Capture probe group and kit for measles virus full-length genome and sequencing method of measles virus full-length genome

Abstract

The invention provides a capturing probe group and a kit for a measles virus full-length genome and a sequencing method of the measles virus full-length genome, and belongs to the technical field of virus detection. The invention relates to an inactive probe group of a measles virus full-length genome. The inactive probe group comprises probes with nucleotide sequences as shown in SEQ ID NO: 1-SEQ ID NO: 742. The capturing probe group and next-generation sequencing are combined to obtain a measles full-length genome sequence. Compared with a primer or a probe used in a sequencing method in the prior art, the single probe in the probe group is short and high in specificity, the same site is designed by using the imbricate probe, all variations of the site are covered, and when a virus sequence is captured, the probe group has the characteristics of high sensitivity, high coverage rate and high flux.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and particularly relates to a capture probe set for the full-length genome of measles virus, a kit thereof, and a sequencing method thereof. Background Art

[0002] Measles is a highly contagious acute febrile exanthematous disease caused by measles virus, which is classified as a Class B infectious disease in China. The full-length genome of measles virus is about 16 kb; measles virus has 6 structural genes, encoding 6 major structural proteins. Starting from the 3'-end, they are N protein, P protein, M protein, F protein, H protein, and L protein in sequence. The 450-nucleotide sequence at the COOH-terminal of the measles virus N gene is the minimum target gene sequence specified by the WHO for identifying the genotypes of measles virus. Although measles virus has only one serotype, it has multiple genotypes. Currently, a total of 24 genotypes in 8 genomes from A to H have been discovered. As of 2024, only genotypes B3 and D8 are prevalent in populations around the world. The genotypes of measles virus have geographical distribution characteristics, with different local epidemic strains or dominant epidemic strains in different regions. At the same time, the global epidemic of wild measles virus is also somewhat correlated with the era. Currently, the N450 sequences of the monitored measles virus have a relatively high consistency, lacking specific epidemiological information. Relying solely on the N450 sequence window, it is impossible to distinguish whether the same N450 virus transmitted between provinces is continuous local transmission or new importation. In this case, it is necessary to introduce an extended sequence window, such as the full genome, in countries and regions where measles is approaching elimination or has been eliminated, so as to improve the molecular surveillance resolution of measles virus, better trace the transmission path of the virus, determine whether the virus is continuous local transmission or new importation, and provide data support for measles elimination.

[0003] Currently, the methods for obtaining the full-length genome sequence of measles virus mainly include the following: 1) RT-PCR method: Specific primers are designed for specific amplification of a certain fragment length, and then Sanger sequencing is used. After splicing, the full-length sequence is obtained. However, in the MF non-coding region, due to the high G + C content of up to 70%, it is difficult to obtain good sequence results. At the same time, this method requires a high virus load, high cost, and low throughput; 2) Next-generation sequencing: By sequencing all nucleic acids of the sample using universal primers, the cost is reduced, high accuracy is maintained, the sequencing duration is reduced, but the read length is short. Affected by the sequencing depth, the subsequent output data volume is huge, and the requirements for data analysis are relatively high. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a capture probe set for the full-length genome of measles virus, which uses the overlapping probe design method to design short-fragment capture probes with high specificity and covering all variant sites, ensuring the accuracy and reliability of the sequencing results.

[0005] The present invention provides a set of capture probes for the full-length genome of measles virus, and the set of capture probes includes probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 742.

[0006] Preferably, one end of the capture probe is modified with biotin.

[0007] The present invention provides a kit for sequencing and detecting the full-length genome of measles virus, which includes the set of capture probes, streptavidin-labeled magnetic beads, and library hybridization reaction reagents.

[0008] Preferably, the library hybridization reaction reagents include at least one of the following reagents: TargetSeq HybBufferv2, Hyb Human Block, Blocking Oligo, and RNase enzyme inhibitor.

[0009] Preferably, the kit further includes at least one of the following: RNA fragmentation reagent, reverse transcription reaction reagent, cDNA second-strand synthesis reaction reagent, adapter ligation reagent, purification reagent, PCR pre-reaction reagent, and post-capture PCR amplification reaction reagent.

[0010] Preferably, the PCR pre-reaction reagent includes PCR MasterMix containing UDG enzyme and / or UDI primer;

[0011] The post-capture PCR amplification reaction reagent includes PCR primers and / or post-capture PCR reaction premix.

[0012] The present invention provides the application of the set of capture probes or the kit in the sequencing of the full-length genome of measles virus.

[0013] Preferably, the sequencing includes next-generation sequencing.

[0014] The present invention provides a method for next-generation sequencing of the full-length genome of measles virus based on the set of capture probes, which includes the following steps:

[0015] Hybridize the measles virus genome library of the sample to be tested with the set of capture probes to obtain a hybridization product;

[0016] Isolate the DNA fragments hybridized with the probes from the hybridization product;

[0017] Perform post-capture PCR amplification using the DNA fragments hybridized with the probes as templates, perform next-generation sequencing on the obtained PCR products, and perform data analysis on the sequencing results to obtain the full-length genome of measles virus.

[0018] The present invention provides a set of capture probes for the full-length genome of measles virus, and the set of capture probes includes probes with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 742. The set of capture probes of the present invention is designed based on the full-length genome sequence of measles virus in GenBank data, and has the characteristics of short length of a single probe, high specificity, and the use of the tiling probe design method at the same locus to cover all variations at this locus, thereby forming a set of capture probes. Compared with the conventional sequencing method of designing primers in fragments, when the set of capture probes binds to viral RNA, it has the characteristics of high sensitivity, high coverage rate, and high throughput, and can detect new mutations. The measles virus full-length genome capture technology (next-generation sequencing) based on the set of probes has the advantages of high throughput and short time consumption, and can improve the detection accuracy, specificity, and timeliness of sequence acquisition of the sample to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of probe design and probe capture principle;

[0020] Figure 2 Comparison chart of sequencing results of sample 2 by first-generation sequencing and second-generation sequencing based on capture probes of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The present invention provides a set of capture probes for the full-length genome of measles virus, including probes with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 742.

