A method for detecting familial thoracic aortic aneurysm and dissection related mutant genes based on high-throughput sequencing technology

Abstract

The present invention discloses a method for detecting familial thoracic aortic aneurysm and dissection related mutant genes based on high-throughput sequencing technology. The steps include: (1) sample collection; (2) panel design; (3) library construction; (4) sequencing on the machine; (5) data analysis and annotation. This patent invention is based on the high-throughput sequencing technology process of target region capture. By selecting a set of 12 pathogenic genes related to FTAAD and using a multiplex PCR capture process to detect 12 pathogenic genes at one time, compared with the prior art, it saves the number of detections and detection time. Compared with the prior art, the present invention has the advantages of strong pertinence, low cost and fast process.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a method for detecting genes related to familial thoracic aortic aneurysm and dissection based on the illumina sequencing platform. Background Art

[0002] Thoracic aortic aneurysm is a very dangerous cardiovascular critical illness and usually has no symptoms. However, if the aortic aneurysm is not fixed, it will become larger and larger over time and may even tear the aorta. Aortic rupture will cause blood to flow to the wrong parts of the body and is very likely to endanger life. Approximately one-fifth of patients with thoracic aortic aneurysm are caused by defective genes inherited from their parents, and this type of thoracic aortic aneurysm is called familial thoracic aortic aneurysm. If one of the parents carries a defective gene related to familial thoracic aortic aneurysm and dissection (FTAAD), each of their children has a 50% chance of inheriting this gene, and siblings or parents of those carrying the FTAAD risk gene also have a 50% chance of inheriting the gene. If genetic testing shows that a person is at risk of developing FTAAD, then they have a 50%-80% probability of having FTAAD, which is 50 times higher than those without the defective gene. These patients should undergo regular imaging examinations to monitor their aorta. Currently, blood pressure-lowering drugs are usually used for FTAAD patients to relieve the pressure on the aorta; but if the aorta becomes too large, surgery is required. Currently, several genes have been found to be related to FTAAD. Alterations in some genes, including ACTA2, MYLK, and MYH11, have been shown to cause FTAAD; other genes such as TGFBR1, TGFBR2, SMAD3, PRKG1, LOX, COL3A1, TGFB2, TGFB3, and FBN-1 are also related to the risk of developing FTAAD. Therefore, through genetic testing, we can identify people at risk of FTAAD.

[0003] The first Chinese edition of the "Gene Diagnosis Guidelines for Monogenic Hereditary Cardiovascular Diseases" formulated by the Precision Cardiovascular Medicine Group of the Chinese Society of Cardiology and others recommended results related to twelve genes, such as ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, and TGFB3. Among them, gene mutations in ACTA2 can explain 10-15% of familial TAAD. Patients carrying pathogenic gene mutations but without clinical symptoms can undergo early disease prediction and intervention, such as regular (once a year before aortic dilation and once every six months after dilation) imaging examinations to detect and monitor early diseases. Individualized treatment can be carried out according to genotypes and phenotypes. For example, surgery in advance (such as when the maximum internal diameter of the thoracic aorta reaches 4.0-5.0 cm) can prevent the occurrence of dissection and rupture; for carriers of pathogenic gene mutations in TGFBR1 and TGFBR2, surgery can be considered when the maximum internal diameter of the thoracic aorta reaches 4.2 cm; for patients with ACTA2 gene mutations, surgery can be considered when the maximum internal diameter of the thoracic aorta reaches 4.5 cm. Patients with ACTA2 gene mutations have a higher risk of early stroke and coronary artery disease and should be taken seriously. If there is one or more first-degree relatives of the proband with phenotypes of thoracic aortic dilation, aneurysm, or dissection, imaging examinations and gene testing can be performed on their second-degree relatives.

[0004] Currently, the commonly used gene detection technologies in clinical practice include sequencing technologies such as Sanger sequencing and high-throughput sequencing, probe technologies such as fluorescence PCR, gene chips, suspension arrays, mass spectrometry technologies, and ARMs, and mass spectrometry such as DHPLC. Among them, probe and mass spectrometry technologies can also detect mutations, but they can only detect known variations of single nucleotides such as SNPs. Sequencing technology is the gold standard for gene mutation detection, the most advanced level that can be achieved by existing technologies, the closest to objective facts, or even reflects the objective facts themselves; it is like pathological diagnosis. In sequencing, Sanger sequencing, as a widely used technology, has the advantages of being fast, accurate, and simple, but has problems such as low throughput, relatively high cost, and low degree of automation, making it difficult to meet the requirements of detecting multiple genes and multiple variations at one time. High-throughput sequencing achieves the goal of simultaneously detecting unknown gene mutations in multiple genes and is divided into whole-genome sequencing and targeted sequencing. Targeted sequencing attempts to obtain different combinations from millions of sequences, so an enrichment step is indispensable. Among them, the hybridization capture method uses probes complementary to the target of interest, the amplicon-based method relies on multiplex PCR reactions, and selective circularization relies on molecular inversion probes; whole-genome sequencing can simultaneously detect all types of variations on the genome, but it lacks specificity and has a large amount of data, and various costs are relatively high. However, clinical applications usually pursue rapidity, economy, and simplicity. Selecting a set of effective gene sets for high-throughput sequencing to reduce costs has become an effective method for detecting mutant genes in FTAAD.

[0005] Other names for FTAAD are: thoracic aortic aneurysm (TAA), thoracic aortic aneurysm and dissection (TAAD), familial aortic aneurysm (FAA), and aortic root dilation. Summary of the Invention

[0006] The technical problem to be solved by the present invention is the current lack of an effective and low-cost method for detecting gene mutations in FTAAD. For this technical problem, a multiplex PCR capture technique is adopted. The set of pathogenic genes of FTAAD is captured and enriched by the amplification principle, and then detected through a high-throughput sequencing platform. Using a bioinformatics analysis process and the ACMG genetic interpretation guidelines, gene mutations related to FTAAD are detected and interpreted to assist clinical diagnosis and treatment of FTAAD. This method has high throughput, can detect up to dozens of mutation sites in a single-tube reaction, is easy to operate, and has low cost.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for detecting mutation genes related to familial thoracic aortic aneurysm and dissection based on high-throughput sequencing technology, the steps of which include,

[0009] (1) Sample collection:

[0010] Select 12 pathogenic genes related to familial thoracic aortic aneurysm and dissection, namely ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, and TGFB3;

[0011] (2) Panel design:

[0012] The kit consists of 7 tubes of reagents,

[0013] Among them, 5 tubes of reagents are stored at -20°C, including 1 tube of IGT-I7 Index (10uM) reagent, 1 tube of IGT-I5 Index (10uM) reagent, 1 tube of Primer pool reagent, 1 tube of IGT-EM808 polymerase mixture reagent, and 1 tube of Enhancer buffer NB (1N) reagent;

[0014] The other 2 tubes of reagents are stored at 4°C, including 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent;

[0015] In the designed kit, the Primer pool reagent contains a total of 367 amplicons. The sequences of the amplicons are 1 - 367. Each amplicon includes a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are SEQ ID No.1 - SEQ ID No.734;

[0016] (3) Library construction:

[0017] (3.1) The first round of multiplex PCR reaction

[0018] Prepare the reaction solution in a PCR tube according to the following formula, and gently pipette up and down to mix evenly;

[0019]

[0020] Run the PCR instrument, place the above PCR tube, and perform the reaction according to the following program: first at 95°C for 210 s, then execute the cycling program: 98°C for 10 s, 60°C for 5 min, for a total of 18 cycles, and finally extend at 72°C for 5 minutes;

[0021] (3.2) Magnetic bead purification of the product

[0022] (3.2.1) Add 27 μl of AMPure XP magnetic beads equilibrated at room temperature to 30 μl of the PCR product processed in step (3.1), and pipette and mix 20 times with a pipette;

[0023] (3.2.2) After incubating at room temperature for 5 min, place the PCR tube on a DynaMag - 96 Side magnetic rack for 3 min;

[0024] (3.2.3) Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μl of YF buffer B to the tube, and pipette and mix 20 times with a pipette;

[0025] (3.2.4) After incubating at room temperature for 5 min, place the PCR tube on a DynaMag - 96 Side magnetic rack for 3 min;

[0026] (3.2.5) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μl of an 80% ethanol solution by volume percentage to the tube, and let it stand for 30 s;

[0027] (3.2.6) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μl of an 80% ethanol solution by volume percentage to the tube, let it stand for 30 s, and then thoroughly remove the supernatant;

[0028] (3.2.7) Let it stand at room temperature for 3 min to completely volatilize the residual ethanol;

[0029] (3.2.8) Remove the PCR tube from the magnetic stand, add 24 μl of Nuclease-free water, gently pipette to resuspend the magnetic beads, avoiding the generation of bubbles, and then let it stand at room temperature for 2 min;

[0030] (3.2.9) Place the PCR tube back on the magnetic stand and let it stand for 3 min;

[0031] (3.2.10) Use a pipette to aspirate 13.5 μl of the supernatant and transfer it to a new 200 μl PCR tube. The transferred supernatant in the tube is the multiplex PCR product;

[0032] (3.3) Second-round adapter sequence PCR reaction

[0033] Prepare the reaction solution in the PCR tube according to the following formula and gently pipette up and down to mix well. Among them, the PCR product mixture is the purified multiplex PCR product obtained in step (3.2.10);

[0034]

[0035]

[0036] Run the PCR instrument, place the above PCR tube, and perform the reaction according to the following program: first at 95 °C for 210 s, and then execute the cycling program: 98 °C for 20 s, 68 °C for 1 min, 72 °C for 30 s for 9 cycles, and finally extend at 72 °C for 5 min;

[0037] (3.4) Second-round magnetic bead purification

[0038] (3.4.1) Take 30 μl of the PCR reaction system processed in step (3.3), and add 27 μl of AMPure XP magnetic beads equilibrated at room temperature, and pipette and mix well 20 times;

[0039] (3.4.2) After incubating at room temperature for 5 min, place the PCR tube on the DynaMag-96 Side magnetic stand for 3 min;

[0040] (3.4.3) Thoroughly remove the supernatant, remove the PCR tube from the magnetic stand, add 50 μl of YF buffer B to the tube, and pipette and mix well 20 times;

[0041] (3.4.4) After incubating at room temperature for 5 min, place the PCR tube on the DynaMag-96 Side magnetic stand for 3 min;

[0042] (3.4.5) Remove the supernatant, keep the PCR tube on the magnetic stand, add 180 μl of 80% ethanol solution by volume to the tube, and let it stand for 30 s;

[0043] (3.4.6) Remove the supernatant. Keep the PCR tube on the magnetic stand and add 180 μl of 80% ethanol solution by volume. After standing for 30 s, completely remove the supernatant.

