CN114958991A - Ultrahigh-flux multiple PCR amplicon capturing method - Google Patents

Ultrahigh-flux multiple PCR amplicon capturing method Download PDF

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CN114958991A
CN114958991A CN202210687838.7A CN202210687838A CN114958991A CN 114958991 A CN114958991 A CN 114958991A CN 202210687838 A CN202210687838 A CN 202210687838A CN 114958991 A CN114958991 A CN 114958991A
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孔育权
李耕慧
胡征
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Shanghai Diying Biotechnology Co ltd
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Abstract

The invention relates to the field of second-generation sequencing, in particular to an ultrahigh-flux multiplex PCR amplicon capturing method for second-generation sequencing, which comprises the following steps: 1) carrying out multiple PCR amplification on a sample to be detected by using a digestible composite modified primer to obtain a PCR product; 2) performing enzyme digestion reaction on the PCR product by using a second enzyme mixed solution containing primer digestive enzyme, error correction enzyme and end repair enzyme, wherein the enzyme digestion reaction comprises the steps of removing redundant primer parts on the amplification product by using digestive enzyme and removing a non-specific amplification product by using mismatch error correction enzyme; 3) the full-length linker containing Index is used for connection reaction to directly obtain a library for sequencing or the library for sequencing is obtained after amplification. The method provided by the invention has the advantages that the amplification efficiency and specificity are obviously improved, the steps are simple, the cost is low, the time is short, and the method has wide application value in the fields of genetic disease detection, tumor accompanying diagnosis and other gene detection and scientific research.

Description

Ultrahigh-flux multiplex PCR amplicon capturing method
Technical Field
The invention relates to the field of second-generation sequencing, in particular to a capture method based on an ultrahigh-flux multiple PCR amplicon, which is particularly suitable for second-generation sequencing application with more PCR amplicons.
Background
High throughput Sequencing, referred to in some literature as Next Generation Sequencing (NGS), is an epoch-making revolutionary change to traditional Sequencing (Sanger Sequencing), which can sequence hundreds of thousands to millions of DNA molecules.
The targeted capture technologies in NGS include probe-based solution phase hybrid capture and multiplex PCR-based amplicon capture technologies. Multiplex PCR (multiplex PCR) capture, also called multiplex primer PCR capture, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments. The reaction principle, reaction reagents and operation process are the same as those of general PCR, but the multiplex PCR has great advantages in many applications: 1. higher throughput (more fragments can be analyzed per reaction well); 2. lower sample dosage and reagent dosage (especially suitable for rare samples, and reduces cost).
With the development of high-throughput sequencing technology in recent years, the requirement of scientific research and clinical application on the number of primer pairs for a single reaction is increasing, and one problem faced by the traditional multiplex PCR technology is that the hybridization kinetic properties are different between different primer pairs. Primers with higher binding efficiency can make full use of PCR reagents, thereby leading to reduction of other PCR products. This will usually result in some of the products that should have been amplified not being amplified. This problem is a major limiting factor in the increase of the number of single-tube amplicons in multiplex PCR, and the mainstream solution in the industry is to divide multiple amplicons into multiple tubes for amplification reaction, which will result in the increase of the amount of sample and reagents. In addition, the traditional multiplex PCR amplicon has poor amplification uniformity and needs to be adjusted repeatedly, and serious interference can be caused by excessive primer pairs in a single tube. Therefore, it is necessary to develop a method capable of increasing the number of amplicons in a single tube of a multiplex PCR while ensuring or improving the uniformity of amplification.
Patent document CN110592200A discloses a multiplex PCR method that improves amplification specificity and uniformity. The method comprises the following steps: extracting genomic DNA from a sample; fragmenting the extracted genomic DNA and purifying; and carrying out PCR amplification reaction by using the purified genome DNA as a template and using a plurality of specific primer pairs, wherein in the PCR amplification reaction, annealing is carried out by using different temperatures from low to high. The invention fragments and purifies DNA, can combine template and primer quickly, and improves the usage rate of primer. Firstly, under a lower annealing temperature mode, fully combining the amplification primers to the template DNA, and then gradually increasing the annealing temperature to improve the combination specificity of each primer; this results in a significant improvement in amplification specificity and uniformity of multiplex PCR.
At present, the capture method of the ultrahigh-flux multiplex PCR amplicon is not seen.
Disclosure of Invention
The invention aims to provide an ultrahigh-flux multiplex PCR amplicon capturing method which has the advantages of large number of multiplex PCR single-tube amplicons, good amplification uniformity and capability of obviously improving the amplification efficiency.