[0022] In the present invention, the set of probes includes a total of 742 probes, which can comprehensively cover the measles virus genome sequence, and has the characteristics of uniformity, specificity, and high capture efficiency for the captured region. The capture probes are preferably synthesized by high-throughput electrochemical synthesis. In the embodiments of the present invention, the capture probes are commissioned to be synthesized by Beijing E-Genomics Co., Ltd.

[0023] In the present invention, one end of the probe is preferably labeled with biotin. The modification of biotin facilitates the formation of a complex after the subsequent hybridization of the capture probe with the target region DNA, and then the amplification system of biotin-streptavidin is used to separate the target sequence.

[0024] The present invention provides a kit for measles virus full-length genome sequencing detection, including the capture probe, streptavidin-labeled magnetic beads, and library hybridization reaction reagents.

[0025] In the present invention, the streptavidin-labeled magnetic beads bind to the biotin on the capture probe through streptavidin, and then the capture probe and the target region DNA sequence that specifically binds to the capture probe are separated from the system by using magnetic force. The library hybridization reaction reagent preferably includes at least one of the following reagents: TargetSeq Hyb Bufferv2, Hyb Human Block, Blocking Oligo and RNase enzyme inhibitor. The library hybridization reaction reagent is used for the hybridization reaction of the capture probe and for the capture probe to bind to a specific DNA sequence in the genomic library.

[0026] In the present invention, the kit preferably further includes an RNA fragmentation reagent, a reverse transcription reaction reagent, a cDNA second-strand synthesis reaction reagent, an adapter ligation reagent, a purification reagent, a PCR pre-reaction reagent, and a post-capture PCR amplification reaction reagent. The PCR pre-reaction reagent preferably includes at least one of the following: a PCR MasterMix containing UDG enzyme and a UDI primer. The RNA fragmentation reagent, the reverse transcription reaction reagent, the cDNA second-strand synthesis reaction reagent, the adapter ligation reagent, and the purification reagent are used for constructing a measles virus genomic library. The present invention places no special restrictions on the reagents for constructing the measles virus genomic library, and well-known reagents in the art can be used. The post-capture PCR amplification reaction reagent preferably includes at least one of the following: a PCR primer and a post-capture PCR reaction premix. The post-capture PCR amplification reaction reagent is used to amplify the captured DNA sequence of the target region to obtain a large amount of DNA sequences of the target region to meet the sequencing requirements.

[0027] The present invention provides the application of the capture probe set or the kit in the full-length genomic sequencing of measles virus.

[0028] In the present invention, the genotype of the measles virus preferably includes at least one of the following: H1, D8, and B3. The sequencing preferably includes next-generation sequencing. The method for detecting the full-length genome of the measles virus preferably involves hybridizing the measles virus genomic library with the capture probe, separating the target region DNA sequence bound to the capture probe in the hybridization solution. Since the capture probe can cover 100% of the full-length genome of the measles virus, a sequence covering the full-length genome of the measles virus is obtained. After PCR amplification and next-generation sequencing, the full-length genome of the measles virus is obtained. The full-length genome of the measles virus obtained by sequencing can be used for measles virus genome variation monitoring and / or measles virus genome traceability analysis. By using the kit for next-generation sequencing to obtain the full-length genome of the measles virus and comparing it with the reference genome, the variation information of the measles virus genome is obtained. The variation information includes single-base mutations and / or insertion / deletion mutations. By comparing and analyzing the variation information with the genomic sequences in the database, the purpose of measles virus genome variation monitoring and / or traceability analysis is achieved. The method for measles virus genome traceability analysis is based on obtaining the full-length genome sequence information of the measles virus, a full-genome database of a large number of sample strains can be established, and based on this database, the virus type in the measles virus outbreak area can be traced. The method for measles virus genome variation monitoring preferably enables the effective detection of mutations at any locus on the measles virus genome by the capture probe, accurately achieving the monitoring of measles virus variation.

[0029] The present invention provides a method for next-generation sequencing of the full-length genome of the measles virus based on the capture probe, comprising the following steps:

[0030] Hybridize the measles virus genomic library of the test sample with the capture probe to obtain a hybridization reaction solution;

[0031] Separate the DNA fragment hybridized with the probe from the hybridization reaction solution;

[0032] Perform post-capture PCR amplification using the DNA fragment hybridized with the probe as a template, subject the obtained PCR product to next-generation sequencing, and perform data analysis on the sequencing results to obtain the full-length genome of the measles virus.

[0033] In the present invention, the measles virus genomic library of the test sample is hybridized with the capture probe to obtain a hybridization solution.

[0034] The present invention has no special restrictions on the method for constructing a measles virus genomic library of a sample to be tested, and a genomic library construction method well-known in the art can be used, such as extraction and fragmentation of total RNA, reverse transcription and cDNA second-strand synthesis, addition of adapters and pre-PCR reaction, and fragment purification to obtain a genomic library. When extracting total RNA of the sample to be tested, it is not necessary to culture pathogenic microorganisms, and the RNA extracted directly from clinical samples (such as blood, oral swabs, throat swabs, alveolar lavage fluid, feces, etc.) can be used. In the examples of the present invention, the construction of the genomic library adopts the instructions of a commercial kit ( FastStranded RNA Library Prep Kit v2.0, TargetSeq Hyb&Wash Kit v2.0).

[0035] In the present invention, the reaction system for the hybridization is preferably 30 μL, and preferably includes the following reagents: TargetSeq Hyb Buffer v2 13 μL, Hyb Human Block 5 μL, Blocking Oligo 2 μL, RNase Block 5 μL, Nuclease-Free Water 3 μL, and Target Probes 2 μL. The reaction program for the hybridization is preferably to treat at 80 °C for 5 min and at 50 °C for 12 - 18 h.

