Probe, primer, kit and method for human adenovirus whole genome sequencing and application

Abstract

The invention provides a probe, a primer, a kit, a method and application for human adenovirus whole genome sequencing. Aiming at a conserved sequence in an adenovirus genome, a plurality of pairs of primer probes are explored. According to the primer pair and the probe, the coverage degree of different adenovirus type genomes is greatly improved, and meanwhile, the amplification capacity of the primer aiming at targets with different lengths is enhanced. Eight adenovirus whole-genome sequences can be uniformly amplified, so that not only is the coverage degree of the whole-genome sequences improved, but also the capturing capacity for targets with different lengths is enhanced. In addition, the method has high tolerance for samples with different qualities, samples with low concentration or samples degraded to a certain degree can be subjected to high-uniformity total-sequence capture, and the method has high sensitivity and uniformity.

Description

Technical Field

[0001] The present application relates to the field of adenovirus technology, and in particular to probes, primers, kits, methods and applications for whole genome sequencing of human adenovirus. Background Art

[0002] Adenovirus is a non-enveloped particle with a diameter of 70 to 90 nm, composed of 252 capsomers arranged in an icosahedron. The diameter of each virus particle is 7 to 9 nm. The viral capsid contains a linear double-stranded DNA molecule containing about 30-47 kb, with inverted repeat sequences of about 100 bp at each end.

[0003] At present, there are 52 known types of human adenoviruses, and the means of monitoring these viruses include nucleic acid testing, virus isolation and culture, sequencing analysis, etc. Nucleic acid testing and virus isolation and culture can only perform basic typing of the virus and cannot obtain more sequence variation information. Therefore, whole genome sequencing analysis of human adenovirus is currently the most effective means of tracking virus variation analysis, new mutation types, and changes in epidemic situation. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a probe, primer, kit, method and application for whole genome sequencing of human adenovirus.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows: One of the purposes of the present invention is to provide a sequencing composition, comprising a universal upstream primer, a universal downstream primer and a universal probe.

[0006] The sequence of the universal upstream primer is 5'-T[barcode1]CAAA catcatcaat-3', wherein barcode1 is a sequence of 3-5 nucleotides, and the base is selected from at least one of A, T, C and G; The sequence of the universal downstream primer includes: 5'-T[barcode2]T[barcode3]T[barcode4]-3'; wherein barcode2 is a sequence of 2 to 7 nucleotides, and the base is selected from at least one of A, T, C and G; barcode3 is a sequence of 1 to 7 nucleotides, and the base is selected from at least one of A, T, C and G. Barcode4 is a sequence of 1 to 5 nucleotides, and the base is selected from at least one of A, T, C and G; The sequence of the probe includes: 5'-ATGAGRSAYYTRMGHBWYMTG; wherein R, S, Y, M, H, B, and W are all degenerate bases.

[0007] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:1, an upstream primer as shown in SEQ ID NO:2, and a probe as shown in SEQ ID NO:3.

[0008] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:4, an upstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6.

[0009] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:7, an upstream primer as shown in SEQ ID NO:8 and a probe as shown in SEQ ID NO:9.

[0010] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:10, an upstream primer as shown in SEQ ID NO:11 and a probe as shown in SEQ ID NO:12.

[0011] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:13, an upstream primer as shown in SEQ ID NO:14, and a probe as shown in SEQ ID NO:15.

[0012] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:16, an upstream primer as shown in SEQ ID NO:17, and a probe as shown in SEQ ID NO:18.

[0013] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:19, an upstream primer as shown in SEQ ID NO:20, and a probe as shown in SEQ ID NO:21.

[0014] Specifically, the sequencing composition includes an upstream primer as shown in SEQ ID NO:22, an upstream primer as shown in SEQ ID NO:23, and a probe as shown in SEQ ID NO:24.

[0015] One of the purposes of the present invention is to provide a kit, including a PCR amplification kit, wherein the PCR amplification kit includes the sequencing composition.

[0016] Specifically, the kit also includes a nucleic acid extraction kit and a double-stranded fluorescence quantification kit.

[0017] Specifically, the nucleic acid extraction kit is a DNeasy 96 Plant Kit (QIAGEN) kit, and the double-stranded fluorescent quantification kit is a Qubit® dsDNA HS Assay Kit.

[0018] Specifically, the kit also includes Nextera XT Library Preparation Kit.