The technical scheme adopted by the invention is as follows:
an ultra-high throughput multiplex PCR amplicon capture method, comprising the steps of:
1) carrying out multiple PCR amplification on a sample to be detected by using an amplification primer pool containing digestible complex modification and a first enzyme mixed solution to obtain an amplicon product; at least one amplification primer in the amplification primer pool comprises compound modified base nucleotides, and the modification mode is one or more of the following conditions: a T base is replaced with a U (uracil) modified base, a T base is replaced with a + T (locked nucleotide thymine) modified base, a T base is replaced with a 5mC (5 methylcytosine) modified base, a T base is replaced with a + a (locked nucleotide adenine) modified base, a G base is replaced with an 8-oxo G (8-hydroxydeoxyguanosine) modified base, and a T base is replaced with an I (hypoxanthine) modified base; the first enzyme mixed solution is QuartTaq HotStart mixed solution;
2) performing enzyme digestion reaction on the amplicon product by using a second enzyme mixed solution containing primer digestive enzyme, mismatch error correcting enzyme and end repairing enzyme, wherein the enzyme digestion reaction comprises removing redundant primer parts on the amplification product by using the primer digestive enzyme and removing non-specific amplification products by using the mismatch error correcting enzyme; the second enzyme mixed solution comprises primer digestive enzyme, mismatch error correcting enzyme, T4 polynucleotide kinase, Taq polymerase and QuarPrep end repair tailing buffer solution; the primer digestive enzyme is selected from one or two of UDG enzyme and FPG enzyme; the mismatch error correcting enzyme is selected from one or more of T7 endonuclease I, T4 endonuclease VII, E coli endonuclease V, Surveror endonuclease and CEL I endonuclease;
3) performing a connection reaction by using the full-length joint containing the Index, the third enzyme solution and the second buffer solution to directly obtain a library for sequencing or obtain a library for sequencing after amplification; the third enzyme solution is DNA ligase.
Preferably, for any one amplification primer containing a complex modified base nucleotide, the number of modified bases is any integer between 0 and n, and n represents the total length of the amplification primer.
Preferably, the library for sequencing is obtained directly after ligation using a full-length linker comprising Index and a third enzyme solution.
Preferably, the mismatch error correcting enzyme is used to reduce non-specificity resulting from PCR amplification.
Preferably, the full-length linker comprising Index replaces the universal short linker for ligation to construct a library for sequencing.
Preferably, the sample to be detected is selected from the group consisting of a prokaryotic or eukaryotic microorganism genome, an animal or plant genome or a human genome.
Preferably, the pool of amplification primers comprises primers as shown in SEQ ID NO 1-262. Preferably, the SEQ ID NO 1 and the SEQ ID NO 2 are primer pairs, the SEQ ID NO 3 and the SEQ ID NO 4 are primer pairs, and the like.
The invention also provides the application of the ultrahigh-flux multiplex PCR amplicon capturing method in any one of the following aspects: pathogen detection, genetic disease detection, tumor early screening detection, tumor-associated diagnosis detection, susceptibility gene detection and the like.
The invention has the beneficial effects that:
(1) the method of the invention carries out special modification design treatment on the part of amplification primers, each amplification primer comprises compound modified base nucleotides, and the nucleotides of four bases of A, T, C and G are modified and replaced as follows, including: a T base is replaced by a U (uracil) modified base, + T (locked nucleotide thymine) modified base, 5mC (5 methylcytosine) modified base or + A (locked nucleotide adenine) modified base, and a G base is replaced by an 8-oxo G (8-hydroxydeoxyguanosine) modified base or an I (hypoxanthine) modified base, and the like. The number of substitutions is between 0 and n, where n represents the total length of the primer. The primers containing modified bases improve the specificity of primer binding, and can greatly improve the number of amplified primer pairs in each reaction. Is more suitable for the construction of an ultra-high-flux NGS amplicon library.
(2) The partially digested product is used for constructing an NGS library through ligase, and the overall uniformity is improved compared with a two-step amplification method.
(3) The full-length adaptor containing Index is used for replacing the conventional universal truncated adaptor to carry out the connection reaction, so that the waste caused by the filtration of sequencing data due to short amplification products is avoided.
(4) The amplification primer is digested by primer digestive enzyme, so that sequencing waste of invalid data generated on the amplification primer part in the sequencing process is avoided.
(5) And the mispairing error correcting enzyme is introduced in the step of partially digesting the primer, so that the mispairing of the PCR amplification product is repaired, and the accuracy and the specificity of the multiple amplification are improved.
(6) Compared with a method for capturing target fragments by liquid phase probe hybridization, the method provided by the invention improves the amplification efficiency of primers, reduces the steps of hybridization capture, saves reagent cost and greatly shortens experiment time.
(7) The invention has wide application value in the fields of pathogen detection, genetic disease detection, tumor companion diagnosis, tumor early screening and other gene detection and scientific research.
Drawings
FIG. 1: the sequencing quality of the data was verified.
FIG. 2: analyzing the map of the fragment captured by the ultra-multiplex amplicon method.
FIG. 3: sequencing alignment and capture efficiency map by ultra multiplex amplicon method.
FIG. 4: the depth of coverage (1X and 100X) was determined by the multiplex amplicon method.
FIG. 5: the sequencing homogeneity is determined by a super multiplex amplicon method.
FIG. 6: the uniformity of the ultra-multiplex amplicon method was compared to the conventional two-step PCR capture method.
FIG. 7 is a schematic view of: and comparing the effect of the error correcting enzyme in the ultra-multiplex amplicon method. Error correcting enzymes can reduce non-specific amplification products. The concentration of 5U and 15min incubation gave the best effect, T7E1 representing T7 endonuclease I.
FIG. 8: lift-up plot of full-length linker with Index for the effective data scale.