[0036] In the present invention, the method for separating the DNA fragment hybridized with the probe from the hybridization solution is preferably to mix the hybridization solution with streptavidin-modified magnetic beads, and utilize the binding property of streptavidin and biotin to bind the magnetic beads to the hybridization complex to form a ternary complex, and separate the ternary complex from the hybridization solution under the action of magnetic field force.

[0037] After obtaining the DNA fragment hybridized with the probe, the present invention uses the DNA fragment hybridized with the probe as a template for post-capture PCR amplification, performs next-generation sequencing on the obtained PCR product, and performs data analysis on the sequencing results to obtain the full-length genome of the measles virus.

[0038] In the present invention, the reaction system for the post-capture PCR amplification is preferably 50 μL, and preferably includes the following reagents: 24 μL of the DNA fragment suspension hybridized with the probe, 1 μL of Post PCR primer, and 25 μL of Post PCR Master Mix. The reaction program for the post-capture PCR amplification is preferably 95 °C for 1 min; 98 °C for 20 s, 60 °C for 30 s, 72 °C for 30 s, 7 - 12 cycles; 72 °C for 5 min.

[0039] In the present invention, the method for analyzing the sequencing results preferably includes preliminarily filtering the raw sequencing data to obtain Cleanreads;

[0040] Removing the host gene sequences from the Clean reads, aligning the obtained sequences to the viral genome, and assembling to obtain contig sequences;

[0041] After removing the host gene sequences from the contig sequences again, aligning them to the viral genome sequence to obtain data for viral typing and sorting;

[0042] Aligning the data for viral typing and sorting to the reference genome, and correcting the variant sites to obtain the full-length genome of the measles virus.

[0043] In the present invention, since there are adapter information and low-quality sequence reads in the raw sequences obtained by sequencing, in order to improve the quality of the sequencing data, the raw sequencing data is preliminarily filtered. The method of the preliminary filtering is preferably to cut off the sequences with an average base quality value less than 20 bp in a sliding window of 8 bp; remove the adapter sequences at the end of the sequences; if the first or last base of the sequence is less than 20 bp, then directly cut off the base; usually, if the remaining sequence length is less than 40 bp (paired-end) after the removal, then discard the pair of sequences.

[0044] In the present invention, the method for removing the host gene sequences is preferably to use the Bowtie2 software to align the sequencing data to the reference genome of the host and collect the sequences that cannot be aligned.

[0045] In the present invention, it is preferably to use the bwa software to align the collected sequences that cannot be aligned to the viral genome. The viral genome herein refers to the measles virus genome. The method for assembling the contig sequences is preferably to use the MEGAHIT software for assembly.

[0046] In the present invention, after removing the host gene sequences from the contig sequences again, aligning them to the viral genome to obtain data for viral typing and sorting. The viral genome herein refers to the measles virus genome.

[0047] In the present invention, aligning the data for viral typing and sorting to the reference genome, and correcting the variant sites to obtain the full-length genome of the measles virus.

[0048] In the present invention, the reference genome is preferably the measles virus typing sequence ranked first in the virus typing sorting data. The alignment is preferably performed using the bwa software. The variant site correction is preferably to perform mutation analysis using the samtools software, and after obtaining the mutation sites, the iVar software is used to correct the variant sites. After the variant site correction, sequence integration is preferably further included. The method of sequence integration is preferably as follows: taking the measles virus typing sequence ranked first in the virus typing sorting data as the reference genome, extracting the reads that can be aligned to both the host and the virus genome from the Cleanreads, finding the integration site coordinates on the host genome according to these sequences, and integrating the corrected sequences into the full-length measles virus genome sequence. In the embodiments of the present invention, both the second-generation sequencing and the sequencing data analysis are entrusted to Aiji Taikang Company to complete.

[0049] In the present invention, by adopting the above detection method and combining the probe hybridization capture and the second-generation sequencing technology, the full-length measles virus genome sequence can be obtained at low cost and quickly.

[0050] The following will describe in detail a capture probe set and a kit for the full-length genome of a measles virus provided by the present invention with reference to the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] The capture probe Panel for the full-length sequence of the measles virus (Measles morbillivirus, taxid: 11234) genome was evaluated and designed using the intelligent probe design platform AIdesign independently developed by Aiji Taikang. This design platform is designed and optimized based on thermodynamic stability and AI artificial intelligence learning, fully considering the balance of coverage, uniformity, and capture efficiency of each virus in the database, and can achieve customized design for any species and any region, ensuring accurate and efficient design.

[0053] According to 889 full-length reference genome sequences of the measles virus (Measles morbillivirus, taxid: 11234) downloaded from the NCBI database, oligonucleotide probes complementary to the sequences in the database were designed using the base complementary pairing principle. Then, all the probes designed from the variants were merged to remove redundancy to prevent the situation of too high depth in highly conserved regions. After comprehensive evaluation, a total of 742 probes were designed and synthesized, with a coverage of up to 100% for all sequences in the database, and ensuring the uniformity and effectiveness of the capture depth in each region of each genome.

[0054] Due to the complex genomic sequence structure with irregular regions, such as high-GC, high-AT regions, Alu sequences, etc., and at the same time, due to the limitation of the read length of next-generation sequencing and the need to obtain full-length sequences, probe design becomes particularly crucial, and various factors such as probe coverage, uniformity, and capture efficiency need to be comprehensively weighed. The following are the principles of probe design in liquid-phase capture technology:

[0055] 1. Probe length: The probes designed by Aiji Taikang are 100-nt long probes.

[0056] 2. Probe layer number: That is, the average number of probes covering the target region. Aiji Taikang adopts an overlapping probe sequence design method to perform multi-layer probe coverage on the target region. At the same time, during the probe sequence design process, the thermodynamic stability is evaluated, and probes are compensated for regions with low thermodynamic stability. For pathogens with large variations such as viruses, a probe density design of more than 10 layers is usually carried out; for pathogens with relatively small variations such as bacteria, the average probe density is also set to 3 to 10 layers. In this way, even if relatively complex variations or new variations occur in the pathogen genome, the probe tolerance can still stably capture its genome.

