity is poor, making it difficult to assemble reliable full sequences, and data analysis and storage costs are high. Therefore, despite its powerful capabilities, it suffers from low efficiency and unstable quality in specifically acquiring the whole genomes of known low-abundance viruses.

[0006] Furthermore, traditional multiplex PCR amplification methods remain the mainstream technique for obtaining viral sequences. This method typically involves designing specific primers for targeted amplification of target regions. However, when applied to HEV whole-genome sequencing, it suffers from limitations such as high primer design complexity and difficulty in ensuring coverage. Since primer design relies on a reference sequence, amplification loss is easily achieved for highly variable regions due to decreased primer binding efficiency. Simultaneously, the amplification bias of different primer pairs leads to severely uneven sequencing depth, and this method has stringent requirements for sample quality, especially for degraded viral RNA samples, where long amplicon fragments are difficult to generate. Moreover, multiplex PCR systems covering the entire genome require complex reaction optimization, increasing experimental time and cost. In summary, existing multiplex PCR amplification and splicing strategies, when applied to HEV whole-genome sequencing, suffer from incomplete coverage, poor uniformity, low compatibility with degraded samples, and system complexity, thus limiting their application in precise source tracing research. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention provides a capture probe combo, a kit, and its application in hepatitis E virus whole-genome sequencing. This probe combo can capture the HEV virus genome, including highly variable and conserved regions, and can specifically enrich ultra-low copy numbers of the viral genome from high background host nucleic acids. It is suitable for clinical and environmental samples with low viral loads and has broad application prospects.

[0008] In a first aspect, the present invention provides a probe assembly comprising: probes with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.951.

[0009] Furthermore, at least one of the probes is connected to a detectable marker; Preferably, the detectable marker includes one or more of fluorescent dyes, radioactive isotopes, enzymes, chemiluminescent groups, or biotin.

[0010] Secondly, the present invention provides a kit comprising the aforementioned probe combination.

[0011] Furthermore, it also includes one or more of the following: hybridization buffer reagents, human genome blocking reagents, adapter blocking reagents, ribonuclease inhibitors, or water.

[0012] Furthermore, it also includes: reagents for constructing genome libraries; Preferably, the genomic library construction reagents include one or more of the following: RNA fragmentation reagents, reverse transcription reagents, cDNA two-strand synthesis reagents, adapter ligation reagents, purification reagents, PCR pre-reaction reagents, and post-capture PCR amplification reaction reagents.

[0013] Thirdly, the present invention provides a gene chip, the gene chip comprising: a solid support and a probe assembly as described above fixed on the solid support.

[0014] Fourthly, the present invention provides the application of the aforementioned probe combination, or the aforementioned kit, or the aforementioned gene chip in any of the following: (1) Detecting hepatitis E virus, or preparing products for detecting hepatitis E virus; (2) Whole genome sequencing of hepatitis E virus, or preparation of products for whole genome sequencing of hepatitis E virus.

[0015] The hepatitis E virus detection described in this invention includes detection in multiple scenarios, such as clinical diagnosis, epidemic tracing, virus mutation monitoring, food safety testing, and basic research.

[0016] Furthermore, the detection of hepatitis E virus described in this invention is based on non-disease diagnosis and treatment purposes.

[0017] Fifthly, the present invention provides a method for whole-genome sequencing of hepatitis E virus, comprising: performing whole-genome sequencing on the sample to be tested using the aforementioned probe combination, or the aforementioned kit, or the aforementioned gene chip.

[0018] Furthermore, including: (1) Extract RNA from the sample to be tested and construct a cDNA library; (2) Hybridize the probe combination with the cDNA library; (3) Capture the DNA fragment that hybridizes with the probe combination in the product of the hybridization reaction; (4) Perform PCR amplification, purification and sequencing on the captured products.

[0019] Furthermore, the conditions for the hybridization reaction include: Treat at 75~85℃ for 3~10 min, then perform hybridization reaction at 45~55℃ for 8~16 h.

[0020] The present invention has the following beneficial effects: 1. The probe combo of the present invention has high efficiency and broad spectrum: the probe combo is designed based on all whole genome reference sequences in NCBI, which can efficiently capture the major HEV genotypes (types 3 and 4) known in the prior art, cover 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.

[0021] 2. The probe combination 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.

[0022] 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.

[0023] 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, food safety testing, and basic research, providing key technical support for the precise prevention and in-depth research of hepatitis E. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a diagram of the capture probe design scheme provided in an embodiment of the present invention.

[0026] Figure 2 This is a flowchart of the whole genome sequencing process for hepatitis E virus provided in an embodiment of the present invention.