[0044] (3.4.7) Let it stand at room temperature for 3 min to completely evaporate the residual ethanol.

[0045] (3.4.8) Remove the centrifuge tube from the magnetic stand, add 24 μl of Nuclease-free water or 1×TE buffer (pH 8.0), pipette and mix 20 times to resuspend the magnetic beads, avoiding the generation of bubbles, and let it stand at room temperature for 2 min.

[0046] (3.4.9) Place the PCR tube back on the magnetic stand and let it stand for 3 min.

[0047] (3.4.10) Use a pipette to aspirate 20 μl of the supernatant and transfer it to a new 200 μl PCR tube. The transferred supernatant in the tube is the prepared multiplex PCR library.

[0048] (3.5) Library quantification

[0049] Take 2 μl of the multiplex PCR library obtained in step (3.4.10) and use a 3.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration. The concentration range of a normal library constructed from human genomic DNA is 5 - 40 ng / μl.

[0050] (4) Sequencing on the machine

[0051] Use Hiseq Xten PE150 to sequence the qualified multiplex PCR library obtained in step (3.6) to obtain a gene sequence list, and the data volume for uploading to the machine is 300 Mb.

[0052] (5) Data analysis and annotation: After the sequencing data is downloaded, perform bioinformatics analysis on the downloaded data to detect mutations on the genes.

[0053] (5.1) Quality control of the downloaded data in Fastq format: For each pair of original Fastq sequences (raw reads) generated from each sample library and lane, use the software cutAdapt to remove the sequencing adapters and sequences reads containing a large number of 'N' bases to obtain sequences (Clean reads) that meet the quality control requirements. Use the software fastqc to count the basic information of the sequences Clean reads, including the sequence and base quantity, GC content and distribution, sequencing error rate and distribution, and base quality distribution.

[0054] (5.2) Alignment: Using the software bwa, align the sequence Clean reads with the human reference genome GRCh38 to obtain the alignment information file BAM of the sequence Clean reads on the reference genome;

[0055] (5.3) Preprocessing of the alignment information file BAM: Include using picard and GATK software to merge the alignment information files BAM generated from different libraries and lanes of the same sample, sort them according to the genomic coordinates, verify the alignment information file BAM, remove the duplicate sequences generated during the PCR process, correct the base quality values, and obtain the preprocessed alignment file CleanBAM; Use picard software to count the alignment file Clean BAM to obtain the quality control information; The quality control information includes the coverage, depth, capture efficiency, uniformity of the target region capture chip, the number of alignable sequences and bases, and the length distribution of the inserted fragments;

[0056] (5.4) Variant detection: Use GATK software to detect small fragment variations on genes, including single nucleotide polymorphisms SNP and insertions and deletions Indel; Filter the detected variations, the SNP filtering conditions are: QD < 3.37 || FS > 31.397 || SOR > 10.419 || MQ < 20.0 || MQRankSum < -12.49 || ReadPosRankSum < -3.721, and the Indel filtering conditions are: QD < 5.2 || FS > 52.254 || SOR > 9.044 || ReadPosRankSum < -5.504;

[0057] Annotate the variations that meet the filtering conditions, and mark the corresponding genes, transcripts, variant types, functions, and frequencies in the normal population;

[0058] Statistical analysis of the variant results to obtain the variant quantity, length distribution, the quantity of each type of variant, and population variant frequency information.

[0059] To obtain better technical effects, first dilute the concentration of the gDNA to be detected to the same concentration before step (3.1), and then transfer it to a PCR 8-tube strip.

[0060] To obtain better technical effects, there is also step (3.6) library quality detection after step (3.5),

[0061] Take 1ul of the multiplex PCR library obtained in step (3.4), and use the Qsep100 fully automatic nucleic acid and protein analysis system to measure the library fragment length and purity. The target fragment distribution range of a normal library is between 300bp - 420bp;

[0062] Use the multiplex PCR library obtained in step (3.4) for library detection using Aligent 2100 and QPCR;

[0063] The multiplex PCR library obtained in step (3.4) is considered qualified if the Aligent 2100 fragment result is between 250bp - 350bp, and the QPCR result is greater than 10nM;

[0064] Use the qualified multiplex PCR library for step (4) to perform sequencing to obtain a gene sequence list.

[0065] To achieve better technical effects, it also includes step (6) variant interpretation: explaining the relationship between variant SNPs and variant Indels and diseases;

[0066] According to the ACMG / AMP guidelines, use the software REO-HIT to classify the variants into 5 categories: pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign;

[0067] To achieve better technical effects, it also includes step (7) issuing a report, using Sanger sequencing to verify the sequencing results of pathogenic, likely pathogenic, variant of uncertain significance, and likely benign detected above one by one, and issuing a report.

[0068] This patent invention is based on the technical process of target region capture high-throughput sequencing. By selecting 12 pathogenic gene sets related to FTAAD, using the multiplex PCR capture process and the following implementation methods for high-throughput capture sequencing analysis, it aims to interpret the gene variation situation related to FTAAD through gene detection technology, and the results of multi-gene variation analysis refer to the guidelines issued by the American College of Medical Genetics (ACMG), the Association for Molecular Pathology (AMP), and the Precision Cardiovascular Disease Group of the Chinese Society of Cardiology, etc. to interpret the variants.

[0069] Compared with the prior art, the present invention has advantages such as strong pertinence, low cost, and fast process.

[0070] Reagent manufacturer: Illumina. Brief Description of the Drawings

[0071] Figure 1 It is the genetic variation analysis and interpretation process of the embodiment of the present invention;

[0072] Figure 2 It is the quality inspection library result of the Qsep100 fully automatic nucleic acid and protein analysis system of the embodiment of the present invention;

[0073] Figure 3 It is the detection technical process of the embodiment of the present invention. Detailed Embodiments

[0074] The content of the present invention will be further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments.

[0075] A method for detecting genes related to familial thoracic aortic aneurysm and dissection based on high-throughput sequencing technology, the steps of which include

[0076] (1) Sample collection:

[0077] Selecting a specific and effective gene set is crucial for screening disease mutation genes. Omitting genes will reduce the detection sensitivity, while selecting too many genes will increase the cost and operation difficulty. Based on the above situation, in this patent invention, by querying a large number of disease databases and research records on FTAAD in the literature, including OMIM, ClinVar, CGD, Orphanet, DISEASE, GeneReviews, and the "Guidelines for Gene Detection of Monogenic Hereditary Cardiovascular Diseases", genes / variations that can cause FTAAD with sufficient evidence are selected. These evidences include gene and protein function research, family heredity research, population variation analysis, etc. Finally, 12 pathogenic genes for FTAAD are selected, and the gene list is shown in Table 1;

[0078] Table 1

[0079]

[0080] Finally, 12 pathogenic genes related to familial thoracic aortic aneurysm and dissection, namely ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, and TGFB3, are selected;

[0081] (2) Panel design:

[0082] The kit consists of 7 tubes of reagents,

[0083] Among them, 5 tubes of reagents are stored at -20 °C, including 1 tube of IGT-I7 Index (10 uM) reagent, 1 tube of IGT-I5 Index (10 uM) reagent, 1 tube of Primer pool reagent, 1 tube of IGT-EM808 polymerase mixture reagent, and 1 tube of Enhancer buffer NB (1N) reagent;

[0084] The other 2 tubes of reagents are stored at 4 °C, including 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent;

[0085] The designed region is the exon region of each gene, and 15 bp is allowed to extend on both sides of the exon region;

[0086] In the designed kit, the Primer pool reagent contains a total of 367 amplicons, and the sequences of the amplicons are 1 - 367. Each amplicon includes a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are SEQ ID No.1 - SEQ ID No.734. For the detailed primer sequences, please refer to Appendix 3;

[0087] For the position information of IGT-I7 Index, please refer to Appendix 1 "Position Information of 96 IGT-I7 Indexes";

[0088] For the information of IGT-I5 Index, please refer to Appendix 2;

[0089] Reagent manufacturer: Illumina;

[0090] The kit uses two rounds of PCR reactions for library construction, with the advantages of short library construction cycle, high alignment rate, high capture rate, good uniformity, good repeatability, and simple operation. The kit supports the construction of libraries from human genomic DNA extracted from various samples, such as saliva, blood, etc.;

[0091] (3) Library construction:

[0092] (3.0) Dilute the concentration of all gDNA to be detected to the same concentration and transfer it to a PCR 8-strip tube. On the one hand, it is convenient for pipetting operation, and on the other hand, it is to add the same starting amount of gDNA to reduce the concentration difference of the final library and facilitate the mixing of libraries; Please mark the reaction number on the upper part of the PCR tube wall or the tube cap to prevent the mark from disappearing due to high temperature or other reasons, and avoid mistakes in subsequent product mixing operations and sample cross-contamination;

[0093] (3.1) The first round of multiplex PCR reaction

[0094] Prepare the reaction solution in a PCR tube (or strip tube, or PCR plate) according to the following formula, and gently pipette up and down to mix evenly;

[0095]

[0096] Run the PCR instrument, put the above PCR tube into it, and perform the reaction according to the following program: first at 95°C for 210 s, then execute the cycling program: 98°C for 10 s, 60°C for 5 min for a total of 18 cycles, and finally extend at 72°C for 5 min;