[0057] 3. Specificity: That is, the uniqueness of the probe in the genomic range. The higher the specificity, the higher the capture efficiency of the probe. Probe design in low-complexity regions should be avoided; however, in special cases, such as when certain hotspots happen to be located in low-complexity regions, the sequence conditions of this region and adjacent regions need to be comprehensively evaluated, and probes should be placed carefully.

[0058] 4. Binding ability: In regions with a GC content of about 50%, the probe capture ability is the strongest. In high-GC regions, the probe binding ability is strong, but the DNA fragment itself has a stronger binding ability (longer length), and the probe competition resistance is relatively large; in high-AT regions, the DNA fragment itself has a weak binding ability, and the probe binding ability to it is also weak. Therefore, in high-GC and high-AT regions, the number of probes needs to be appropriately increased to make up for the disadvantages in terms of competition resistance and binding ability.

[0059] 5. Secondary structure: The formation of hairpin structures by the probe itself and dimers between probes should be avoided as much as possible, otherwise it will affect the binding of the probe to the target DNA.

[0060] 6. For cases where microorganisms have multiple highly variable sequences, etc., attention should be paid to:

[0061] (a) Design probes for all virus whole-genome sequences that can be downloaded from the database to avoid omission;

[0062] Merge and remove redundancy for all probes to prevent the situation of too high sequencing depth in highly conserved regions. 6. For cases where microorganisms have multiple highly variable sequences, etc., attention should be paid to:

[0063] (b) Design probes for all the viral whole-genome sequences that can be downloaded from the database to avoid omission.

[0064] Merge and remove redundancy from all the probes to prevent the situation of too high sequencing depth in highly conserved regions. After comprehensive evaluation, a total of 742 probes were designed and synthesized, with a coverage of up to 100% for all the sequences in the database, and the capture depth of each region of each genome was ensured to be uniform and effective. The probe sequences are shown in Table 1.

[0065] Table 1 Probe sequences

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Example 2

[0100] A method for detecting the whole genome of measles based on the combination of capture probe sets and next-generation sequencing

[0101] 1. RNA extraction from the sample to be tested

[0102] Clinical samples GS16-35 (H1 genotype), HLJ18-2 (D8 genotype), and AH19-1 (B3 genotype) positive for measles collected from 2016 to 2019 were used to extract RNA using the QIAamp Viral RNA Minikit (product number 52906, Qiagen, Germany). The RNA concentration was quantified using the Qubit RNA HS assay kit and the Qubit Fluorometer (Life Technologies, USA).

[0103] 2. Library preparation

[0104] 2.1. RNA fragmentation

[0105] 2.1.1 Take out the RNA sample and FastFrag Buffer from the -80°C and -20°C refrigerators respectively, melt them on an ice box, briefly vortex, centrifuge instantaneously, and place them on the ice box for standby.

[0106] 2.1.2 Prepare the reaction system according to Table 2 below, mix well and centrifuge instantaneously:

[0107] Table 2 Fragmentation reaction system

[0108] Reagent Volume RNA sample 13 μL (total 10 ng - 1 μg) FastFragBuffer 4 μL Total volume 17 μL

[0109] 2.1.3 Set the PCR instrument running program according to Table 3, and place the reaction solution on the PCR instrument:

[0110] Table 3 Reaction program

[0111]

[0112] 2.2. Reverse transcription

[0113] 2.2.1 Take out the FastFirst Strand Buffer in the kit from the -20°C refrigerator, melt it on an ice box, briefly vortex after melting, centrifuge instantaneously, and place it on the ice box for standby.

[0114] 2.2.2 Take out the Fast First Strand Enzyme in the kit from the -20°C refrigerator, invert and mix well, centrifuge instantaneously, and place it on the ice box for standby.

[0115] 2.2.3 Prepare the reaction system according to Table 4 below, pipette and mix well (avoid vigorous shaking), centrifuge instantaneously:

[0116] Table 4 Reverse transcription reaction system

[0117] Reagent Volume Sample from the end of the reaction in Step 1 17 μL FastFirstStrandBuffer 6 μL FastFirstStrandEnzyme 2 μL Total volume 25 μL

[0118] 2.2.4 Set the PCR instrument running program according to Table 5, and place the reaction solution on the PCR instrument:

[0119] Table 5 Reverse transcription reaction system

[0120]

[0121] 2.3. cDNA second strand synthesis, 3'-end addition of "A"

[0122] 2.3.1 Take out the Fast Second Strand Buffer with dUTP in the kit from the -20°C refrigerator in advance, melt it on an ice box, briefly vortex after melting, centrifuge instantaneously, and place it on the ice box for standby.

[0123] 2.3.2 Take out the Fast Second Strand Enzyme in the kit from the -20°C refrigerator, invert and mix well, then centrifuge briefly, and place it on an ice box for standby.

[0124] 2.3.3 Prepare the reaction system according to Table 6 below, pipette and mix well (avoid vigorous shaking), and centrifuge briefly:

[0125] Table 6 cDNA second-strand synthesis system

[0126] Reagent Volume Sample from the end of the reaction in Step 2 25 μL FastSecondStrandBuffer with dUTP 30 μL FastSecondStrandEnzyme 5 μL Total volume 60 μL

[0127] 2.3.4 Set the PCR instrument running program according to Table 7, and place the reaction solution on the PCR instrument to run the program:

[0128] Table 7 cDNA second-strand synthesis reaction program

[0129]

[0130] 2.4. Ligation of adapters

[0131] 2.4.1 Take out the Adapter from the -20°C refrigerator in advance, place it on an ice box to melt, briefly vortex and mix well after melting, then centrifuge briefly, and place it on an ice box for standby.