FIG. 9: an example of the application of the ultra-multiplex amplicon method in the detection of neonatal genetic disease (α -thalassemia) was detected.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
In view of the problem mentioned in the background section that the interaction between multiple PCR primers results in reduced amplification efficiency, in an exemplary embodiment of the present invention, a method for constructing a high throughput sequencing library is provided, the method comprising: performing multiple PCR amplification on the genome DNA by using a first enzyme mixed solution, a first buffer solution and a specially designed amplification primer to obtain a PCR product; carrying out enzyme digestion reaction on the PCR product by using the second enzyme mixed solution, and carrying out primer digestion, mismatch correction and end A addition to obtain an amplicon product; performing joint connection on the amplicon product by using a third enzyme solution, a second buffer solution and a full-length joint containing index to obtain an NGS library; amplification of the NGS library using a fourth enzyme cocktail and P5/P7 universal primers (optional step).
In the above construction method of the present invention, the genomic DNA refers to a sample to be detected. For example, the sample to be detected can be various prokaryotic or eukaryotic microorganism genomes, animal or plant genomes, human or mouse genomes and the like, and the concentration is controlled to be about 10 ng/mu l.
The invention will now be further illustrated with reference to specific examples.
Example 1
In this example, human genomic DNA was used as the experimental material, and the specific experimental steps were as follows:
1. sample preparation: the concentration of genomic DNA was accurately determined using a Qubit. The genomic DNA was diluted to 10 ng/. mu.l for use in accordance with the concentration measurement.
2. The ultrahigh-flux PCR amplicon capture scheme verification experiment adopts 131 pairs of amplification primers, covers all exons of BRCA1 and BRCA2, and is divided into 2 pool for amplification reaction, and the sequences of the primers are shown as SEQ ID NO. 1-262. In addition, a high throughput protocol validation was performed using neonatal genetic disease screening Panel containing 4780 pairs (2 pool) of primers.
3. Specific PCR amplification: performing PCR amplification by using a first enzyme mixed solution (2 XQuartTaq HotStart mixed solution), a first buffer solution (QuartTaq PCR enhancer) and an amplification primer to obtain a PCR product. Specific reaction systems are shown in the following table.
Components Volume of
Genomic DNA 2μl
First enzyme mixture 10μl
Amplification primers 5μl
A first buffer solution 2μl
Nuclease-free water 1μl
Total of 20μl
After the system is prepared, the reaction tube is placed in a PCR instrument or other thermal incubation reaction instruments, and the reaction is carried out according to the following procedures:
Figure BDA0003700360910000041
4. primer digestion treatment and addition of A: the PCR product was water-supplemented to 50. mu.l, purified with 1.5X purified magnetic beads, eluted with 35. mu.l nuclease-free water, and 9.5. mu.l of a second enzyme mixture (T4 polynucleotide kinase, Taq polymerase, FPG enzyme, UDG enzyme, T7 endonuclease I and QuarPrep end repair tailing buffer) was added thereto and subjected to the following reaction:
temperature of Time
37℃ 10min
50℃ 10min
65℃ 5min
4℃ ∞
5. Connecting a joint: taking out the third enzyme solution (T4 DNA ligase) and the second buffer solution (Tris-HCl, MgCl) 2 DTT, ATP, PEG6000 and 1, 2-propanediol), and linker primers with index, according to the following table, a linker-linked reaction system was prepared:
components Volume (μ l)
The product of the last step 44.5
Full-length joint with Index 2.5
Third enzyme solution 10
Second buffer solution 16
Nuclease-free water 6.9
Blowing, beating, mixing and instantaneous centrifuging.
The following procedure was performed on a PCR instrument while the beads were removed and incubated at room temperature for 30min, and sufficient 80% ethanol was prepared with nuclease-free water.
Step (ii) of Temperature of Time
1 25℃ 15min
2 4℃ Hold
And purifying the reaction system after the connection is finished by using 0.8 multiplied by magnetic beads, and then respectively rinsing by 80% ethanol and eluting by nuclease-free water to obtain purified fragments with the connectors.
6. Second round PCR amplification (optional step): and performing PCR enrichment on the to-be-spliced fragment by using a fourth enzyme mixed solution (2 XQuartTaq HiFi HotStart mixed solution) and a P5/P7 universal primer to obtain a DNA library. In particular, see the following table:
components Volume (μ l)
The product of the last step 17
Fourth enzyme mixture 25
Universal primer 8
Total of 50
Wherein the working concentration of the fourth enzyme mixture is 1 ×.
The PCR amplification was then performed according to the PCR program of the following table:
Figure BDA0003700360910000051
and (3) post-PCR purification: after the reaction is finished, taking out the PCR tube, performing instantaneous centrifugation, collecting all liquid at the bottom of the tube, purifying with magnetic beads with the same volume, and eluting with 21 mu l of nuclease-free water after purification is finished, thus obtaining the PCR tube.
7. Performing quality inspection on the library: use of
Figure BDA0003700360910000052
The concentration of the library was measured by dsDNA HS Assay Kit and the size of the library fragment was measured using a 2100 bioanalyzer, all according to the instructions. The first step of specific PCR amplification adopts 22 cycles, the second round of PCR amplification adopts 7 cycles, and the library yield is about 150-160 ng.
8. Performing on-machine sequencing on the library qualified by quality inspection, entrusting the sequencing service to Beijing Nuo standing grain genesis science and technology GmbH for specific implementation, adopting an Illumina gene sequencer, and setting the sequencing mode to be PE150 (namely double-end sequencing and 150 bases for each sequence) and the sequencing data amount of each sample to be 1G base. In order to ensure the quality of the library before loading, the concentration detection of the library, the size detection of the insert fragment and the accurate detection of the molar concentration of the library are required.