[0097] (3.2) Magnetic bead purification and merging of products

[0098] (3.2.1) Add 27 μl of AMPure XP magnetic beads balanced at room temperature to 30 μl of the PCR product processed in step (3.1), and pipette and mix 20 times with a pipette;

[0099] (3.2.2) Incubate the PCR tube at room temperature for 5 min, and then place the PCR tube on the DynaMag-96Side magnetic stand for 3 min;

[0100] (3.2.3) Thoroughly remove the supernatant, take the PCR tube off the magnetic stand, add 50 μl of YF buffer B into the tube, and pipette and mix well 20 times;

[0101] (3.2.4) Incubate the PCR tube at room temperature for 5 min, and then place the PCR tube on the DynaMag-96Side magnetic stand for 3 min;

[0102] (3.2.5) Remove the supernatant, keep the PCR tube on the magnetic stand, add 180 μl of ethanol solution with a volume percentage concentration of 80% into the tube, and let it stand for 30 s;

[0103] (3.2.6) Remove the supernatant, keep the PCR tube on the magnetic stand, add 180 μl of ethanol solution with a volume percentage concentration of 80% into the tube, let it stand for 30 s, and then thoroughly remove the supernatant. It is recommended to use a 10-μl pipette to remove the residual ethanol solution at the bottom;

[0104] (3.2.7) Let it stand at room temperature for 3 min to completely volatilize the residual ethanol;

[0105] (3.2.8) Take the PCR tube off the magnetic stand, add 24 μl of Nuclease-free water, gently pipette and resuspend the magnetic beads, avoiding generating bubbles, and then let it stand at room temperature for 2 min;

[0106] (3.2.9) Place the PCR tube back on the magnetic stand and let it stand for 3 min;

[0107] (3.2.10) Use a pipette to aspirate 13.5 μl of the supernatant and transfer it into a new 200-μl PCR tube. The transferred supernatant in the tube is the purified multiplex PCR product;

[0108] (3.3) The second-round adapter sequence PCR reaction

[0109] Prepare the reaction solution in a PCR tube (or strip tube, or PCR plate) according to the following formula, and gently pipette up and down to mix well. Among them, the PCR product mixture is the purified multiplex PCR product obtained in step (3.2.10);

[0110]

[0111] Operate the PCR instrument, place the above-mentioned PCR tubes, and perform the reaction according to the following procedure: First, incubate at 95°C for 210 s, and then execute the cycling program: cycle 9 times at 98°C for 20 s, 68°C for 1 min, and 72°C for 30 s, and finally extend at 72°C for 5 min;

[0112] (3.4) Second-round magnetic bead purification

[0113] (3.4.1) Take 30 μl of the PCR reaction system processed in step (3.3), and add 27 μl of AMPure XP magnetic beads equilibrated at room temperature. Pipette and mix well 20 times;

[0114] (3.4.2) After incubating at room temperature for 5 min, place the PCR tube on a DynaMag-96 Side magnetic stand for 3 min;

[0115] (3.4.3) Thoroughly remove the supernatant, remove the PCR tube from the magnetic stand, add 50 μl of YF buffer B to the tube, and pipette and mix well 20 times;

[0116] (3.4.4) After incubating at room temperature for 5 min, place the PCR tube on a DynaMag-96 Side magnetic stand for 3 min;

[0117] (3.4.5) Remove the supernatant, keep the PCR tube on the magnetic stand, add 180 μl of 80% ethanol solution by volume to the tube, and let it stand for 30 s;

[0118] (3.4.6) Remove the supernatant, keep the PCR tube on the magnetic stand, add 180 μl of 80% ethanol solution by volume to the tube, let it stand for 30 s, and then thoroughly remove the supernatant. It is recommended to use a 10 μl pipette to remove the residual ethanol solution at the bottom;

[0119] (3.4.7) Let it stand at room temperature for 3 min to completely volatilize the residual ethanol;

[0120] (3.4.8) Remove the centrifuge tube from the magnetic stand, add 24 μl of Nuclease-free water or 1×TE buffer (pH 8.0), pipette and mix well 20 times to resuspend the magnetic beads, avoid generating bubbles, and let it stand at room temperature for 2 min;

[0121] (3.4.9) Place the PCR tube back on the magnetic stand and let it stand for 3 min;

[0122] (3.4.10) Use a pipette to aspirate 20 μl of the supernatant and transfer it to a new 200 μl PCR tube. The transferred supernatant in the tube is the prepared multiplex PCR library;

[0123] (3.5) Library quantification

[0124] Take 2 μl of the multiplex PCR library obtained in step (3.4.10) and use a 3.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration; the concentration range of a normal library constructed from saliva gDNA or blood gDNA is 5 - 40 ng / μl;

[0125] (3.6) Library quality detection

[0126] Take 1 μl of the multiplex PCR library obtained in step (3.4) and use the Qsep100 fully automatic nucleic acid and protein analysis system to measure the library fragment length and purity. The target fragment distribution range of a normal library is between 300 bp and 420 bp. The detection results are shown in Figure 2 ;

[0127] Use Aligent 2100 and QPCR to detect the multiplex PCR library obtained in step (3.4);

[0128] The Aligent 2100 fragment result of the multiplex PCR library obtained in step (3.4) is qualified if it is between 250 bp and 350 bp, and the QPCR result is qualified if it is greater than 10 nM;

[0129] (4) Sequencing on the machine

[0130] Use Hiseq Xten PE150 to sequence the qualified multiplex PCR library obtained in step (3.6) to obtain a gene sequence list, and the on - machine data volume is 300 Mb;

[0131] (5) Data analysis and annotation: After the sequencing data is downloaded, perform bioinformatics analysis on the downloaded data to detect mutations on the gene;

[0132] (5.1) Quality control of the downloaded data in Fastq format: For each pair of original Fastq sequences (raw reads) generated from each sample library and lane, use the software cutAdapt to remove sequencing adapters and sequences (reads) containing a large number of 'N' bases to obtain sequences (Clean reads) that meet the quality control requirements. Use the software fastqc to count the basic information of the sequences Clean reads, including the sequence and base quantity, GC content and distribution, sequencing error rate and distribution, and base quality distribution;

[0133] (5.2) Alignment: Use the software bwa to align the sequences Clean reads with the human reference genome GRCh38 to obtain the alignment information file BAM of the sequences Clean reads on the reference genome;

[0134] (5.3) Preprocessing of the alignment information file BAM: It includes using the picard and GATK software to merge the BAM files of the alignment information generated from different libraries and lanes of the same sample, sorting them according to the genomic coordinates, verifying the BAM files of the alignment information, removing the duplicate sequences generated during the PCR process, and correcting the base quality values to obtain the preprocessed alignment file CleanBAM. Use the picard software to count the Clean BAM of the alignment file to obtain the quality control information; the quality control information includes the coverage, depth, capture efficiency, uniformity of the target region capture chip, the number of alignable sequences and bases, and the length distribution of the inserted fragments;

[0135] (5.4) Variant detection: Use the GATK software to detect small fragment variations on genes, including single nucleotide polymorphisms SNP and insertions and deletions Indel;

[0136] Filter the detected variations. The SNP filtering conditions are: QD < 3.37 || FS > 31.397 || SOR > 10.419 || MQ < 20.0 || MQRankSum < -12.49 || ReadPosRankSum < -3.721, and the Indel filtering conditions are: QD < 5.2 || FS > 52.254 || SOR > 9.044 || ReadPosRankSum < -5.504;

[0137] Annotate the variations that meet the filtering conditions to obtain the variant information, and the variant information includes genes, transcripts, variant types, functions, and frequencies in the normal population;

[0138] Statistically analyze the variant results to obtain the number of variations, length distribution, the number of each type of variation, and the proportion that appears in the population;

[0139] (6) Variant interpretation: Explain the relationship between variant SNPs and variant Indels and diseases,

[0140] According to the ACMG / AMP guidelines (Genet Med. 2015 May; 17(5): 405–424.), use the software REO-HIT to classify the variations into 5 categories: pathogenic, likely pathogenic, of uncertain significance, likely benign, and benign;

[0141] The processing process of the software REO-HIT is as Figure 1 shown.

[0142] The specific steps are as follows,

[0143] Load the database: Load the ClinVar, CGD, and OMIM databases;

[0144] Collect variant information: Read the variant information obtained in step (5.4) one by one to obtain population variant frequency information, disease variant information, and variant function prediction information;

[0145] Process and interpret evidence:

[0146] i) Compare the collected variant information with the database, and sequentially determine whether each variant meets the pathogenic evidence or benign evidence described in the ACMG / AMP guidelines (Genet Med. 2015 May; 17(5): 405–424.);

[0147] Among them, pathogenic evidence includes: very strong pathogenic PVS, strong pathogenic PS, moderate pathogenic PM, weak pathogenic PP;

[0148] Benign evidence includes: independent benign BA, strong benign BS, weak benign BP;

[0149] ii) Combine variant evidence and classify each variant as pathogenic, likely pathogenic, of uncertain significance, likely benign, or benign according to the ACMG / AMP combination rules;

[0150] (7) Issue a report

[0151] Verify the sequencing results of pathogenic, likely pathogenic, of uncertain significance, and likely benign detected above one by one by Sanger sequencing and issue a report.

[0152] Comparative example

[0153] Sanger sequencing is the gold standard for detecting DNA mutations, but due to its limited sensitivity and inability to simultaneously detect multiple targets in parallel.

[0154] If Sanger sequencing is used to detect the ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, and TGFB3 genes, Sanger sequencing needs to be performed on each exon separately, which is costly.

[0155] The target region capture high-throughput sequencing used in the present invention involves the targeted enrichment of the exons of the ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, and TGFB3 genes. With the parallel sequencing of Illumina XTen, the detection of all the above gene exons can be achieved only once.