[0132] 2.4.2 According to the amount of RNA input for library construction, dilute the Adapter (15 μM) to an appropriate concentration in advance according to Table 8:

[0133] Table 8 Relationship between the amount of input RNA and the ratio of adapters

[0134] RNA input amount Adapter concentration Dilution factor 100 ng - 500 ng 7.5 μM 2-fold 50 ng 3.75 μM 4-fold 25 ng 1.5 μM 10-fold 10 ng 0.75 μM 20-fold

[0135] 2.4.3 Take out the Fast Ligation Buffer and Fast Ligase Mix in the kit from the -20°C refrigerator in advance, place them on an ice box to melt for standby.

[0136] 2.4.4 Prepare the reaction system on an ice box according to Table 9 below:

[0137] Table 9 Reaction system for adding adapters

[0138] Reagent Volume Sample from the end of the reaction in Step 3 60 μL FastLigationBuffer 30 μL FastLigaseMix 5 μL Adapter (after dilution) 5 μL Total volume 100 μL

[0139] First, mix the diluted Adapter and the sample after the reaction in step 3, then add the pre-mixed reaction solution to reduce adapter self-ligation. Pipette and mix well (avoid vigorous shaking) and centrifuge briefly.

[0140] 2.4.5 Set the PCR instrument parameters according to Table 10, and place the PCR tube on the PCR instrument to run the program:

[0141] Table 10 Ligation Reaction Procedure

[0142]

[0143] 2.5. Post-ligation Purification

[0144] 2.5.1 Use freshly prepared 80% ethanol (prepared with absolute ethanol and Nuclease-Free Water) for magnetic bead purification. (The magnetic beads used for purification are IGT TM Pure Beads or Agencourt AMPure XP).

[0145] 2.5.2 Take out the purified magnetic beads from the 4°C refrigerator. After mixing and equilibrating at room temperature for 30 min, vortex and mix for later use.

[0146] 2.5.3 Add 0.45 times the volume of the purified magnetic beads to the 100 μL reaction solution in Step 4. Pipette and mix well, then let it stand for 5 min.

[0147] 2.5.4 Centrifuge briefly, place the PCR tube on the magnetic stand for 3 min until the solution is clear.

[0148] 2.5.5 Keep the PCR tube on the magnetic stand, discard the supernatant, add 200 μL of 80% ethanol solution to the PCR tube, let it stand for 30 s, and then discard the supernatant.

[0149] 2.5.6 Repeat the previous step.

[0150] 2.5.7 Cover the tube lid, centrifuge briefly, place it on the magnetic stand, and carefully use a 10 μL pipette to discard the residual ethanol at the bottom.

[0151] 2.5.8 Keep the PCR tube on the magnetic stand and let it stand at room temperature for 3 - 5 min to dry the magnetic beads.

[0152] 2.5.9 Remove the PCR tube from the magnetic stand, add 22 μL of Nuclease-Free Water, pipette and mix well, then let it stand for 2 min.

[0153] 2.5.10 Centrifuge briefly, place the PCR tube on the magnetic stand for 2 min until the solution is clear.

[0154] 2.5.11 Use a pipette to aspirate 20 μL of the supernatant and transfer it to a new PCR tube, make a mark, and prepare for Step 6.

[0155] 2.6. Pre-PCR Reaction

[0156] 2.6.1 Take out the PCR Master Mix with UDG and UDI Primer in the kit from the -20°C refrigerator in advance, place it on an ice box to melt, mix well and then centrifuge instantaneously, and place it on the ice box for standby.

[0157] 2.6.2 Prepare the PCR reaction solution according to Table 11 below, and record the Index number used:

[0158] Table 11 Pre-PCR reaction system

[0159] Reagent Volume Sample from the end of the reaction in Step 5 20 μL PCR Master Mix with UDG 25 μL UDI Primer 5 μL Total volume 50 μL

[0160] The UDI Primer sequence is as follows:

[0161] 5’-AATGATACGGCGACCACCGAGATCTACACNACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO: 743), where N in this sequence represents "i5 Index", and the i5 Index adapter sequence refers to the Index sequence adapter located at the P5 end (close to the P5 primer end of the Flow-cell) during library construction on the Illumina sequencing platform.

[0162] 5’-CAAGCAGAAGACGGCATACGAGATNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 744), where N in this sequence represents "i7 Index", and the i7 Index adapter sequence is used to distinguish different samples on the Illumina sequencing platform. The i7 Index adapter sequence is usually located at the end of the sequencing library and binds to the sequencing primer to ensure that the data of each sample can be correctly distinguished during multiplexed sequencing. Mix well by pipetting and centrifuge instantaneously.

[0163] 2.6.5 Set the PCR instrument and run it according to the records in Table 12 and Table 13:

[0164] Table 12 Pre-PCR reaction program

[0165]

[0166]

[0167] Table 13 Relationship between the input sample volume and the number of PCR cycles

[0168] Input sample amount PCR cycle number (library output 500 ng 7 250 ng 7 100 ng 9 50 ng 10 25 ng 11 10 ng 12

[0169] 2.7. Purification after PCR amplification

[0170] 2.7.1 Use freshly prepared 80% ethanol (prepared from anhydrous ethanol and Nuclease-Free Water) for magnetic bead purification. (The magnetic beads used for purification are IGT TM Pure Beads or Agencourt AMPure XP).

[0171] 2.7.2 Take out the purified magnetic beads from the 4°C refrigerator, mix well, equilibrate at room temperature for 30 min, then vortex and mix well for standby.

[0172] 2.7.3 Add 0.9 times the volume of the purified magnetic beads to the 50 μL reaction solution after step 6, pipette and mix well, and let stand for 5 min.

[0173] 2.7.4 Centrifuge briefly, place the PCR tube on the magnetic stand for 3 min until the solution is clear.

[0174] 2.7.5 - 2.7.8 The steps are the same as 2.5.5 - 2.5.8.