Library detection mainly comprises 3 methods:
(1) the Qubit 2.0 carries out primary quantification on the concentration of the library;
(2) agilent 2100 detects the integrity of the library DNA fragments and the size of the insert;
(3) the Q-PCR method accurately quantifies the effective concentration of the library.
After the library is qualified, different libraries are poolling to flowcell and cBOT are clustered according to the requirements of effective concentration and target off-machine data volume, and then an Illumina high-throughput sequencing platform (HiSeq/MiSeq) is used for sequencing.
9. For the data returned by the sequencing company, we performed the analysis according to standard procedures, specifically, the following steps:
(1) and (3) data quality control: if the sequencing error rate is represented by e, and the base quality value of the data measured by the Illumina platform is represented by Qphrred, then the following are: qphred ═ 10log10 (e). The concise correspondence between the correct rate of base recognition and the Phred score in Illumina software is shown in the following table:
phred score Incorrect base recognition Base correct discrimination rate Q-score
10 1/10 90％ Q10
20 1/100 99％ Q20
30 1/1000 99.9％ Q30
40 1/10000 99.99％ Q40
The sequencing error rate is related to the quality of bases, and according to the characteristics of a sequencing technology, the error rate of a plurality of cycles at the front end and the error rate of the tail end of a sequencing fragment are high. Generally, Q20 ≥ 99%, Q30 ≥ 95% represent ideal sequencing quality, where Q20, Q30 represent the percentage of bases with Phred values > 20, 30, respectively, over the total. The sequencing quality distribution of the verification data is shown in FIG. 1, and it can be seen that the Q-score values of all bases are greater than 30, and the sequencing quality meets the analysis requirement. For quality-qualified data, linker content testing is also required to remove the exogenous non-human genomic sequences introduced by the experiment. Joint detection uses the professional open source letter software TrimGalore (https:// www.bioinformatics.babraham.ac.uk/projects/trim _ bulk /), which automatically identifies the type of joint by a built-in algorithm (Illumina universal adapter/Nextera transposase adapter/Illumina small RNA adapter, etc.). By detecting and removing the linker sequence, we obtained processed data clean data for further analysis.
(2) Data comparison and analysis: to further identify clean data and to evaluate the efficiency of amplification of the present ultramultiplex PCR primers, the data needs to be aligned with the human standard reference genome (alignment). Specifically, we used the mem algorithm of the open source tool bwa (http:// bio-bw. sourceform. net/bw. shtml), using the paired end alignment model, and the alignment object was the human standard reference genome GRCh37(hg 19). And the comparison result is in a sam (Sequence Alignment/Map format) format, an open source tool samtools (http:// samtools, source for. net /) is used for sequencing the result according to the position on the genome by comparison, an index is established, and the result is converted into a bam (Binary Alignment/Map format) format with a larger file compression ratio so as to save the storage space.
(3) And (4) quality control of comparison results: in general, the experimental protocol for capture of the ultramultiplex PCR amplicon has several indicators for evaluating the amplification efficiency, which are:
a. the contrast ratio (MappingRate) represents the proportion of human sources in the data after pretreatment, and the higher the ratio, the better the ratio;
b. the capture efficiency (TargetRate) represents the data proportion of the expected amplification region of the multiple primers in the human data, and the higher the capture efficiency, the better the capture efficiency;
c. coverage (Coverage) represents the proportion of actual amplification Coverage of the target amplification region, 100% indicates that each pair of primers completes amplification;
d. uniformity (Uniformity) indicates the degree of Uniformity of the amplification ability of each pair of primers, and higher Uniformity indicates better amplification Uniformity.
The sample results of the test are shown in the attached figures 3-5, and it can be seen that each evaluation index under the method is ideal and has very high sample-to-sample consistency.
Compared with the most common two-step PCR method on the market at present, the uniformity is obviously improved. See figure 6.
(4) Mutation detection analysis: and (3) carrying out mutation analysis on the result of the bam file generated by comparison by using an open source tool VarScan (http:// v. source form. net /), so as to obtain a clear mutation site result file, wherein the result is stored in a vcf (variable calling format) format and comprises information such as mutation position/mutation form/site depth/mutation frequency and the like. Based on known positive results, the results of variation for the actual assay are shown in FIG. 9: the positive site is alpha-thalassemia SEA, the variant form is Deletion of 20kb from HBA2 to HBA1, and the positive site has definite pathogenicity and familial inheritance and is consistent with the situation of a known clinical sample.