[0156] FTAAD has the same clinical symptoms as sporadic TAAD, and FTAAD-related gene mutations need to be detected for diagnosis. FTAAD is an autosomal dominant genetic disease, but the penetrance varies in different families. Carriers have a 50%-80% chance of developing TAAD, with significant genetic heterogeneity. Currently, 12 pathogenic genes have been reported, including ACTA2, FBN1, MYH11, MYLK, TGFBR1, TGFBR2, and COL3A1. Traditional technology makes it difficult to screen for mutations in these genes at one time, and the next-generation sequencing technology provides a new solution.

[0157] The present invention provides an experimental process and interpretation method for detecting 12 pathogenic genes related to FTAAD at one time, including the screening of all FTAAD-related genes and the capture of related genes using multiplex PCR technology. Accurate interpretation of the relationship between mutations and diseases provides an important basis for FTAAD screening, early diagnosis, differential diagnosis, personalized medication, fertility guidance, and exercise recommendations.

[0158] The technology of the present invention adopts multiplex PCR capture technology to capture the selected specific effective gene set using the amplification principle, and then detects it through a high-throughput sequencing platform. It uses a self-built bioinformatics analysis process and ACMG genetic interpretation guidelines to detect the gene mutation of FTAAD, so as to assist the clinic in accurately diagnosing and treating the disease.

[0159] A method and application for detecting FTAAD pathogenic gene mutations based on high-throughput sequencing technology, characterized by ① multiplex PCR targeted enrichment of all 12 pathogenic genes of FTAAD described in the "Guidelines for Genetic Testing of Single Gene Inherited Cardiovascular Diseases". ② The designed 367 pairs of primers can target and enrich the above 12 pathogenic gene exons and the 15bp intervals on both sides from the human genome through a single PCR reaction. ③ The experimental process and technical parameters for constructing libraries in conjunction with the labeling technology used by MiniSeq, NextSeq, HiSeq 3000 / 4000, Hiseq X Ten, MiSeq, HiSeq 2000 / 2500 and NovaSeq sequencers announced by Illumina; the use of labeling adapters in the capture library construction process reduces sequencing noise pollution, corrects PCR errors, and improves the accuracy of the results, especially for low-frequency mutations that are easier to detect. ④ The library constructed by the invention kit is used for the sequencing technical parameters and post-sequencing data quality requirements of MiniSeq, NextSeq, HiSeq 3000 / 4000, Hiseq X Ten, MiSeq, HiSeq 2000 / 2500 and NovaSeq sequencers published by Illumina. ⑤ The bioinformatics analysis methods, parameters and processes of the sequencing sequences corresponding to this invention. ⑥ The pathogenicity interpretation process and clinical interpretation of sequencing variant results.

[0160] Possible variant schemes of the present invention under the high-throughput sequencing technology include: 1. The method for specifically enriching the pathogenic gene to be detected in the genome is changed from multiplex PCR to hybridization capture or selective circularization; 2. The adapter, PCR amplification conditions and experimental procedures are changed for the probes used on sequencing platforms other than Illumina; 3. The sequencing depth and sequencing quality parameters are changed for the sequencing methods used on sequencing platforms other than Illumina, etc.; 4. The bioinformatics algorithms and the corresponding sequencing data interpretation methods and variant interpretation means for detection accuracy; 5. The pathogenic interpretation method and clinical application explanation.

[0161] The present invention detects gene sequences based on high-throughput targeted sequencing technology, and interprets gene variations and their pathogenic probabilities through bioinformatics analysis. It is applicable to medical and third-party testing institutions equipped with sequencers, large servers, bioinformatics teams, etc. The possible variant schemes for the clinical application of this invention may be based on probe technologies such as fluorescence PCR, gene chips, suspension arrays, mass spectrometry technology and ARMs. Mass spectrometry such as DHPLC directly detects pathogenic hot spot SNPs and the corresponding result interpretation methods.

[0162] Appendix 1:

[0163] Position information of IGT-I7 Index, using BOX1, 96-well plate

[0164] 1 2 3 4 5 6 7 8 9 10 11 12 A A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 C C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12 D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12 F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12 G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12 H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12

[0165] Appendix 2: IGT-I5 Index sequence information

[0166] The aliquoting sequence at the IGT-I5 Index end is related to the sequencing platform:

[0167] 1. For MiniSeq, NextSeq, HiSeq 3000 / 4000 and Hiseq X Ten sequencing platforms, please use the aliquoting sequence in the Index column;

[0168] 2. For MiSeq, HiSeq 2000 / 2500 and NovaSeq sequencing platforms, please use the aliquoting sequence in the Inprimer column;

[0169] 3. If it is single-end sequencing, only split the data with the aliquoting sequence in the IGT-I7 Index column, as shown in the following table.

[0170] Name Indcx Sub-sampling Sequence Inprimcr Sub-sampling Sequence IGT-15-31# GTAGAGGA TCCTCTAC IGT-15-32# CCGCCTTA TAAGGCGG IGT-15-33# ATAGTACG CGTACTAT lGT-15-34# TTCTGCCT AGGCAGAA

[0171] Appendix 3:

[0172] The primer pool reagent provides 367 amplicons with sequences from 1 to 367. Each amplicon includes a forward primer and a reverse primer, and the sequences of the forward and reverse primers are SEQ ID No. 1 - SEQ ID No. 734. See the following table for details.