[0175] 2.7.9 Remove the PCR tube from the magnetic stand, add 30 μL of Nuclease-Free Water, pipette and mix well, and let stand for 2 min.

[0176] 2.7.10 Centrifuge briefly, place the PCR tube on the magnetic stand for 2 min until the solution is clear. Pipette 28 μL of the supernatant and transfer it to a new PCR tube, and make a mark.

[0177] 2.7.12 Take 1 μL of the library and use the Qubit ds DNA HS Assay Kit reagent to measure the library concentration on a Qubit 4.0 Fluorometer, and record the library concentration.

[0178] 2.7.13 Take 1 μL of the sample and use a fragment analyzer to measure the fragment length. After the experiment, arrange for sequencing on the machine.

[0179] 3. Targeted capture

[0180] 3.1. Preparation before hybridization capture experiment

[0181] This experiment requires approximately two rounds of liquid-phase probe hybridization capture experiments.

[0182] 3.1.1 Take out Hyb Human Block, RNase Block and the supporting Blocking Oligo from the -20°C refrigerator, place it on an ice box to melt, briefly vortex and mix well, then centrifuge briefly, and place it on the ice box for temporary storage;

[0183] 3.1.3 Take out the probes and the library for hybridization capture from the -80°C and -20°C refrigerators, place them on an ice box to melt, briefly vortex and centrifuge instantaneously, and store them temporarily on the ice box;

[0184] 3.1.5 Take out TargetSeq Hyb Bufferv2, melt it at room temperature, briefly vortex and centrifuge instantaneously. If there is precipitation, heat TargetSeq Hyb Bufferv2 in a 37°C water bath until the reagent is completely dissolved before use.

[0185] 3.2. Library hybridization with probes

[0186] 3.2.1 When hybridizing a single library, take 750 ng of the library and add it to a PCR tube, and make a mark; when hybridizing multiple libraries mixed, add 500 ng for each library.

[0187] 3.2.2 Place the PCR tube in a vacuum concentrator centrifuge, open the lid of the PCR tube, and concentrate it to a dry state. Prepare the hybridization reaction solution according to Table 14 below:

[0188] Table 14 Hybridization reaction system

[0189]

[0190] 3.2.4 Add the hybridization reaction solution to the dried library, vortex for 30 s to ensure that the DNA dried at the bottom of the tube dissolves, and centrifuge briefly.

[0191] 3.2.5 Set the reaction program according to Table 15, and run the hybridization reaction solution on a PCR instrument:

[0192] Table 15 Hybridization reaction program

[0193]

[0194] 3.2.6 It is recommended that the hybridization time be 12 - 18 h, and perform step 3 30 min before the end of the program.

[0195] 3.3. Preparation before capture experiment

[0196] 3.3.1 Take out Cap Beads from the 4°C refrigerator in advance, mix well and equilibrate at room temperature for 30 min.

[0197] 3.3.2 Take out Wash Buffer 1. If precipitation occurs, heat Wash Buffer1 in a 37°C water bath until the precipitation is completely dissolved before use.

[0198] 3.3.3 Take out TargetSeq Take out Wash Buffer2v2 and preheat it on a water bath at 50 °C.

[0199] 3.3.4 Take 50 μL Add Cap Beads into a new PCR tube, place it on a magnetic stand for 1 min. Wait until the solution becomes clear, then discard the supernatant.

[0200] 3.3.5 Remove the PCR tube from the magnetic stand, add 180 μL of Binding Buffer, pipette or vortex to mix well to resuspend the magnetic beads. After a brief centrifugation, place the PCR tube on the magnetic stand for 1 min. Wait until the solution becomes clear, then discard the supernatant;

[0201] 3.3.7 Repeat steps 3.5 - 3.6 twice, using Binding Buffer to wash the magnetic beads three times in total;

[0202] 3.3.8 Remove the PCR tube from the magnetic stand, add 180 μL of Binding Buffer, pipette or vortex to mix well, and immediately proceed to step 4.

[0203] 3.4. Target Region DNA Capture

[0204] 3.4.1 Keep the hybridization product from step 2 on the PCR instrument, and add the 180 μL CapBeads prepared in step 3 to the hybridization product, and pipette to mix well.

[0205] 3.4.2 Close the tube cap, remove the PCR tube from the PCR instrument, place it on a vertical rotating mixer with a rotation speed not exceeding 10 rpm, and bind at room temperature for 30 min (if there is no vertical rotating mixer in the laboratory, it can be bound at room temperature for 30 min, and mix by inverting the tube up and down several times every 5 min during this period).

[0206] 3.4.3 Remove the PCR tube, briefly centrifuge it, place it on the magnetic stand for 2 min. After the solution becomes clear, discard the supernatant.

[0207] 3.4.4 Remove the PCR tube from the magnetic stand, add 150 μL of Wash Buffer 1 to the PCR tube, gently pipette to mix well to resuspend the magnetic beads, replace the tube cap with a new one, and then place it on a vertical rotating mixer to wash at room temperature for 15 min with a rotation speed not exceeding 10 rpm.

[0208] 3.4.5 Remove the PCR tube, briefly centrifuge it, place it on the magnetic stand for 2 min. After the solution becomes clear, discard the supernatant.

[0209] 3.4.6 Remove the PCR tube from the magnetic stand, add 150 μL of TargetSeq preheated at 50 °C WashBuffer2v2, gently pipette to mix well, centrifuge briefly, place on a metal bath, and incubate at 50 °C for 10 min.

[0210] 3.4.7 Remove the PCR tube, centrifuge briefly, place on a magnetic stand for 2 min. After the solution becomes clear, discard the supernatant.

[0211] 3.4.8 Repeat steps 4.6 - 4.7 twice, and use TargetSeq in total Wash Buffer 2v2 Wash the magnetic beads three times at 50 °C.