The embodiments described above are some, but not all embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
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<213> Artificial sequence (Artificial sequence)
<400> 26
tgattggcaa cacgaaaggt aaaaat 26
<210> 27
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
cgctgatgaa tgtgaaaaat ctaaaaacca 30
<210> 28
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
attttctcca tctgggctcc attt 24
<210> 29
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
ttcagtgatg gaggaaatgt tggtt 25
<210> 30
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
gtggtggctc agctacttga g 21
<210> 31
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
gctgatttct gttgtatgct tgtactg 27
<210> 32
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
gtaatcggct ctaaagaaac atgatgc 27
<210> 33
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
tccaggaaga ctttgtttat agacctca 28
<210> 34
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
ctgaaagaga aatgggaaat gagaacattc 30
<210> 35
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
tgagctctaa ttttgttgta tttgtcctgt 30
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
acttccacac ggttgtgaca tc 22
<210> 37
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
gcaataaaac tagtagtgca gataccca 28
<210> 38
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
caatgactga tttttaccaa gagtgcaa 28
<210> 39
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
aggtcccaaa tggtcttcag aataatc 27
<210> 40
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
gctttctgta atcgaaagag ctaaaatgtt 30
<210> 41
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
tgatgaatgg ttttatagga acgctatgt 29
<210> 42
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
cagttgtgag attatctttt catggctatt 30
<210> 43
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
gtttccagca gctgaaattt gtga 24
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
acttatctct ttgtggtgtt acatgtgtac 30
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
atctgagaaa ccccacagcc 20
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
tgtccctccc atcctctgat 20
<210> 47
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
tttcaggaag gaatgttccc aatagtag 28
<210> 48
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
ccaggctctt agccaaaata ttagca 26
<210> 49
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
ccccagagca tcacttggg 19
<210> 50
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
attctgagag gctgctgctt ag 22
<210> 51
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
agtgacttgt ttaaacagtg gaattctaga 30
<210> 52
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
gcataccacc catctgtaag ttcaataa 28
<210> 53
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 53
ccagtggtat gtgggagttt gtt 23
<210> 54
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 54
agatttgtaa atctcagggc aaaggt 26
<210> 55
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 55
gtttattgca ttcttctgtg aaaagaagc 29
<210> 56
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 56
tgctttttgg atcattttca cactgtc 27
<210> 57
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 57
agcctaatct tactagacat gtcttttctt 30
<210> 58
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 58
cgcgttgaag aagtacaaaa tgtcattaat 30
<210> 59
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 59
ttcccatgga aaagaatcaa gatgtatgt 29
<210> 60
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 60
tcctttcatt agctacttgg aagacaaaat 30
<210> 61
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 61
ccattctagg acttgcccct ttc 23
<210> 62
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 62
cagaaaacac agaaaaatct ccagcaaa 28
<210> 63
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 63
gaccagctca caagagaaga aaatact 27
<210> 64
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 64
ggtttctctt atcaacacga ggaagtattt 30
<210> 65
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 65
gcctgtgaat ggtctcaact aacc 24
<210> 66
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 66
gcaagaatgc agtctgtatg agattcaa 28
<210> 67
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 67
tctcagccca gatgacttca aag 23
<210> 68
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 68
gagtcatctg aggagaattc agttcttttt 30
<210> 69
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 69
gggaatcagg ctttactaga agaaca 26
<210> 70
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 70
caatgtggtc tttgcagcta tttactt 27
<210> 71
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 71
cccttcactt cagcaaattt ttagatcc 28
<210> 72
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 72
ctggtagctc caactaatca taagagattt 30
<210> 73
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 73
gtgaaagtat ctagcactgt gtatgtatgt 30
<210> 74
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 74
acttccattg aaggaagctt ctctt 25
<210> 75
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 75
ggtacagcag actgtggaat gt 22
<210> 76
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 76
aaagactctg catttttgct gttaattttt 30
<210> 77
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 77
cttcctttat ttcaccatca tctaacaggt 30
<210> 78
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 78
ccaaaattga atgctatgct tagattaggg 30
<210> 79
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 79
tttagtagag acagggtttc tccatt 26
<210> 80
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 80
gcgttgcctt tgtttcttct taattc 26
<210> 81
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 81
ctgtaatgag ctggcatgag tatttg 26
<210> 82
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 82
cacctccaag gtgtatgaag tatgtatttt 30
<210> 83
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 83
caaatagtag atgtgctttt tgatgtctga 30
<210> 84
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 84
ggtttagaga ctttctcaaa ggcttagat 29
<210> 85
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 85
atgacaaaaa tcatctctcc gaaaaacaag 30
<210> 86
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 86
caagttcctc aacgcaaata tcttcatt 28
<210> 87
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 87
atttggagta atgagtccag tttcgtt 27
<210> 88
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 88
agaagaggaa tgtgcaacat tctctg 26
<210> 89
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 89
aaaatcaaag tgtttgttcc aatacagca 29
<210> 90
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 90
caattggtgg cgatggtttt ctc 23
<210> 91
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 91
tcagcttact ccggccaaaa aa 22
<210> 92
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 92
agccgcggca gcgaccc 17
<210> 93
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 93
tatgtgggtt tgcaatttat aaagcagc 28
<210> 94
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 94
tatacaacag aatatacgat ggcctccata 30
<210> 95
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 95
cccatcgtgg gatcttgctt at 22
<210> 96