[0019] One of the purposes of the present invention is to provide a method for whole genome sequencing of human adenovirus, characterized in that it comprises the following steps: (1) Extract DNA from the sample to be tested; (2) Providing a sequencing composition as described in claim 1 or 2; (3) using the sequencing tissue to perform PCR amplification on the DNA of the sample to be tested to obtain an amplification product; (4) constructing a sequencing library using the amplified product; (5) Perform sequencing analysis using the constructed sequencing library.

[0020] Specifically, the PCR amplification reaction system includes 20 μL of 10 μL 2×NEB Taq Master Mix, 0.6 μL 5 μM upstream primer, 0.6 μL 5 μM downstream primer, 0.6 μL 5 μM probe, 20 ng, 1 μL sample DNA and the remainder of water.

[0021] Specifically, the steps of constructing the library include: (1) Add the above amplified products to a total volume of 100 μL, and then purify with 180 μL of purification magnetic beads; (2) adding the purified products from the previous round to ATM and TD components for fragmentation; (3) The fragmented sequences were further amplified using the Nextera XT Index Kit (FC-131-1001, Illumina) to complete library construction; (4) Library quality control: Use the dsDNA HS Assay Kit (Qubit® dsDNA HS Assay Kit, Invitrogen / Q33230) to quantify the library. The yield of a qualified library should be above 50 ng.

[0022] One of the purposes of the present invention is to provide the use of the sequencing composition or the kit in adenovirus whole genome sequencing.

[0023] The sequencing composition provided by the present invention includes a universal upstream primer, a universal downstream primer and a probe of a specific sequence. Among them, the universal upstream primer and the universal downstream primer respectively include four Barcode sequences. It can be changed according to the changes in the adenovirus genome to be tested, and according to the difference in bases, it can amplify different sequence fragments on different genomes. The primer scheme can be optimized according to the sequence of the adenovirus genome, and the size of the genome and the sample amount can be arbitrarily adjusted. At the same time, by adding a probe sequence, the specific region of the adenovirus can be effectively amplified, which is of great significance for identifying and distinguishing different types of adenoviruses. It not only has a good enrichment effect, but also makes library construction and sequencing more concise and accurate, and reduces sequencing costs.

[0024] The sequencing composition and kit provided by the present invention can uniformly amplify the full gene sequences of eight adenoviruses, which not only improves the coverage of the full sequence, but also enhances the capture ability for targets of different lengths. It has a strong tolerance for samples of different qualities, and can achieve high uniformity full sequence capture for samples with lower concentrations or samples that have been degraded to a certain extent. The method has strong sensitivity and uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The Hadv-4 whole genome sequencing coverage map for the experimental group (upper panel) and the control group (lower panel).

[0026] Figure 2 The Hadv-7 whole genome sequencing coverage map for the experimental group (upper figure) and the control group (lower figure).

[0027] Figure 3 The Hadv-11 whole genome sequencing coverage map for the experimental group (upper panel) and the control group (lower panel).

[0028] Figure 4 The Hadv-14 whole genome sequencing coverage map for the experimental group (upper figure) and the control group (lower figure).

[0029] Figure 5 The Hadv-17 whole genome sequencing coverage map for the experimental group (upper figure) and the control group (lower figure).

[0030] Figure 6 The Hadv-34 whole genome sequencing coverage map for the experimental group (upper panel) and the control group (lower panel).

[0031] Figure 7 The Hadv-35 whole genome sequencing coverage map for the experimental group (upper panel) and the control group (lower panel).

[0032] Figure 8 The Hadv-37 whole genome sequencing coverage map for the experimental group (upper panel) and the control group (lower panel). DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0034] An example of the present invention provides a sequencing primer set, which includes a universal upstream primer, a universal downstream primer and a probe.

[0035] The sequence of the universal upstream primer is 5'-T[barcode1]CAAA catcatcaat-3', wherein barcode1 is a sequence of 3-5 nucleotides, and the base is selected from at least one of A, T, C and G.

[0036] The sequence of the universal downstream primer includes: 5'-T[barcode2]T[barcode3]T[barcode4]-3'. Wherein, barcode2 is a sequence of 2 to 7 nucleotides, and the base is selected from at least one of A, T, C and G. Barcode3 is a sequence of 1 to 7 nucleotides, and the base is selected from at least one of A, T, C and G. Barcode4 is a sequence of 1 to 5 nucleotides, and the base is selected from at least one of A, T, C and G.

[0037] The sequence of the probe includes: 5'-ATGAGRSAYYTRMGHBWYMTG, wherein R, S, Y, M, H, B, and W are all degenerate bases.