[0212] 3.4.9 Keep the PCR tube on the magnetic stand, add 200 μL of 80% ethanol to the PCR tube, let it stand for 30 s, and then completely discard the ethanol solution (residual ethanol can be removed with a 10 μL pipette). Air-dry the magnetic beads at room temperature to completely volatilize the residual ethanol.

[0213] 3.4.10 Add 24 μL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic stand, briefly vortex to resuspend the magnetic beads, and proceed to the amplification reaction in step 5.

[0214] 3.4.11 Keep the PCR tube on the magnetic stand, add 200 μL of 80% ethanol to the PCR tube, let it stand for 30 s, and then completely discard the ethanol solution (residual ethanol can be removed with a 10 μL pipette). Air-dry the magnetic beads at room temperature to completely volatilize the residual ethanol (during the drying process, observe the surface of the magnetic beads to avoid over-drying the magnetic beads).

[0215] 3.4.12 Add 24 μL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic stand, briefly vortex to resuspend and mix the magnetic beads well, and proceed to the amplification reaction in step 5.

[0216] 3.5. Post-PCR amplification after capture

[0217] 3.5.1 Take out the Post PCR MasterMix and Post PCR Primer from the -20 °C refrigerator in advance, place them on an ice box to melt, and store them temporarily on the ice box after melting.

[0218] 3.5.2 Please double-check whether the PostPCR Primer is used correctly before the experiment. Briefly vortex the PostPCR Master Mix and PostPCR Primer, and centrifuge briefly.

[0219] 3.5.3 Prepare the PCR reaction solution according to Table 16 below. Note that this step is a PCR reaction with magnetic beads:

[0220] Table 16 Post-PCR reaction system after capture

[0221] Reagent Volume Magnetic bead suspension obtained in Step 4 24 μL PostPCRPrimer 1 μL PostPCRMasterMix 25 μL Total volume 50 μL

[0222] Among them, the specific sequences of the Post PCR Primer are as follows:

[0223] 5'-AATGATACGGCGACCACCGA(SEQ ID NO: 745)

[0224] 5'-CAAGCAGAAGACGGCATACGA(SEQ ID NO: 746)

[0225] After the preparation in 3.5.4, use a pipette to aspirate and mix well, and then quickly transfer it to the PCR instrument. Do not use the method of vortexing and centrifuging to mix.

[0226] Set the PCR instrument program as follows. Place the PCR reaction solution on the PCR instrument and run the program according to Table 17:

[0227] Table 17 PCR Reaction Program

[0228]

[0229]

[0230] After the program is completed, perform the magnetic bead purification in step 6.

[0231] The number of PCR cycles can refer to the parameters on the tube wall label of the probe Target Prober or the probe specification document. The number of Post-PCR cycles is related to the total input amount of the library during hybridization. When the input amount of the library during hybridization is large, the number of Post-PCR cycles can be appropriately reduced. Since the MGI platform requires a relatively large amount of library for on-machine sequencing, it is recommended to add two more cycles.

[0232] 3.6. Post-amplification Purification

[0233] 3.6.1 Take out the purification magnetic beads, mix well and place them at room temperature for 30 min for equilibration.

[0234] In the PCR product of step 5, add 1.1 times the volume of magnetic beads (55 μL), aspirate and mix well by pipetting or vortexing, and let it stand at room temperature for 5 min.

[0235] Steps 3.6.3 - 3.6.7 are the same as 2.7.4 - 2.7.8.

[0236] Remove the PCR tube from the magnetic rack, add 25 μL of Nuclease-Free Water, aspirate and mix well, and let it stand for 2 min. Centrifuge briefly, place the PCR tube on the magnetic rack for 2 min until the solution becomes clear.

[0237] 3.6.9 Use a pipette to aspirate 23 μL of the supernatant and transfer it to a new PCR tube; store the captured library in a -20 °C refrigerator. The captured library can be stored in a -20 °C refrigerator for one month.

[0238] 3.6.10 Take 1 μL of the library and measure the library concentration using the Qubit dsDNA HS Assay Kit reagent on a Qubit 4.0 Fluorometer, and record the library concentration.

[0239] 3.6.11 Take 1 μL of the library and perform fragment quality inspection using a Fragment Analyzer. The fragment size should be basically the same as the pre-library size.

[0240] 3.7 Sequencing on the machine.

[0241] Use the high-throughput sequencing platform NovaSeq 6000 to sequence the captured library. The original image data file obtained by sequencing is converted into the original sequencing sequence (Sequenced reads) through base calling analysis, which is called Raw data or Raw reads. The results are stored in the FASTQ (abbreviated as fq) file format, which contains sequence information and its corresponding sequencing quality information, etc.

[0242] 2. Remove host cell genes to obtain clean reads:

[0243] In the original data obtained by sequencing, there will be a small number of reads containing adapter information, low-quality bases, etc. To ensure the quality of information analysis, it is necessary to perform preliminary filtering on the Raw reads to obtain Clean reads, and subsequent analysis is based on Clean reads. The main content of data filtering is as follows:

[0244] (1) In a sliding window of 8 bp, shear off the sequences with an average base quality value less than 20.

[0245] (2) Remove the adapter sequences at the end of the sequence.

[0246] (3) If the first or last base of the sequence is less than 20, the base will be directly sheared off.

[0247] (4) Usually, if the remaining sequence length is less than 40 (paired-end) after removal, the pair of sequences will be discarded.

[0248] 3. Virus typing

[0249] ① Remove host sequences

[0250] Since there will still be residual host genomic sequences in the sequencing data, Bowtie2 is first used to align the sequencing data to the host reference genome to obtain the sequences that cannot be aligned (unmapR1&unmapR2). This part of the sequences that cannot be aligned are the sequences that may actually belong to the virus.