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 96
aaagctcttc ctttttgaaa gtctgttttt 30
<210> 97
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 97
ctgaagcctc tgaaagtgga ct 22
<210> 98
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 98
catttgcttc aaactgggct gaa 23
<210> 99
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 99
caacaagaca aacaacagtt ggtattagg 29
<210> 100
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 100
tgtcagttca tcatcttcca taaaagctt 29
<210> 101
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 101
tggtattgaa attttagcac tgtaagcaac 30
<210> 102
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 102
ttaaggtcta tccaaaactt tattgccagt 30
<210> 103
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 103
aggatgttaa agctcattca gtcaaagat 29
<210> 104
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 104
aggaacgggc ttggaagaaa ataat 25
<210> 105
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 105
ttttacctca gtcacataat aaggaatgca 30
<210> 106
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 106
ggttctaagc aacactgtga cgta 24
<210> 107
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 107
ataggaaaat accagcttca tagacaaagg 30
<210> 108
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 108
gccacagtag atgctcagta aatatttcta 30
<210> 109
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 109
tgggtgtttt atgcttggtt ctttagt 27
<210> 110
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 110
agagagtcta aaacagcttc tcacct 26
<210> 111
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 111
tgaggaaaca gtggtaaata agagagatga 30
<210> 112
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 112
tcctccttct gtgagcaaac ag 22
<210> 113
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 113
aatactgcta ctctctacag atctttcagt 30
<210> 114
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 114
gccaaatgaa cagacaagta aaagacat 28
<210> 115
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 115
caaaatatgt ggaggcccaa caa 23
<210> 116
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 116
tgttgctatt ctttgtctaa caccaaaaa 29
<210> 117
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 117
gagttgtggc accaaatacg aaac 24
<210> 118
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 118
agtcctagtg gattcactga cagatataaa 30
<210> 119
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 119
gtgacttttg gactttgttt ctttaagga 29
<210> 120
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 120
ccaagggact aattcatggt tgttc 25
<210> 121
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 121
caagctcttt tgtctggttc aacag 25
<210> 122
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 122
tcttataaac tggaaaggtt aagcgtcaat 30
<210> 123
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 123
atgtagtata gggaagcttc ataagtcagt 30
<210> 124
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 124
agcctttttg ggatattaaa tgttctgga 29
<210> 125
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 125
cccattgcag cacaactaag ga 22
<210> 126
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 126
ctaacacact gttcaactct gtgaaaatg 29
<210> 127
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 127
actgtgccca aacactacct tt 22
<210> 128
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 128
ttaaggacaa agttggttct tcagaatca 29
<210> 129
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 129
cccaaagtgt aaagaaatgc agaattct 28
<210> 130
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 130
ggctgaattt tcaatgactg aataaggg 28
<210> 131
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 131
acaggatttg gaaaaacatc agggaat 27
<210> 132
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 132
tttggttcca cttcagatac aaatgagt 28
<210> 133
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 133
gaaaaatatt agtgtcgcca aagagtcatt 30
<210> 134
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 134
caactgggac actttctttc agtattttg 29
<210> 135
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 135
tgataatcac ttcttccatt gcatctttct 30
<210> 136
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 136
atggagattc cataaactaa caagcactt 29
<210> 137
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 137
atgccttaac aaaagtaatc catagtcaag 30
<210> 138
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 138
tgtggagttt taaataggtt tggttcgtaa 30
<210> 139
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 139
tccaatgtaa aagatgcaaa tgcatacc 28
<210> 140
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 140
ggtggcccta cctcaaaatt attactatta 30
<210> 141
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 141
tggatactta aagccttctg tgtcatttc 29
<210> 142
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 142
cccaaagatc tcatgttaag tggagaaa 28
<210> 143
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 143
gtaactcaga ctcagcatca gca 23
<210> 144
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 144
aaaactgagg ctctttagct tcttagg 27
<210> 145
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 145
gaacccattt tcaagaactc taccatg 27
<210> 146
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 146
ctgaagctac ctccaaaact gtga 24
<210> 147
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 147
cagccttagc tttttacaca agttgt 26
<210> 148
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 148
gaatagctgt tagacatgct actgttact 29
<210> 149
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 149
ggttttcata cagctagcgg gaaa 24
<210> 150
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 150
accacagtct caatagaaac aaggttttta 30
<210> 151
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 151
ttgtttccta ggcacaataa aagatcga 28
<210> 152
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 152
ggaagtgtta acttcttaac gttagtgtca 30
<210> 153
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 153
gaagataaca aatatactgc tgccagtaga 30
<210> 154
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 154
ttgagctttc gcaacttcca aaaa 24
<210> 155
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 155
ttctgcagag gtacatccaa taagtttatc 30
<210> 156
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 156
tacttgaatc actgccatca aattctaagt 30
<210> 157
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 157
caaatgggca ggactcttag gt 22
<210> 158
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 158
ctgaggcttg ctcagtttct tttg 24
<210> 159
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 159
aactagtttt tgccagtttt ttaaaataac ct 32
<210> 160
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 160
gaaacaaact cccacatacc actg 24
<210> 161
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 161
cctcagatgt tattttccaa gcaggat 27
<210> 162
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 162
gcattcatta tgacatgaag atcagcatct 30
<210> 163
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 163
gggaaggaaa gaattttgct taagatatca 30
<210> 164
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 164
ctcaaagtat ttcattttct tggtgccat 29