[0038] The sequencing composition provided by the present invention includes a universal upstream primer, a universal downstream primer and a probe of a specific sequence. Among them, the universal upstream primer and the universal downstream primer respectively include four Barcode sequences. It can be changed according to the changes in the adenovirus genome to be tested, and according to the difference in bases, it can amplify different sequence fragments on different genomes. The primer scheme can be optimized according to the sequence of the adenovirus genome, and the size of the genome and the sample amount can be arbitrarily adjusted. At the same time, by adding a probe sequence, the specific region of the adenovirus can be effectively amplified, which is of great significance for identifying and distinguishing different types of adenoviruses. It not only has a good enrichment effect, but also makes library construction and sequencing more concise and accurate, and reduces sequencing costs.

[0039] Accordingly, an embodiment of the present invention further provides a method for whole genome sequencing of human adenovirus, which comprises the following steps: (1) Extract DNA from the sample to be tested; (2) providing the sequencing composition; (3) using the sequencing tissue to perform PCR amplification on the DNA of the sample to be tested to obtain an amplification product; (4) constructing a sequencing library using the amplified product; (5) Perform sequencing analysis using the constructed sequencing library.

[0040] Preferably, the PCR amplification products are subjected to second-generation library construction sequencing and the sequencing results are analyzed. Preferably, the second-generation library construction sequencing analysis usually requires Illumina sequencing. In a preferred embodiment of the present invention, the obtained sequencing results are analyzed, and the [barcodes] of the designed different primers can be used to distinguish the samples for analysis, not only for detection but also for mining population variation sites.

[0041] The following is a full gene amplification, library construction and sequencing analysis of each person's adenovirus throat swab sample, as follows: The following is a full gene amplification, library construction and sequencing analysis of each person's adenovirus throat swab sample, as follows: 1. Sample Throat swab samples from patients clinically confirmed to be infected with human adenovirus Hadv-4, Hadv-7, Hadv-11, Hadv-14, Hadv-17, Hadv-34, Hadv-35, and Hadv-37 were stored at -20°C.

[0042] 2. Sample DNA Extraction The genomic DNA of human adenovirus from each throat swab sample was extracted using the DNeasy 96 Plant Kit (QIAGEN).

[0043] The quality of the extracted genomic DNA was detected and quantified: agarose gel detection was marked with λ marker, 1 μL DNA was taken, 2 μL 10× bromophenol blue loading buffer was added, mixed, and spotted into 0.8% agarose gel containing 0.5 μg / ml Goldview dye, and electrophoresed at 90V for 40 min using 1×TAE buffer; DNA bands were observed using a gel imaging analysis system (Tanon4100).

[0044] Take 1-2 μL DNA sample and detect the genomic DNA with NANODROP 2000C. Calculate the DNA concentration based on the light absorption value at 260nm, and judge the presence of polysaccharides, proteins, RNA and other impurities based on the OD260 / OD280 and OD260 / OD230 ratios to determine the purity of the DNA.

[0045] 3. PCR amplification PCR amplification was performed using the primer pairs and probes shown in Table 1.

[0046] Table 1

[0047] The PCR amplification system is shown in Table 2. The amplification program is: 95°C, 10 min; (95°C 30 s, 58°C 2 min; 60°C 3 min; 62°C 1 min; 72°C 1 min) × 5 cycles, (95°C 30 s; 66°C 3 min) × 30 cycles.

[0048] Table 2 (20 μL)

[0049] 4. Library construction The library was constructed using Illumina's Nextera XT Library Preparation Kit (Cat. No.: FC-131-1024). The specific process is as follows: (1) Add the above amplified products to a total volume of 100 μL, and then purify with 180 μL of purification magnetic beads; (2) adding the purified products from the previous round to ATM and TD components for fragmentation; (3) The fragmented sequences were further amplified using the Nextera XT Index Kit (FC-131-1001, Illumina) to complete library construction; (4) Library quality control: Use the double-stranded fluorescent quantitative kit (Qubit® dsDNA HS Assay Kit, Invitrogen / Q33230) to quantify the library. The output of a qualified library should be above 50 ng.

[0050] 5. Bioinformatics analysis and assembly Bioinformatics methods were used to align and assemble with the reference genomes of various types of human adenoviruses, and then the coverage depth of each genomic site was calculated (i.e., how many sequencing fragments covered each site). The number of genomic sites with a sequencing depth of more than 30× was counted as X bp, and the coverage was calculated by 100%×X / genome. The coverage of each adenovirus subtype was ≥99.9%, and the full sequence coverage greater than 30% of the average depth could reach more than 90%, and the amplification efficiency was uniform. This shows that the technical method has good detection sensitivity and accuracy. The results are shown in Table 3 below: Table 3

[0051] As a control, the system shown in Table 2 was used without adding probes, and the same primers as in Table 1 were used for library construction, and the library was sequenced and assembled for bioinformatics analysis. The whole genome sequencing results of the obtained samples are shown in Table 4. As can be seen from Table 4, the library amplification system using only primer pairs but no probes cannot obtain the whole genome sequence of the sample with high uniformity, and is prone to uneven amplification, resulting in the inability to obtain a complete whole genome sequence after the final sequencing. Part of the sequence is lost or the depth is extremely low, resulting in unreliable accuracy of the results.