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] SEQUENCE LISTING <110> Key Laboratory of Cardiovascular and Cerebrovascular Diseases Prevention and Treatment of the Ministry of Education The First Affiliated Hospital of Gannan Medical University Duan, Yanyu Liu, Ziyou <120> Method for Detecting Familial Thoracic Aortic Aneurysm and Dissection - Related Mutant Genes Based on High - Throughput Sequencing Technology <130> 2020 <160> 734 <170> PatentIn version 3.5 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <400> 1 cagttgtgtg ctagagacag agag 24 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <400> 2 tgtcctaccg cactaacgac t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 ccaggaaaag ggcgtttgtt g 21 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <400> 4 cagaccctaa tgtttgccac aatg 24 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 ttcaatccca tctctggcag tg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 gtgtgctctc ctgaccattc tt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <400> 7 cagcagtagt gtggtgttct gt 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <400> 8 cacaagtttt gggagatgct gac 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 attgccactg ggtctgtcac 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <400> 10 ctctccactt aaggaccatg gc 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <400> 11 tggtcctgga agttactgag ca 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <400> 12 catggtgact tatcacgccc aa 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 cttctctctg ctgtgctgca t 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <400> 14 ggaaatgcag gacctcatga ca 22 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <400> 15 acagaggaac ctaatctgtg tcct 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <400> 16 cacaaatgcc caattacagc tgag 24 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <400> 17 gtttttcttt taattatttg gtctctggat gg 32 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <400> 18 aagggatcag ctacctccac tt 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <400> 19 agcaaagatg gctgtcttct ca 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <400> 20 tgtgtatgca gcataaggca ga 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <400> 21 ctccaaccat gaccaggaag ag 22 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <400> 22 aacgaatgcc tcagcgctc 19 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <400> 23 agccatgcat cttgagagtg ag 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <400> 24 taggatgtgt aggggccaga tt 22 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <400> 25 ttcttatccc aacagcagag gaa 23 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <400> 26 gttggcttga ctcaaatgcc tc 22 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <400> 27 ccacttgagg ataagccatc aga 23 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <400> 28 acactgaagt gaccccctac ata 23 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <400> 29 cacagcagca ttccgatttg g 21 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <400> 30 gctttcccct cttgcttctt ct 22 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <400> 31 gtcacttctg atgcactcaa agc 23 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <400> 32 agtcaggtaa ttaaggcaga tatatgca 28 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <400> 33 tgttcccagg atcagtacac gta 23 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <400> 34 catcatgttt tggacacatt cctgg 25 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <400> 35 aaagcctggg ccctaaacta c 21 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <400> 36 gagtattgga ggggacagac atc 23 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <400> 37 ttccctatga ggttcacgca ac 22 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <400> 38 ccataacaca gagggaagtt accg 24 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <400> 39 gccatcaaag cttcatggaa tcc 23 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <400> 40 aaggctgtcc tgagactcat ttg 23 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <400> 41 ggcaattggc catggaaaac g 21 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <400> 42 atccgcctgg aaacctgctt 20 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <400> 43 ccctatcgga catgctgaat tttg 24 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <400> 44 agatatagat gaatgtgaag tgttccc 27 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <400> 45 gacagcttta tccagtccga gt 22 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <400> 46 tgtcagaact gcaaagtctg ga 22 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <400> 47 ttcttttgca ggaaaagctg aca 23 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <400> 48 taggcccaag actagatttt agcag 25 <210> 49 <211> 25 <212> DNA <213> Artificial Sequence <400> 49 caggcaatgt ttcagaaaat gggta 25 <210> 50 <211> 34 <212> DNA <213> Artificial Sequence <400> 50 cctaaggtca ttacatttat tgtagtgtta tatt 34 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <400> 51 aagacctcaa tggtggcaga ag 22 <210> 52 <211> 22 <212> DNA <213> Artificial Sequence <400> 52 gtgacagtgt gatgacagat gc 22 <210> 53 <211> 23 <212> DNA <213> Artificial Sequence <400> 53 cataaggagg agaaaaggca cgt 23 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <400> 54 ccttcgtaag cttactcttc tggtc 25 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <400> 55 tatgtcccac attccacgtc ag 22 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <400> 56 ggaacccaga aagtcttaga attatgag 28 <210> 57 <211> 31 <212> DNA <213> Artificial Sequence <400> 57 acttgaacaa tgcaagaaaa ataactagat g 31 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <400> 58 ctgttgtgtt ttgttttgtt gtgtttt 27 <210> 59 <211> 24 <212> DNA <213> Artificial Sequence <400> 59 ggaactgact tacacaaacc atgc 24 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <400> 60 ctgagtcctt ctactgacga atgg 24 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <400> 61 gagaatggct ctccagagca a 21 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <400> 62 acaggacagg cccatgtttt 20 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <400> 63 ttcaggaagt agccatgcag ac 22 <210> 64 <211> 22 <212> DNA <213> Artificial Sequence <400> 64 aagcgtctca gctctctcct ta 22 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <400> 65 tgcaggaaag aggaaagcca a 21 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <400> 66 catgtgctaa cagacctctg gt 22 <210> 67 <211> 22 <212> DNA <213> Artificial Sequence <400> 67 cttgaaactt gggagaccca ca 22 <210> 68 <211> 16 <212> DNA <213> Artificial Sequence <400> 68 caagaggcgg cgggag 16 <210> 69 <211> 22 <212> DNA <213> Artificial Sequence <400> 69 aataggttcc agccactggc tt 22 <210> 70 <211> 22 <212> DNA <213> Artificial Sequence <400> 70 cagaagccaa tgtgagtctt gc 22 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <400> 71 agacatcagg agaaactaac ttctgac 27 <210> 72 <211> 28 <212> DNA <213> Artificial Sequence <400> 72 ccttgtcttc ccattctaat gaaaaaca 28 <210> 73 <211> 22 <212> DNA <213> Artificial Sequence <400> 73 cgcagagcca cattcattga tg 22 <210> 74 <211> 21 <212> DNA <213> Artificial Sequence <400> 74 agcaagtggc cagatccaat g 21 <210> 75 <211> 22 <212> DNA <213> Artificial Sequence <400> 75 acaaggattc accagctgga tc 22 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <400> 76 ggaattttaa cccctctttg ccc 23 <210> 77 <211> 23 <212> DNA <213> Artificial Sequence <400> 77 gtggagttct tacaggcaaa gga 23 <210> 78 <211> 23 <212> DNA <213> Artificial Sequence <400> 78 gatgcacagt cacgctgtat ttc 23 <210> 79 <211> 24 <212> DNA <213> Artificial Sequence <400> 79 agattccctg caagtatttt tgga 24 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <400> 80 cagacaatcg ggaagggtac t 21 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <400> 81 ttaggaaagt gcggtgccaa 20 <210> 82 <211> 22 <212> DNA <213> Artificial Sequence <400> 82 aaggaaggag ctccatcctc ta 22 <210> 83 <211> 22 <212> DNA <213> Artificial Sequence <400> 83 aagggaagct ttgagggaca tc 22 <210> 84 <211> 22 <212> DNA <213> Artificial Sequence <400> 84 ggtaggttcc cttttgttgc tg 22 <210> 85 <211> 28 <212> DNA <213> Artificial Sequence <400> 85 tggtacctat attcatggct atacagtg 28 <210> 86 <211> 32 <212> DNA <213> Artificial Sequence <400> 86 cttcacgttt aaaaaatacc ttgttattca ct 32 <210> 87 <211> 22 <212> DNA <213> Artificial Sequence <400> 87 cttacatcat ggccagtctg ca 22 <210> 88 <211> 22 <212> DNA <213> Artificial Sequence <400> 88 aggaaagcaa ctgaagggtg tc 22 <210> 89 <211> 22 <212> DNA <213> Artificial Sequence <400> 89 ttctgctaag tccagtggac ac 22 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <400> 90 catgccagtg ggaacctctt 20 <210> 91 <211> 22 <212> DNA <213> Artificial Sequence <400> 91 actgacttcc tttgctgatg ca 22 <210> 92 <211> 22 <212> DNA <213> Artificial Sequence <400> 92 tgctctttag