[0251] ② Pathogen genome alignment

[0252] The sequences that cannot be aligned are aligned to the virus genome using the bwa software (designing the reference species sequence information as the alignment template). The MEGAHIT software assembles the reads aligned to the genome into contig sequences. Then, the blastn is used to align the contig sequences with the host genome to remove the host sequences again, obtaining non-human contig sequences. These non-human contig sequences are then aligned to the virus genome sequence to obtain the possible virus typing ranking results-virus.txt.

[0253] 4. Consensus sequence

[0254] The typing sequence ranked first in the virus typing ranking is used as the reference genome. The cleanreads are aligned to this reference genome using the bwa software to obtain the aligned bam file. The samtools software is used for mutation analysis to obtain the mutation sites. The iVar software is used to correct the mutation sites in the genome to obtain the final consensus sequence.

[0255] 5. Integration analysis

[0256] The typing sequence ranked first in the virus typing ranking is used as the reference genome. The lumpy software is used to analyze the cleanreads, extract the reads that can be aligned to both the host and the virus genome. Based on these sequences, the coordinates of the integration sites on the host genome are found, and the final consensus sequence is integrated, and the depth of the reads is statistically analyzed to obtain the full-length genome sequence of the measles virus.

[0257] The results are shown in Table 18.

[0258] Table 18 Full-length genome sequencing results of measles virus in the test samples

[0259]

[0260]

[0261] Comparative Example 1

[0262] Comparison of the full-length genome sequencing results of measles based on the first-generation sequencing method

[0263] The target nucleotide sequence of measles virus for genotyping was amplified by RT-PCR method, and the amplified product was sequenced by first-generation sequencing technology. The full genome of the sample was obtained after splicing the sequencing results.

[0264] 1. Sample description: Sample 1 is measles virus of genotype H1, Sample 2 is measles virus of genotype D8, and Sample 3 is measles virus of genotype B3.

[0265] 2. Kit: TaKaRa One Step RNAPCRKit(AMV)(CatNo:DRR057A).

[0266] 3. The primer pair sequences are shown in Table 19.

[0267] Table 19 Primer pair sequences

[0268]

[0269] 4. Prepare the PCR amplification reaction system according to Table 20:

[0270] Table 20 Reaction system

[0271] Reagent Volume 2xBuffer 25 <![CDATA[RNaseFreeH2O]]> 14 UpstreamPrimer (20 μmol / l) 2 DownstreamPrime (20 μmol / l) 2 Taq (5 U / μl) 2 RNA template 5 Total 50

[0272] 5. Perform PCR amplification according to the reaction program in Table 21:

[0273] Table 21 Reaction program for PCR amplification

[0274]

[0275] 5. Sequence determination: The obtained PCR product was sent to a biotechnology company for sequence determination.

[0276] 6. Sequence splicing and arrangement: The Sequencher 5.0 software was used to arrange and splice the sequences. To ensure accuracy, each fragment was sequenced bidirectionally in both forward and reverse directions.

[0277] 7. The obtained sample fragments were sequenced, arranged, and spliced to obtain the first-generation full genome sequence.

[0278] Sequence alignment was performed with the full genome sequences obtained by probe hybridization capture and second-generation sequencing technology. The full lengths of the genomes obtained by first-generation sequencing of the three samples were all shorter than the full-length sequences obtained by this method. There were no differences in the genomic sequence loci obtained by the two sequencing methods for Sample 1 and Sample 3. No sequence map was obtained for the first-generation sequencing result of Sample 2 between loci 4772 - 5344 (see Figure 2)。The measles virus 4446-5457 is the MF non-coding region, which is one of the regions with the largest variations in the genome. The G+C content is as high as 70%, and it is difficult to obtain good sequence results by first-generation sequencing. The specific results are shown in Table 22. This indicates that the present invention can achieve the purpose of accurately and efficiently detecting the genome of the measles virus based on probe hybridization capture and second-generation sequencing technology.

[0279] Table 22 Comparison table of first-generation sequencing and second-generation sequencing results of the same sample

[0280]

[0281] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A capture probe set for the full-length genome of measles virus, characterized in that, The capture probe set includes probes with nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO:

742.

2. The capture probe set according to claim 1, wherein One end of the probe is modified with biotin.

3. A kit for full-length genome sequencing of measles virus, characterized in that, It includes the capture probe set described in claim 1 or 2, streptavidin-labeled magnetic beads, and library hybridization reaction reagents.

4. The kit for measles virus genome sequencing according to claim 3, wherein The library hybridization reaction reagent includes at least one of the following reagents: TargetSeq Hyb Bufferv2, HybHuman Block, Blocking Oligo, and RNase enzyme inhibitor.

5. The kit for measles virus genome sequencing according to claim 3 or 4, characterized in that, The kit further includes at least one of the following: RNA fragmentation reagent, reverse transcription reaction reagent, cDNA second-strand synthesis reaction reagent, adapter ligation reagent, purification reagent, PCR pre-reaction reagent, and post-capture PCR amplification reaction reagent.

6. The kit for full-length genome sequencing of measles virus according to claim 5, characterized in that, The PCR pre-reaction reagent includes PCR Master Mix containing UDG enzyme and / or UDI primer.

7. The kit for full-length genome sequencing of measles virus according to claim 5, characterized in that, The post-capture PCR amplification reaction reagent includes PCR primers and / or post-capture PCR reaction premix.

8. Use of the capture probe set described in claim 1 or 2 or the kit described in any one of claims 3 to 7 in the full-length genome sequencing of measles virus.

9. The application according to claim 8, wherein The sequencing includes next-generation sequencing.

10. A method for performing next-generation sequencing of the full-length genome of measles virus based on the capture probe set described in claim 1 or 2, characterized in that, It includes the following steps: Hybridize the measles virus genomic library of the sample to be tested with the probe set to obtain a hybridization product; Isolate the DNA fragment hybridized with the probe from the hybridization product; Perform post-capture PCR amplification using the DNA fragment hybridized with the probe as a template, perform next-generation sequencing on the obtained PCR product, and perform data analysis on the sequencing results to obtain the full-length genome of measles virus.

Images