[0052] Table 4

[0053] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A sequencing composition, characterized in that It includes a universal upstream primer, a universal downstream primer and a universal probe; The sequence of the universal upstream primer is 5'-T[barcode1]CAAA catcatcaat-3', wherein barcode1 is a sequence of 3-5 nucleotides, and the base is selected from at least one of A, T, C and G; The sequence of the universal downstream primer includes: 5'-T[barcode2]T[barcode3]T[barcode4]-3'; wherein barcode2 is a sequence of 2 to 7 nucleotides, and the base is selected from at least one of A, T, C and G; barcode3 is a sequence of 1 to 7 nucleotides, and the base is selected from at least one of A, T, C and G; barcode4 is a sequence of 1 to 5 nucleotides, and the base is selected from at least one of A, T, C and G; The sequence of the probe includes: 5'-ATGAGRSAYYTRMGHBWYMTG; wherein R, S, Y, M, H, B, and W are all degenerate bases.

2. The sequencing composition according to claim 1, wherein comprising an upstream primer as shown in SEQ ID NO: 1, an upstream primer as shown in SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3; or an upstream primer as shown in SEQ ID NO:4, an upstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6; or an upstream primer as shown in SEQ ID NO:7, an upstream primer as shown in SEQ ID NO:8, and a probe as shown in SEQ ID NO:9; or an upstream primer as shown in SEQ ID NO: 10, an upstream primer as shown in SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12; or an upstream primer as shown in SEQ ID NO: 13, an upstream primer as shown in SEQ ID NO: 14, and a probe as shown in SEQ ID NO: 15; or an upstream primer as shown in SEQ ID NO: 16, an upstream primer as shown in SEQ ID NO: 17, and a probe as shown in SEQ ID NO: 18; or an upstream primer as shown in SEQ ID NO: 19, an upstream primer as shown in SEQ ID NO: 20, and a probe as shown in SEQ ID NO: 21; Or an upstream primer as shown in SEQ ID NO:22, an upstream primer as shown in SEQ ID NO:23, and a probe as shown in SEQ ID NO:

24.

3. A kit, characterized in that: It comprises a PCR amplification kit, wherein the PCR amplification kit comprises the sequencing composition as described in claim 1.

4. The kit according to claim 2, characterized in that It also includes a nucleic acid extraction kit and a double-stranded fluorescence quantification kit.

5. The kit according to claim 3, characterized in that The nucleic acid extraction kit is a DNeasy 96 Plant Kit, and the double-stranded fluorescent quantitative kit is a Qubit® dsDNA HS Assay Kit.

6. The kit according to claim 3, characterized in that The kit also includes a Nextera XT Library Preparation Kit.

7. A method for whole genome sequencing of human adenovirus, characterized in that: The steps include: (1) Extract DNA from the sample to be tested; (2) Providing a sequencing composition as described in claim 1 or 2; (3) using the sequencing tissue to perform PCR amplification on the DNA of the sample to be tested to obtain an amplification product; (4) constructing a sequencing library using the amplified product; (5) Perform sequencing analysis using the constructed sequencing library.

8. The method according to claim 7, characterized in that The PCR amplification reaction system contained 20 μL of: 10 μL 2×NEB Taq Master Mix, 0.6 μL 5 μM upstream primer, 0.6 μL 5 μM downstream primer, 0.6 μL 5 μM probe, 20 ng, 1 μL sample DNA and the balance of water.

9. The method according to claim 7, characterized in that The steps of constructing the library include: (1) Add the above amplified products to a total volume of 100 μL, and then purify them with 180 μL of purification magnetic beads; (2) adding the purified products from the previous round to ATM and TD components for fragmentation; (3) The fragmented sequences were further amplified using the Nextera XT Index Kit to complete library construction; (4) Perform library quality inspection: Use a double-stranded fluorescence quantitative kit to quantify the library. The output of a qualified library should be above 50 ng.

10. Use of the sequencing composition according to claim 1 or 2, or the kit according to any one of claims 3 to 6 in adenovirus whole genome sequencing.

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