<210> 165
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 165
tccagactct gaagaacttt tctcaga 27
<210> 166
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 166
ggttgcttgt ttatcacctg tgtct 25
<210> 167
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 167
aaaagtggaa tacagtgata ctgactttca 30
<210> 168
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 168
tggcaacagc tcaacgtttt tataattt 28
<210> 169
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 169
gcctcgcctc atgtggtttt at 22
<210> 170
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 170
taactagtat tctgagctgt gtgctaga 28
<210> 171
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 171
gggagggaga ctgtgtgtaa tattt 25
<210> 172
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 172
agttcttttg gtcatcaatc tctttctcc 29
<210> 173
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 173
cttctataaa gttaggtgtt tcctgggtt 29
<210> 174
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 174
tcagtcataa cagctcaaag ttgaacttat 30
<210> 175
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 175
gcttttattc tgctcatggc acaa 24
<210> 176
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 176
caacaaaagt gccagtagtc atttcaatat 30
<210> 177
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 177
ggaatctgct gaacaaaagg aacaag 26
<210> 178
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 178
gcattagtag tggattttgc ttctctgata 30
<210> 179
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 179
aaaaataccg aaagaccaaa aatcagaact 30
<210> 180
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 180
cctaaacaat catgtataca gatgatgcct 30
<210> 181
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 181
catttgttaa cttcagctct gggaaa 26
<210> 182
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 182
caaattgata gttgttctag cagtgaagag 30
<210> 183
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 183
aaatctgaaa gagctaacat acagttagca 30
<210> 184
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 184
ctattaggtc cacctcagaa caagatg 27
<210> 185
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 185
aggagaaccc tcaatcaaaa gaaacttatt 30
<210> 186
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 186
tataaagagg tccttgatta ggcacagt 28
<210> 187
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 187
caaccaaagt ctttgttcca cctttta 27
<210> 188
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 188
gaaaggcaaa aattcatcac acaaattgt 29
<210> 189
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 189
tcttcactat tcacctacgt ctagaca 27
<210> 190
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 190
cccaaaacat gaatgttctc aacaagtg 28
<210> 191
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 191
gtgaggtaga ttgtaaagtc aaaggct 27
<210> 192
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 192
aggtgcggta aaatttggat tctgta 26
<210> 193
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 193
aaatatttaa aatgtgccaa gaactgtgct 30
<210> 194
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 194
aaaaatgatg aagtgacagt tccagtagt 29
<210> 195
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 195
gttgttgaat tcagtatcat cctatgtggt 30
<210> 196
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 196
tgccgtatat gattacgtaa tgtaatgct 29
<210> 197
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 197
ccatcatgtg agtcatcaga acct 24
<210> 198
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 198
caacataaca gatgggctgg aagt 24
<210> 199
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 199
gttccctgat ttatcatttc aggagtct 28
<210> 200
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 200
agagttcact ccaaatcagt agagagtaat 30
<210> 201
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 201
cctcccagag ccctcaaatt ataa 24
<210> 202
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 202
cactgtgcga agacttttat gtctact 27
<210> 203
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 203
ccagaaccac catctttcag taatttg 27
<210> 204
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 204
cttgtttaca gcgatgccaa ca 22
<210> 205
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 205
acgagaataa atcaaaaatt tgccaaacga 30
<210> 206
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 206
tccaatccag acatattttg gttatgttgt 30
<210> 207
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 207
agatgatgtc agcaaaccta agaatgt 27
<210> 208
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 208
ccagtcctgc caatgagaag aa 22
<210> 209
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 209
gcctaccaca aatacaaatt atgaccaag 29
<210> 210
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 210
ttctcttcag gaggaaaagc acag 24
<210> 211
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 211
tgcgtgtgct gcgtgtc 17
<210> 212
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 212
cactaacccg ctccagagg 19
<210> 213
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 213
ttgtgcaaac ttcctgagtt ttcatg 26
<210> 214
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 214
ggttttctac tgttgctgca tcttattttt 30
<210> 215
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 215
cctgcaaaaa taaaaatgca gccattaaa 29
<210> 216
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 216
catccaatgc ctcgtaacaa cc 22
<210> 217
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 217
accagatgac tatcttaaag accacttct 29
<210> 218
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 218
agctttttgc agagcttcag taga 24
<210> 219
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 219
aattcgagtt ccatattgct tatactgct 29
<210> 220
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 220
tcctgaggat tttatcaaga aagcagattt 30
<210> 221
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 221
aagaaaacaa gctagcagaa cattttgtt 29
<210> 222
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 222
caagaaatta gagtcctcag aagagaact 29
<210> 223
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 223
caggcaattc agtaaacgtt aagtgaaa 28
<210> 224
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 224
acaattatca acctcatctg ctctttct 28
<210> 225
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 225
agcttgtgtt gaaattgtaa ataccttgg 29
<210> 226
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 226
gccttttggc taggtgttaa attatgg 27
<210> 227
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 227
ctgactggca tttggttgta cttt 24
<210> 228
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 228
agaatctgct ttcaaaacga aagctg 26
<210> 229
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 229
tgaacctgca gaagaatctg aacat 25
<210> 230
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 230
gtttgcctaa attcctagtt tgtagttctc 30
<210> 231
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 231
gacagtagaa ggactgaaga gtgag 25
<210> 232
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 232
agaatgaatt gacactaatc tctgcttgt 29
<210> 233
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 233