[0027] Figure 3 This is a schematic diagram of HEV1 sequencing coverage and depth provided in an embodiment of the present invention.

[0028] Figure 4 This is a schematic diagram of HEV2 sequencing coverage and depth provided in an embodiment of the present invention.

[0029] Figure 5 This is a schematic diagram of HEV3 sequencing coverage and depth provided in an embodiment of the present invention.

[0030] Figure 6 This is a schematic diagram of HEV4 sequencing coverage and depth provided in an embodiment of the present invention.

[0031] Figure 7 This is a schematic diagram of HEV5 sequencing coverage and depth provided in an embodiment of the present invention.

[0032] Figure 8 This is a schematic diagram of HEV6 sequencing coverage and depth provided in an embodiment of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0035] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0036] Example 1: Design of capture probes for whole-genome sequencing of hepatitis E virus The design scheme of the capture probe set targeting the whole genome of hepatitis E virus (HEV) is as follows: Figure 1 As shown, the design of the capture probe set is based on all 531 complete HEV 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 HEV genome. The probe length is preferably 100 nucleotides to ensure the continuity and uniformity of coverage.

[0037] The probes with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.951 were finally obtained, as well as probe combinations composed of these probes.

[0038] Example 2: Whole genome sequencing of hepatitis E virus This embodiment uses the capture probe set designed in Example 1 for hepatitis E virus whole genome sequencing. The specific process is as follows: Figure 2 As shown, the method includes: 1. Construction of total RNA library.

[0039] (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).

[0040] (2) RNA fragmentation.

[0041] 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 or vortex to mix, and then 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.

[0042] (3) Reverse transcription.

[0043] 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 and run the program. When the PCR instrument temperature drops to 4℃, remove the PCR tube, centrifuge briefly to obtain cDNA, and immediately proceed to step (4).

[0044] (4) cDNA double strand synthesis, addition of “A” to the 3' end.

[0045] 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 and run the program. When the PCR instrument temperature drops to 4℃, remove the PCR tube, centrifuge briefly, and immediately proceed to step (5).

[0046] (5) Connector connection.

[0047] 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℃ Keep warm at 4℃ for 15 minutes, 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 (6).

[0048] (6) Purification after ligation.

[0049] 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).

[0050] (7) Pre-PCR reaction.

[0051] 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, when the temperature of the PCR instrument drops to 4℃, take out the PCR tube, centrifuge briefly, and immediately proceed to step (8).

[0052] (8) Purification after PCR amplification.

[0053] 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.

[0054] 2. High-performance liquid phase hybridization (two rounds of liquid phase capture probe hybridization capture experiments).

[0055] (1) Preparations for the hybridization capture experiment.

[0056] 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 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, heat the TargetSeq One® Hyb Buffer v2 in a 37°C water bath until the reagent is completely dissolved before use).

[0057] (2) Hybridize the total RNA library with the capture probe.

[0058] 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 to 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, TargetSeq® Target Probes (referring to the capture probe set designed in Example 1) 2 μL. After preparation, add the hybridization reaction solution to the dry library, vortex for 30 s to ensure that the DNA at the bottom of the tube is dissolved, and centrifuge briefly; set the PCR instrument parameters as follows: 85℃ hot cap temperature, 80℃ for 5 min, 50℃ incubation for 12 h, place the hybridization reaction solution on the PCR instrument, and run the program; perform step (3) 30 min before the program ends.

[0059] (3) Preparations before the capture experiment.

[0060] 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 by pipetting or vortexing 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 by suction or vortexing, and immediately proceed to step (4).

[0061] (4) Target region DNA capture.

[0062] 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.

[0063] 3. PCR amplification after capture.

[0064] (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: 24 μL of magnetic bead suspension, 1 μL of Post PCR Primer, and 25 μL 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℃ 1 min; 98℃ 20 s, 60℃ 30 s, 72℃ 30 s, 15 cycles; 72℃ 5 min; 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.

[0065] (2) Purification after amplification.

[0066] 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 minutes; briefly centrifuge, place the PCR tube on the magnetic rack for 2 minutes, 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, store the capture library in a -20°C freezer for up to 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 consistent with the size of the whole genome library before capture.

[0067] 4. Sequencing.

[0068] 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.

[0069] In the raw sequencing data, a small number of reads contain adapter information, low-quality bases, etc. To ensure the quality of information analysis, the raw reads are initially filtered to obtain clean reads, and subsequent analyses are based on clean reads. The data filtering mainly includes 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; usually, if the remaining sequence length is less than 40 (paired ends) after removal, the sequence pair is discarded.

[0070] Removing host sequences: Since the host's genome sequence still remains in the sequencing data, the Bowtie2 sequencing data is first aligned to the host's reference genome to obtain the sequences that could not be aligned (unmapR1 & unmapR2). The unmap sequences are the viral sequence information that may actually be needed.