ccttcttccg ataggttttc ccaaatatt 29
<210> 234
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 234
agaaagttaa tgagtggttt tccagaagt 29
<210> 235
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 235
gtagctgtat acgtatggcg tttct 25
<210> 236
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 236
ggatgaggga atacataaaa gttaacacac 30
<210> 237
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 237
gcgttatacc tttgccctga ga 22
<210> 238
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 238
acctcagctc ctagactttc aga 23
<210> 239
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 239
gtgcctggcc tgatacaatt aac 23
<210> 240
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 240
aattcctcct gaattttagt gaataaggct 30
<210> 241
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 241
cataaaactc tttccagaat gttgttaagt c 31
<210> 242
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 242
taccatcttc aacctctgca ttgaaa 26
<210> 243
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 243
tttttggacc taggttgatt gcaga 25
<210> 244
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 244
aaaaacctgt agttcaacta aacagagga 29
<210> 245
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 245
tccaatacct aagtttgaat ccatgctt 28
<210> 246
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 246
caaaggcatc tcaggaacat cac 23
<210> 247
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 247
acataaggtt tttgctgaca ttcagagt 28
<210> 248
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 248
tgctgtgcta aaaatcccac aagtat 26
<210> 249
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 249
aaataaagat gtcagatacc acagcatctt 30
<210> 250
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 250
cctgaattat cactatcaga acaaagcagt 30
<210> 251
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 251
cccagaagct gattctctgt catg 24
<210> 252
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 252
tgctggcatt ttcatgatca tataaaagac 30
<210> 253
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 253
ggaagcaggg aagctcttca tc 22
<210> 254
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 254
tcatgcatct caggtttgtt ctga 24
<210> 255
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 255
aagagaagct gcaagtcatg gt 22
<210> 256
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 256
gctcacgacc atttgagacc a 21
<210> 257
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 257
ccagaatcca aatcaggcct tctt 24
<210> 258
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 258
gctatttcct tgatactgga ctgtca 26
<210> 259
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 259
gctgggagtc cgcctatcat ta 22
<210> 260
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 260
gttctgtttc aaacttgcat gtgga 25
<210> 261
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 261
ggcagagaag acttctgagg cta 23
<210> 262
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 262
cttcatccgg agagtgtagg gta 23

Claims (8)

1. An ultra-high flux multiplex PCR amplicon capture method, comprising the steps of:
1) carrying out multiple PCR amplification on a sample to be detected by using an amplification primer pool containing digestible complex modification and a first enzyme mixed solution to obtain an amplicon product; at least one amplification primer in the amplification primer pool comprises compound modified base nucleotides, and the modification mode is one or more of the following conditions: a T base is replaced with a uracil modified base, a T base is replaced with a locked nucleotide thymine modified base, a T base is replaced with a 5 methylcytosine modified base, a T base is replaced with a locked nucleotide adenine modified base, a G base is replaced with an 8-hydroxydeoxyguanosine modified base, and a T base is replaced with a hypoxanthine modified base; the first enzyme mixed solution is QuartTaq HotStart mixed solution;
2) performing enzyme digestion reaction on the amplicon product by using a second enzyme mixed solution containing primer digestive enzyme, mismatch error correcting enzyme and end repairing enzyme, wherein the enzyme digestion reaction comprises removing redundant primer parts on the amplification product by using the primer digestive enzyme and removing non-specific amplification products by using the mismatch error correcting enzyme; the second enzyme mixed solution comprises primer digestive enzyme, mismatch error correcting enzyme, T4 polynucleotide kinase, Taq polymerase and QuarPrep end repair tailing buffer solution; the primer digestive enzyme is selected from one or two of UDG enzyme and FPG enzyme; the mismatch error correcting enzyme is selected from one or more of T7 endonuclease I, T4 endonuclease VII, E coli endonuclease V, Surveror endonuclease and CEL I endonuclease;
3) performing a connection reaction by using the full-length joint containing the Index and a third enzyme solution to directly obtain a library for sequencing or obtain a library for sequencing after amplification; the third enzyme solution is DNA ligase.
2. The method of claim 1, wherein the number of modified bases of any one of the amplification primers comprising a complex of nucleotides with modified bases is any integer from 0 to n, where n represents the total length of the amplification primer.
3. The method of claim 1, wherein the library for sequencing is directly obtained after ligation reaction using a full-length linker containing Index and a third enzyme solution.
4. The ultra-high throughput multiplex PCR amplicon capture method of claim 1, wherein the mismatch error correcting enzyme is used to reduce non-specificity resulting from PCR amplification.
5. The method for capturing ultra-high throughput multiplex PCR amplicons of claim 1, wherein the full-length adapters comprising Index are used in place of universal short adapters for ligation reactions to construct sequencing libraries.
6. The ultra-high throughput multiplex PCR amplicon capture method of claim 1, wherein said sample to be detected is selected from a prokaryotic or eukaryotic microbial genome, an animal or plant genome, or a human genome.
7. The ultra-high throughput multiplex PCR amplicon capture method of claim 1, wherein said amplification primer pool comprises primers shown in SEQ ID NO. 1-262.
8. The use of the ultra-high throughput multiplex PCR amplicon capture method of claim 1 in any one of: pathogen detection, genetic disease detection, tumor early screening detection, tumor-associated diagnosis detection, susceptibility gene detection and the like.
CN202210687838.7A 2022-06-06 2022-06-17 Ultrahigh-flux multiple PCR amplicon capturing method Pending CN114958991A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210631047 2022-06-06
CN2022106310472 2022-06-06

Publications (1)

Publication Number Publication Date
CN114958991A true CN114958991A (en) 2022-08-30

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN114958991A (en)

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