[0071] 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.

[0072] Obtain the full-length genome sequence of hepatitis E 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 genome sequence of hepatitis E virus.

[0073] Example 3: Validation of Hepatitis E Virus Whole Genome Capture Sequencing This embodiment utilizes the capture probe set designed in Example 1 and the sequencing method in Example 2 to verify the capture sequencing of the entire hepatitis E virus genome, including the following process: (1) Experimental materials.

[0074] Samples: 3 clinically diagnosed hepatitis E HEV RNA positive pig bile samples (HEV1, HEV2, HEV3), 1 clinically diagnosed hepatitis E HEV RNA positive rabbit liver sample (HEV4), 1 clinically diagnosed hepatitis E HEV RNA positive human fecal sample (HEV5), and 1 clinically diagnosed hepatitis E HEV RNA positive human serum sample (HEV6).

[0075] Instruments and reagents: QIAamp Viral RNA extraction kit, Illumina NovaSeq 6000 sequencing platform, capture probe kit of this invention and matching reagents.

[0076] (2) Experimental methods.

[0077] The experimental method in this embodiment is the same as in embodiment 2.

[0078] (3) Results and Analysis: As shown in Table 1, Figures 3-8 As shown, Figure 3 For HEV1 sequencing coverage and depth, Figure 4 For HEV2 sequencing coverage and depth, Figure 5 For HEV3 sequencing coverage and depth, Figure 6 For HEV4 sequencing coverage and depth, Figure 7 For HEV5 sequencing coverage and depth, Figure 8 For HEV6 sequencing coverage and depth.

[0079] The capture coverage results showed that after using the kit of the present invention to capture, sequence and analyze the whole genome of 4 cases of hepatitis E virus according to the above steps, the whole genome coverage can be >99.1%.

[0080] The average sequencing depth results show that after using the kit of the present invention to capture, sequence and analyze the whole genome of 4 cases of hepatitis E virus according to the above steps, the average coverage depth of whole genome sequencing can be >1000x.

[0081] The results of coverage uniformity show that after using the kit of the present invention to capture, sequence and analyze the whole genome of 4 cases of hepatitis E virus according to the above steps, the whole genome depth of ≥30x can be achieved by >98.9%.

[0082] Table 1. Validation of whole-genome sequencing of hepatitis E virus

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probe assembly, characterized in that, include: The nucleotide sequences of the probes are shown in SEQ ID NO.1-SEQ ID NO.

951.

2. The probe assembly according to claim 1, characterized in that, At least one of the probes is connected to a detectable marker; Preferably, the detectable marker includes one or more of fluorescent dyes, radioactive isotopes, enzymes, chemiluminescent groups, or biotin.

3. A reagent kit, characterized in that, Includes the probe combination as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, Also includes: One or more of the following: hybridization buffer, human genome blocking reagent, adapter blocking reagent, ribonuclease inhibitor, or water.

5. The kit according to claim 3 or 4, characterized in that, Also includes: Reagents for genomic library construction; Preferably, the genomic library construction reagents include one or more of the following: RNA fragmentation reagents, reverse transcription reagents, cDNA two-strand synthesis reagents, adapter ligation reagents, purification reagents, PCR pre-reaction reagents, and post-capture PCR amplification reaction reagents.

6. A gene chip, characterized in that, The gene chip includes: a solid support and a probe assembly as described in claim 1 or 2 fixed on the solid support.

7. The use of the probe combination of claim 1 or 2, or the kit of any one of claims 3-5, or the gene chip of claim 6, in any of the following: (1) Detecting hepatitis E virus, or preparing products for detecting hepatitis E virus; (2) Whole genome sequencing of hepatitis E virus, or preparation of products for whole genome sequencing of hepatitis E virus.

8. A method for whole-genome sequencing of hepatitis E virus, characterized in that, include: Whole genome sequencing of the sample to be tested is performed using the probe combination described in claim 1 or 2, the kit described in any one of claims 3-5, or the gene chip described in claim 6.

9. The whole-genome sequencing method according to claim 8, characterized in that, include: (1) Extract RNA from the sample to be tested and construct a cDNA library; (2) Hybridize the probe combination with the cDNA library; (3) Capture the DNA fragment that hybridizes with the probe combination in the product of the hybridization reaction; (4) Perform PCR amplification, purification and sequencing on the captured products.

10. The whole-genome sequencing method according to claim 8 or 9, characterized in that, The conditions for the hybridization reaction include: Treat at 75~85℃ for 3~10 min, then perform hybridization reaction at 45~55℃ for 8~16 h.

Images