ccactgtaac cg 22 <210> 93 <211> 23 <212> DNA <213> Artificial Sequence <400> 93 agacaaactc ttgggtaggc atg 23 <210> 94 <211> 22 <212> DNA <213> Artificial Sequence <400> 94 tgagaggctt tgttgactgg ac 22 <210> 95 <211> 24 <212> DNA <213> Artificial Sequence <400> 95 gaaagttctg acaatgccgt catg 24 <210> 96 <211> 25 <212> DNA <213> Artificial Sequence <400> 96 ccaattattg ttctttgctg acccc 25 <210> 97 <211> 23 <212> DNA <213> Artificial Sequence <400> 97 gaacttgtga gctctcttcc tct 23 <210> 98 <211> 23 <212> DNA <213> Artificial Sequence <400> 98 tcaggccatt ccaaaatgtg aag 23 <210> 99 <211> 24 <212> DNA <213> Artificial Sequence <400> 99 cccaaggaaa ttcaagttgt gtgt 24 <210> 100 <211> 23 <212> DNA <213> Artificial Sequence <400> 100 cagacgggca gagtaacaac taa 23 <210> 101 <211> 23 <212> DNA <213> Artificial Sequence <400> 101 acacagtatg cttgcttctc tga 23 <210> 102 <211> 22 <212> DNA <213> Artificial Sequence <400> 102 ttgggccctg ttcttttatg gt 22 <210> 103 <211> 22 <212> DNA <213> Artificial Sequence <400> 103 cctaatctca tcaagcccag ca 22 <210> 104 <211> 22 <212> DNA <213> Artificial Sequence <400> 104 aaccgaggaa gagtaacgtg tg 22 <210> 105 <211> 23 <212> DNA <213> Artificial Sequence <400> 105 gcagtccttg ataagcaacc tct 23 <210> 106 <211> 23 <212> DNA <213> Artificial Sequence <400> 106 ccaaaagaca tttgtgctga gcc 23 <210> 107 <211> 28 <212> DNA <213> Artificial Sequence <400> 107 cagtgcttat gactaacaag acaagatg 28 <210> 108 <211> 25 <212> DNA <213> Artificial Sequence <400> 108 gactgcggtc agttaatgtt ttctc 25 <210> 109 <211> 28 <212> DNA <213> Artificial Sequence <400> 109 cagagtacat agagtgtttt agggagag 28 <210> 110 <211> 25 <212> DNA <213> Artificial Sequence <400> 110 gacagacatc aatgaatgtg agctg 25 <210> 111 <211> 26 <212> DNA <213> Artificial Sequence <400> 111 gaccacaagt aaatggtgtg aaagtc 26 <210> 112 <211> 22 <212> DNA <213> Artificial Sequence <400> 112 gtcaagatgg acacccagca at 22 <210> 113 <211> 22 <212> DNA <213> Artificial Sequence <400> 113 tttaaaggac gtcccctctc ct 22 <210> 114 <211> 22 <212> DNA <213> Artificial Sequence <400> 114 acctcctgac tgcttgctca ta 22 <210> 115 <211> 22 <212> DNA <213> Artificial Sequence <400> 115 cactcctcgc attcctcagt ac 22 <210> 116 <211> 22 <212> DNA <213> Artificial Sequence <400> 116 ggaagccgtg tggctctatt ta 22 <210> 117 <211> 22 <212> DNA <213> Artificial Sequence <400> 117 cacttgaatg accccctagt gt 22 <210> 118 <211> 22 <212> DNA <213> Artificial Sequence <400> 118 gttctggttg ctattcaggc ac 22 <210> 119 <211> 24 <212> DNA <213> Artificial Sequence <400> 119 cattggagtg gtataggaac caca 24 <210> 120 <211> 29 <212> DNA <213> Artificial Sequence <400> 120 tgctattttt gtctataatt ccaaggtgt 29 <210> 121 <211> 27 <212> DNA <213> Artificial Sequence <400> 121 ggcattccaa aagatagcaa agtacac 27 <210> 122 <211> 26 <212> DNA <213> Artificial Sequence <400> 122 gcaaagtaga tacaggcaaa gtttgg 26 <210> 123 <211> 22 <212> DNA <213> Artificial Sequence <400> 123 catcccagat acatggcaca gt 22 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <400> 124 aagggcagga tctacctgtt ct 22 <210> 125 <211> 26 <212> DNA <213> Artificial Sequence <400> 125 cctctctcat aaggttagcc atgatg 26 <210> 126 <211> 24 <212> DNA <213> Artificial Sequence <400> 126 tcctatcttc cccattttca aggg 24 <210> 127 <211> 22 <212> DNA <213> Artificial Sequence <400> 127 actgcaatgg aaggagagga ct 22 <210> 128 <211> 22 <212> DNA <213> Artificial Sequence <400> 128 tgagtttgca aatggaggga gg 22 <210> 129 <211> 24 <212> DNA <213> Artificial Sequence <400> 129 acagaattac aacagaccct tggt 24 <210> 130 <211> 30 <212> DNA <213> Artificial Sequence <400> 130 tgtcagatta agtactgatg aaagatacca 30 <210> 131 <211> 21 <212> DNA <213> Artificial Sequence <400> 131 gctgggatgg gatattctgc a 21 <210> 132 <211> 20 <212> DNA <213> Artificial Sequence <400> 132 tcagcgatgt gtgtgtgtgt 20 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <400> 133 actacacccc ccaactgcaa 20 <210> 134 <211> 30 <212> DNA <213> Artificial Sequence <400> 134 tggtaacata attgtggaca aattatcaca 30 <210> 135 <211> 23 <212> DNA <213> Artificial Sequence <400> 135 ccgtgcggat atttggaatg aag 23 <210> 136 <211> 22 <212> DNA <213> Artificial Sequence <400> 136 ttcctctgca tgatggttcc tg 22 <210> 137 <211> 25 <212> DNA <213> Artificial Sequence <400> 137 cccaggtaat cgaagaaaat ccatc 25 <210> 138 <211> 23 <212> DNA <213> Artificial Sequence <400> 138 ctgtgagctg ttgcaatcta tgc 23 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <400> 139 ccccatgcaa ccaacacaac 20 <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <400> 140 gtctgccagg attcatcttg ct 22 <210> 141 <211> 20 <212> DNA <213> Artificial Sequence <400> 141 gttttcttgc cgtggtgcaa 20 <210> 142 <211> 25 <212> DNA <213> Artificial Sequence <400> 142 atcccaacca tgaagatttc tttgc 25 <210> 143 <211> 21 <212> DNA <213> Artificial Sequence <400> 143 cagtgctttt ctctggcctg a 21 <210> 144 <211> 21 <212> DNA <213> Artificial Sequence <400> 144 cctaaccacc cctccaactc t 21 <210> 145 <211> 19 <212> DNA <213> Artificial Sequence <400> 145 ggttcggaac tccacaccc 19 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <400> 146 ctcatggcct tcactctcct c 21 <210> 147 <211> 22 <212> DNA <213> Artificial Sequence <400> 147 ataaaatggg ggtcgaggat gc 22 <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <400> 148 gtactttcaa gggtggctga gt 22 <210> 149 <211> 20 <212> DNA <213> Artificial Sequence <400> 149 catgccccaa gctcctagtg 20 <210> 150 <211> 19 <212> DNA <213> Artificial Sequence <400> 150 tgagccaaag gacctccca 19 <210> 151 <211> 23 <212> DNA <213> Artificial Sequence <400> 151 cgaaacatgg acgagaaaaa cca 23 <210> 152 <211> 24 <212> DNA <213> Artificial Sequence <400> 152 gttgtagttg ttagccgagg agaa 24 <210> 153 <211> 18 <212> DNA <213> Artificial Sequence <400> 153 agcgtccatg gccagagt 18 <210> 154 <211> 18 <212> DNA <213> Artificial Sequence <400> 154 ttgccagcac cgtggaag 18 <210> 155 <211> 23 <212> DNA <213> Artificial Sequence <400> 155 gttgagagga cccatgaagg aag 23 <210> 156 <211> 17 <212> DNA <213> Artificial Sequence <400> 156 ccttcctcgg ggcaggt 17 <210> 157 <211> 15 <212> DNA <213> Artificial Sequence <400> 157 accaccgcgc cacct 15 <210> 158 <211> 21 <212> DNA <213> Artificial Sequence <400> 158 ctgttatccc tcccctctgc t 21 <210> 159 <211> 18 <212> DNA <213> Artificial Sequence <400> 159 gtgggattga tgggcccc 18 <210> 160 <211> 18 <212> DNA <213> Artificial Sequence <400> 160 tctgctgggt ctctccct 18 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <400> 161 ggcaacagag caagacctca 20 <210> 162 <211> 27 <212> DNA <213> Artificial Sequence <400> 162 tcctaaagct atctctttct ctttccc 27 <210> 163 <211> 16 <212> DNA <213> Artificial Sequence <400> 163 gggctgcccc tgtgac 16 <210> 164 <211> 16 <212> DNA <213> Artificial Sequence <400> 164 ctccccacca cctgcg 16 <210> 165 <211> 21 <212> DNA <213> Artificial Sequence <400> 165 cccaaatgtg aaggctccag a 21 <210> 166 <211> 19 <212> DNA <213> Artificial Sequence <400> 166 agcagggtgt ggcttcatg 19 <210> 167 <211> 22 <212> DNA <213> Artificial Sequence <400> 167 gtaagaacgg tcccaccaag ag 22 <210> 168 <211> 22 <212> DNA <213> Artificial Sequence <400> 168 caacggtaag tcccaaggtc tg 22 <210> 169 <211> 17 <212> DNA <213> Artificial Sequence <400> 169 tgggagcccc agggtct 17 <210> 170 <211> 19 <212> DNA <213> Artificial Sequence <400> 170 agaggctgat gccaccctg 19 <210> 171 <211> 20 <212> DNA <213> Artificial Sequence <400> 171 gggtcccctg agacagagtc 20 <210> 172 <211> 24 <212> DNA <213> Artificial Sequence <400> 172 ctgattcaag ccctacttgt ctcc 24 <210> 173 <211> 22 <212> DNA <213> Artificial Sequence <400> 173 aagcccatct cagacaacca ag 22 <210> 174 <211> 22 <212> DNA <213> Artificial Sequence <400> 174 tggagcccat tgagaataac cg 22 <210> 175 <211> 22 <212> DNA <213> Artificial Sequence <400> 175 ccaaccccaa agtcattggt ca 22 <210> 176 <211> 22 <212> DNA <213> Artificial Sequence <400> 176 taggctccaa aactccaggg ta 22 <210> 177 <211> 22 <212> DNA <213> Artificial Sequence <400> 177 atcccttaac cccggattct ga 22 <210> 178 <211> 22 <212> DNA <213> Artificial Sequence <400> 178 gtgaattggg tgtgacaggg at 22 <210> 179 <211> 22 <212> DNA <213> Artificial Sequence <400> 179 gcaggagatg acaccaaagc tt 22 <210> 180 <211> 22 <212> DNA <213> Artificial Sequence <400> 180 ctgtgggtta agggcagagt tt 22 <210> 181 <211> 22 <212> DNA <213> Artificial Sequence <400> 181 ggttggctaa atcatggcct ct 22 <210> 182 <211> 22 <212> DNA <213> Artificial Sequence <400> 182 tcaccatgga acgtgtgcat ag 22 <210> 183 <211> 21 <212> DNA <213> Artificial Sequence <400> 183 gaactgcaga caagcaggac a 21 <210> 184 <211> 21 <212> DNA <213> Artificial Sequence <400> 184 aggtctggaa gtaagctggg t 21 <210> 185 <211> 22 <212> DNA <213> Artificial Sequence <400> 185 gtgaccttct tgccattctc ca 22 <210> 186 <211> 24 <212> DNA <213> Artificial Sequence <400> 186 ctgttacctt attccacagg ggac 24 <210> 187 <211> 22 <212> DNA <213> Artificial Sequence <400> 187 ccaagcctcc agattttgca ag 22 <210> 188 <211> 20 <212> DNA <213> Artificial Sequence <400> 188 aaccgggctc cttcttgttt 20 <210> 189 <211> 21 <212> DNA <213> Artificial Sequence <400> 189 agagttggag gggtggttag g 21 <210> 190 <211> 21 <212> DNA <213> Artificial Sequence <400> 190 gtgacacacc agctacaagg g 21 <210> 191 <211> 20 <212> DNA <213> Artificial Sequence <400> 191 agagacagta ggcagcgtga 20 <210> 192 <211> 22 <212> DNA <213> Artificial Sequence <400> 192 ccaaggaggg ggcatctttt aa 22 <210> 193 <211> 23 <212> DNA <213> Artificial Sequence <400> 193 gaaatctggg aatgcacaga ctg 23 <210> 194 <211> 22 <212> DNA <213> Artificial Sequence <400> 194 aaggacatct gagcttgtcc tc 22 <210> 195 <211> 15 <212> DNA <213> Artificial Sequence <400> 195 tggcctcccc ggcag 15 <210> 196 <211> 15 <212> DNA <213> Artificial Sequence <400> 196 gctgcccgct ctgcc 15 <210> 197 <211> 22 <212> DNA <213> Artificial Sequence <400> 197 cctctcatcc gcgtatttgg aa 22 <210> 198 <211> 22 <212> DNA <213> Artificial Sequence <400> 198 aacctacact gcttacacct gc 22 <210> 199 <211> 22 <212> DNA <213> Artificial Sequence <400> 199 tcgatctcct tctggaacct ct 22 <210> 200 <211> 22 <212> DNA <213> Artificial Sequence <400> 200 agacatggtc ttcgcttctc ag 22 <210> 201 <211> 22 <212> DNA <213> Artificial Sequence <400> 201 cctgcaaaag ggatgcaaag ag 22 <210> 202 <211> 22 <212> DNA <213> Artificial Sequence <400> 202 aggatcccct tctagaggct tt 22 <210> 203 <211> 22 <212> DNA <213> Artificial Sequence <400> 203 ctttatgcag accctgacct gt 22 <210> 204 <211> 21 <212> DNA <213> Artificial Sequence <400> 204 ccattcactg tgttcctccc a 21 <210> 205 <211> 22 <212> DNA <213> Artificial Sequence <400> 205 gggatacatg gacacacagc aa 22 <210> 206 <211> 22 <212> DNA <213> Artificial Sequence <400> 206 cttcctgaag agcaccttgg tt 22 <210> 207 <211> 22 <212> DNA <213> Artificial Sequence <400> 207 gcctcaagtg atccacatac ct 22 <210> 208 <211> 24 <212> DNA <213> Artificial Sequence <400> 208 ccaaagctgt tactcttttc cctc 24 <210> 209 <211> 20 <212> DNA <213> Artificial Sequence <400> 209 caagtcctgt tccccagcaa 20 <210> 210 <211> 22 <212> DNA <213> Artificial Sequence <400> 210 tgacaacata tccctttgcc ac 22 <210> 211 <211> 22 <212> DNA <213> Artificial Sequence <400> 211 cgcgtttgag gtattaggat gc 22 <210> 212 <211> 22 <212> DNA <213> Artificial Sequence <400> 212 agtggaaatc aggagctctt cc 22 <210> 213 <211> 21 <212> DNA <213> Artificial Sequence <400> 213 ggtacctcca gcatccattc c 21 <210> 214 <211> 21 <212> DNA <213> Artificial Sequence <400> 214 gatccactgc cctctttgac c 21 <210> 215 <211> 22 <212> DNA <213> Artificial Sequence <400> 215 tccagctcaa agcagaacat gg 22 <210> 216 <211> 24 <212> DNA <213> Artificial Sequence <400> 216 cagaagatct ggaaggacaa tggg 24 <210> 217 <211> 22 <212> DNA <213> Artificial Sequence <400> 217 acacacgtgt acaaggtgtg ac 22 <210> 218 <211> 16 <212> DNA <213> Artificial Sequence <400> 218 gatgctcacg ccccgc 16 <210> 219 <211> 22 <212> DNA <213> Artificial Sequence <400> 219 gggtgcatgg atggataagt ga 22 <210> 220 <211> 22 <212> DNA <213> Artificial Sequence <400> 220 gagaacccgc tgtattcaca ga 22 <210> 221 <211> 22 <212> DNA <213> Artificial Sequence <400> 221 ctaccctcca gactcaaggt gt 22 <210> 222 <211> 22 <212> DNA <213> Artificial Sequence <400> 222 tgaccagatc tcggtctcat gt 22 <210> 223 <211> 23 <212> DNA <213> Artificial Sequence <400> 223 ggaaagatag aggtggctct tgg 23 <210> 224 <211> 21 <212> DNA <213> Artificial Sequence <400> 224 catggggtta acggctgaag t 21 <210> 225 <211> 22 <212> DNA <213> Artificial Sequence <400> 225 accccatttc taccaccagc ta 22 <210> 226 <211> 22 <212> DNA <213> Artificial Sequence <400> 226 ctgtttgggg ggatcaaagt ca 22 <210> 227 <211> 22 <212> DNA <213> Artificial Sequence <400> 227 aagagaggca cttggaacca tg 22 <210> 228 <211> 22 <212> DNA <213> Artificial Sequence <400> 228 agacccttag ctcagagctc at 22 <210> 229 <211> 22 <212> DNA <213> Artificial Sequence <400> 229 tccttatgag gccaggagta gg 22 <210> 230 <211> 20 <212> DNA <213> Artificial Sequence <400> 230 cttcgaggca gccagcatta 20 <210> 231 <211> 32 <212> DNA <213> Artificial Sequence <400> 231 ccttttaata tgacttagaa actgcttttc tc 32 <210> 232 <211> 23 <212> DNA <213> Artificial Sequence <400> 232 gatgaccagt caatcaggga gtc 23 <210> 233 <211> 22 <212> DNA <213> Artificial Sequence <400> 233 atccagccac accctattca tg 22 <210> 234 <211> 23 <212> DNA <213> Artificial Sequence <400> 234 caacgagtga agaaatgacc tgg 23 <210> 235 <211> 23 <212> DNA <213> Artificial Sequence <400> 235 ctgggaatga aagtggcttg aac 23 <210> 236 <211> 23 <212> DNA <213> Artificial Sequence <400> 236 aggtctcctc ctgctcaaaa atc 23 <210> 237 <211> 19 <212> DNA <213> Artificial Sequence <400> 237 tggtccacca gtccaccaa 19 <210> 238 <211> 19 <212> DNA <213> Artificial Sequence <400> 238 acacacagat tcgggcagc 19 <210> 239 <211> 22 <212> DNA <213> Artificial Sequence <400> 239 tgcttgtgga tgtactccac tc 22 <210> 240 <211> 22 <212> DNA <213> Artificial Sequence <400> 240 ggattcagga attgcccatc ac 22 <210> 241 <211> 23 <212> DNA <213> Artificial Sequence <400> 241 agctgaggca ctgaatctaa ctg 23 <210> 242 <211> 21 <212> DNA <213> Artificial Sequence <400> 242 tctgagactc acaccaaccc t 21 <210> 243 <211> 22 <212> DNA <213> Artificial Sequence <400> 243 cgcacatttg tttcaagcca ct 22 <210> 244 <211> 23 <212> DNA <213> Artificial Sequence <400> 244 acagtcctac agcatcgaga tct 23 <210> 245 <211> 18 <212> DNA <213> Artificial Sequence <400> 245 ttgccccaag ctccatgc 18 <210> 246 <211> 20 <212> DNA <213> Artificial Sequence <400> 246 ttcctttcct agcccagggg 20 <210> 247 <211> 22 <212> DNA <213> Artificial Sequence <400> 247 atacccaggt gtcagtctag ca 22 <210> 248 <211> 22 <212> DNA <213> Artificial Sequence <400> 248 agtggacaag actgcatctt cc 22 <210> 249 <211> 21 <212> DNA <213> Artificial Sequence <400> 249 cagtgttgga ggcagttctg t 21 <210> 250 <211> 21 <212> DNA <213> Artificial Sequence <400> 250 cacacctggt tcactccaca a 21 <210> 251 <211> 22 <212> DNA <213> Artificial Sequence <400> 251 tcttgcagct tctgcttgaa gg 22 <210> 252 <211> 19 <212> DNA <213> Artificial Sequence <400> 252 tcagggccct tgaaacccg 19 <210> 253 <211> 20 <212> DNA <213> Artificial Sequence <400> 253 aaaatccggg gtggcaggtt 20 <210> 254 <211> 23 <212> DNA <213> Artificial Sequence <400> 254 tcctggggaa gaaggtgagt aca 23 <210> 255 <211> 20 <212> DNA <213> Artificial Sequence <400> 255 acccatagcg gtcactacca 20 <210> 256 <211> 22 <212> DNA <213> Artificial Sequence <400> 256 ggatttgctc tcccctgaag tt 22 <210> 257 <211> 20 <212> DNA <213> Artificial Sequence <400> 257 ttaagggagg ctggctggac 20 <210> 258 <211> 23 <212> DNA <213> Artificial Sequence <400> 258 gttcatcagt aagcctcgct cag 23 <210> 259 <211> 19 <212> DNA <213> Artificial Sequence <400> 259 actcgctctg agtgggtca 19 <210> 260 <211> 19 <212> DNA <213> Artificial Sequence <400> 260 aaccaatccc ccttcccct 19 <210> 261 <211> 22 <212> DNA <213> Artificial Sequence <400> 261 ctacaaatgt gcccttcctc ca 22 <210> 262 <211> 20 <212> DNA <213> Artificial Sequence <400> 262 ctgagccttc ctgtgcttcc 20 <210> 263 <211> 22 <212> DNA <213> Artificial Sequence <400> 263 ccatgaatgg ttgtcccaca ga 22 <210> 264 <211> 20 <212> DNA <213> Artificial Sequence <400> 264 ttccgggatt ccaaagcctg 20 <210> 265 <211> 23 <212> DNA <213> Artificial Sequence <400> 265 gcaatcggtg accctaatta cct 23 <210> 266 <211> 23 <212> DNA <213> Artificial Sequence <400> 266 tcaccttctg gagaagagag gaa 23 <210> 267 <211> 21 <212> DNA <213> Artificial Sequence <400> 267 cagggatcgt tctgcactag c 21 <210> 268 <211> 21 <212> DNA <213> Artificial Sequence <400> 268 tgaccaagtt accccattgc c 21 <210> 269 <211> 22 <212> DNA <213> Artificial Sequence <400> 269 caagtcagca ggaaagcaat cc 22 <210> 270 <211> 22 <212> DNA <213> Artificial Sequence <400> 270 gatcaccatc actatcccac cc 22 <210> 271 <211> 16 <212> DNA <213> Artificial Sequence <400> 271 tcgggcctcc tgggac 16 <210> 272 <211> 26 <212> DNA <213> Artificial Sequence <400> 272 aggctcttac cctttcttct ttttct 26 <210> 273 <211> 22 <212> DNA <213> Artificial Sequence <400> 273 ggcttctcca agactgttga ca 22 <210> 274 <211> 23 <212> DNA <213> Artificial Sequence <400> 274 caacttgtgt tctctccctg tga 23 <210> 275 <211> 22 <212> DNA <213> Artificial Sequence <400> 275 cctcaaaatg cccctttgtt cc 22 <210> 276 <211> 22 <212> DNA <213> Artificial Sequence <400> 276 tgagtccaac cctcgtgtct at 22 <210> 277 <211> 22 <212> DNA <213> Artificial Sequence <400> 277 agggagtagg gaagcaaaga ct 22 <210> 278 <211> 19 <212> DNA <213> Artificial Sequence <400> 278 tagccacacc tgctgtcct 19 <210> 279 <211> 20 <212> DNA <213> Artificial Sequence <400> 279 acactggtgt ccatgggaga 20 <210> 280 <211> 20 <212> DNA <213> Artificial Sequence <400> 280 tcaggagggg agctgtttga 20 <210> 281 <211> 16 <212> DNA <213> Artificial Sequence <400> 281 acgctgctgg cagtgg 16 <210> 282 <211> 22 <212> DNA <213> Artificial Sequence <400> 282 gcttttctct cctccccctt tc 22 <210> 283 <211> 22 <212> DNA <213> Artificial Sequence <400> 283 tgtcactgtc cggtaatcaa cc 22 <210> 284 <211> 22 <212> DNA <213> Artificial Sequence <400> 284 tgtcactgtc cggtaatcaa cc 22 <210> 285 <211> 22 <212> DNA <213> Artificial Sequence <400> 285 agttccttcc acgtcttgtt gg 22 <210> 286 <211> 22 <212> DNA <213> Artificial Sequence <400> 286 cttcctttgc ctgcaattct gg 22 <210> 287 <211> 22 <212> DNA <213> Artificial Sequence <400> 287 gctgtacgga ttattcccag ca 22 <210> 288 <211> 22 <212> DNA <213> Artificial Sequence <400> 288 gacaatggcg tcgaatgtga tc 22 <210> 289 <211> 22 <212> DNA <213> Artificial Sequence <400> 289 ggattcaggc aagagtgagt ga 22 <210> 290 <211> 22 <212> DNA <213> Artificial Sequence <400> 290 ctcatttcct cagcagctgc ta 22 <210> 291 <211> 18 <212> DNA <213> Artificial Sequence <400> 291 aggccccttc ttccccaa 18 <210> 292 <211> 19 <212> DNA <213> Artificial Sequence <400> 292 actgcaagag aggccatgc 19 <210> 293 <211> 20 <212> DNA <213> Artificial Sequence <400> 293 ttcagggtct cagcaggctt 20 <210> 294 <211> 22 <212> DNA <213> Artificial Sequence <400> 294 ccaagaaggg gacttccaag ac 22 <210> 295 <211> 22 <212> DNA <213> Artificial Sequence <400> 295 cttcaggtcg tcttccgata gg 22 <210> 296 <211> 20 <212> DNA <213> Artificial Sequence <400> 296 acctctgtgg tggaggagtt 20 <210> 297 <211> 23 <212> DNA <213> Artificial Sequence <400> 297 ctgcaggaca caaacaatct agg 23 <210> 298 <211> 23 <212> DNA <213> Artificial Sequence <400> 298 ctgtgatcaa gtccacactg aac 23 <210> 299 <211> 22 <212> DNA <213> Artificial Sequence <400> 299 agggctgcac cttctttaga ac 22 <210> 300 <211> 20 <212> DNA <213> Artificial Sequence <400> 300 agctctgccc tttgccaatt 20 <210> 301 <211> 22 <212> DNA <213> Artificial Sequence <400> 301 gagagcaata cccattgcaa cc 22 <210> 302 <211> 22 <212> DNA <213> Artificial Sequence <400> 302 catgaatgtt caccccacgt tc 22 <210> 303 <211> 22 <212> DNA <213> Artificial Sequence <400> 303 tggctcagag ggataagtga ga 22 <210> 304 <211> 22 <212> DNA <213> Artificial Sequence <400> 304 gatctgagct gacctgtcct tt 22 <210> 305 <211> 22 <212> DNA <213> Artificial Sequence <400> 305 tttcagcagg cagaggtaat gg 22 <210> 306 <211> 22 <212> DNA <213> Artificial Sequence <400> 306 acctacctca caatcacagg gt 22 <210> 307 <211> 20 <212> DNA <213> Artificial Sequence <400> 307 acaacatctt ggctccccag 20 <210> 308 <211> 18 <212> DNA <213> Artificial Sequence <400> 308 ctgcaaacag ccagcctc 18 <210> 309 <211> 22 <212> DNA <213> Artificial Sequence <400> 309 agggcatttc tcggtaacat cc 22 <210> 310 <211> 20 <212> DNA <213> Artificial Sequence <400> 310 aaggccaaag acgttcagca 20 <210> 311 <211> 26 <212> DNA <213> Artificial Sequence <400> 311 gagaacagac taatacaggc tggatc 26 <210> 312 <211> 24 <212> DNA <213> Artificial Sequence <400> 312 gactggattc tctgccaatt tcct 24 <210> 313 <211> 18 <212> DNA <213> Artificial Sequence <400> 313 cctccccatc cagccaca 18 <210> 314 <211> 20 <212> DNA <213> Artificial Sequence <400> 314 agacagggct gtgctttgtc 20 <210> 315 <211> 21 <212> DNA <213> Artificial Sequence <400> 315 gcatgactcc agcttggact c 21 <210> 316 <211> 26 <212> DNA <213> Artificial Sequence <400> 316 gatcattcta ttctcccctc tcctca 26 <210> 317 <211> 22 <212> DNA <213> Artificial Sequence <400> 317 ggaggagaaa tggtgcgaga ag 22 <210> 318 <211> 22 <212> DNA <213> Artificial Sequence <400> 318 tctctccctc ttcccatctc ca 22 <210> 319 <211> 20 <212> DNA <213> Artificial Sequence <400> 319 cctctctttc tgcccctccc 20 <210> 320 <211> 18 <212> DNA <213> Artificial Sequence <400> 320 tgctgtcccc acaggcag 18 <210> 321 <211> 22 <212> DNA <213> Artificial Sequence <400> 321 catggtgtgc atgtgtgatg tc 22 <210> 322 <211> 22 <212> DNA <213> Artificial Sequence <400> 322 aggaagggat ggaagggatg aa 22 <210> 323 <211> 19 <212> DNA <213> Artificial Sequence <400> 323 agtagcccac cctgtgtcc 19 <210> 324 <211> 17 <212> DNA <213> Artificial Sequence <400> 324 gcggggaatg gagccac 17 <210> 325 <211> 22 <212> DNA <213> Artificial Sequence <400> 325 ggtccaggac ttgctttatc ca 22 <210> 326 <211> 22 <212> DNA <213> Artificial Sequence <400> 326 ctgatgtagg cagcacccat aa 22 <210> 327 <211> 23 <212> DNA <213> Artificial Sequence <400> 327 tctaggaagg gctgtattgt cct 23 <210> 328 <211> 24 <212> DNA <213> Artificial Sequence <400> 328 gagaaactct gacagatctc tggc 24 <210> 329 <211> 19 <212> DNA <213> Artificial Sequence <400> 329 actgagccac ctctgctct 19 <210> 330 <211> 19 <212> DNA <213> Artificial Sequence <400> 330 agagattggg gccacagga 19 <210> 331 <211> 22 <212> DNA <213> Artificial Sequence <400> 331 tctttctgct gtgttgggct ac 22 <210> 332 <211> 22 <212> DNA <213> Artificial Sequence <400> 332 ttccagttgt gtgcacaagg ag 22 <210> 333 <211> 24 <212> DNA <213> Artificial Sequence <400> 333 cctgtccagt ctaacctgaa tctc 24 <210> 334 <211> 22 <212> DNA <213> Artificial Sequence <400> 334 tgagaaaggt ggactctctc ca 22 <210> 335 <211> 21 <212> DNA <213> Artificial Sequence <400> 335 acttgtgtaa ccccctggag a 21 <210> 336 <211> 22 <212> DNA <213> Artificial Sequence <400> 336 tccaattttc tccacctcct gc 22 <210> 337 <211> 15 <212> DNA <213> Artificial Sequence <400> 337 ccgtcgagcc cagcc 15 <210> 338 <211> 20 <212> DNA <213> Artificial Sequence <400> 338 ggatggtgat gcacttggtg 20 <210> 339 <211> 19 <212> DNA <213> Artificial Sequence <400> 339 cgaggtttgc tggggtgag 19 <210> 340 <211> 20 <212> DNA <213> Artificial Sequence <400> 340 gagaaagagc aggagcgagc 20 <210> 341 <211> 23 <212> DNA <213> Artificial Sequence <400> 341 tcgagatagg ccgtttgtat gtg 23 <210> 342 <211> 22 <212> DNA <213> Artificial Sequence <400> 342 tggacaagtc acttcttgcc tc 22 <210> 343 <211> 24 <212> DNA <213> Artificial Sequence <400> 343 tgatggtcta tatctgccac aacc 24 <210> 344 <211> 25 <212> DNA <213> Artificial Sequence <400> 344 tctcacattc tagcaagttg gctta 25 <210> 345 <211> 24 <212> DNA <213> Artificial Sequence <400> 345 ctctagagaa gaacgttcgt ggtt 24 <210> 346 <211> 27 <212> DNA <213> Artificial Sequence <400> 346 atgggtctaa tctacatgag agacatc 27 <210> 347 <211> 29 <212> DNA <213> Artificial Sequence <400> 347 tgattggtat taccttttaa gcagtcatg 29 <210> 348 <211> 32 <212> DNA <213> Artificial Sequence <400> 348 aagggaaaaa aggtgatttc agaagatatt aa 32 <210> 349 <211> 26 <212> DNA <213> Artificial Sequence <400> 349 gatcttttaa tgccttggca ttagct 26 <210> 350 <211> 26 <212> DNA <213> Artificial Sequence <400> 350 tgcttactaa gcagaagcag tttaga 26 <210> 351 <211> 32 <212> DNA <213> Artificial Sequence <400> 351 aaccttgaga ttttttctaa gaatctttct ct 32 <210> 352 <211> 23 <212> DNA <213> Artificial Sequence <400> 352 tggtcctgat tgcagcaata tgt 23 <210> 353 <211> 30 <212> DNA <213> Artificial Sequence <400> 353 gtttttgtcg ttgttgatgt ttatttcact 30 <210> 354 <211> 34 <212> DNA <213> Artificial Sequence <400> 354 gttgtcatat cataaattaa gtctttcaac gtag 34 <210> 355 <211> 31 <212> DNA <213> Artificial Sequence <400> 355 gaaacatgta atattgttga ttgtgttgag t 31 <210> 356 <211> 28 <212> DNA <213> Artificial Sequence <400> 356 aggatgtttt catgacgtaa cattacag 28 <210> 357 <211> 24 <212> DNA <213> Artificial Sequence <400> 357 gcccaaccga aatgttaatt ctgt 24 <210> 358 <211> 33 <212> DNA <213> Artificial Sequence <400> 358 tgaaaattta aagcttaaat aatagaactg ctt 33 <210> 359 <211> 22 <212> DNA <213> Artificial Sequence <400> 359 tgtctgaaag gaggttcatc ca 22 <210> 360 <211> 22 <212> DNA <213> Artificial Sequence <400> 360 ctcatgacaa actactgggg ga 22 <210> 361 <211> 26 <212> DNA <213> Artificial Sequence <400> 361 tcttatccag accaatggaa aatggt 26 <210> 362 <211> 26 <212> DNA <213> Artificial Sequence <400> 362 agcagatctg aagaaaaaag gagagt 26 <210> 363 <211> 34 <212> DNA <213> Artificial Sequence <400> 363 tggcagttgg ataatcattt aatatatctt tctc 34 <210> 364 <211> 31 <212> DNA <213> Artificial Sequence <400> 364 tgtaaaaagg ggaaaagaaa gaataacttc t 31 <210> 365 <211> 22 <212> DNA <213> Artificial Sequence <400> 365 acctaccaca tccaactcct tc 22 <210> 366 <211> 21 <212> DNA <213> Artificial Sequence <400> 366 cttccaggat gatggcacag t 21 <210> 367 <211> 22 <212> DNA <213> Artificial Sequence <400> 367 gctgaggtct ataaggccaa gc 22 <210> 368 <211> 22 <212> DNA <213> Artificial Sequence <400> 368 gtactcctgt aggttgccct tg 22 <210> 369 <211> 22 <212> DNA <213> Artificial Sequence <400> 369 tcacctccac agtgatcaca ct 22 <210> 370 <211> 24 <212> DNA <213> Artificial Sequence <400> 370 ggtaaagggg atctagcact agct 24 <210> 371 <211> 22 <212> DNA <213> Artificial Sequence <400> 371 ctgcacatgc cattctcagt ga 22 <210> 372 <211> 22 <212> DNA <213> Artificial Sequence <400> 372 tctgccacct aagaggcaac tt 22 <210> 373 <211> 16 <212> DNA <213> Artificial Sequence <400> 373 gccaggggtc cgggaa 16 <210> 374 <211> 17 <212> DNA <213> Artificial Sequence <400> 374 ctgtcaagcg cagcgga 17 <210> 375 <211> 27 <212> DNA <213> Artificial Sequence <400> 375 tcaaaacagt ttcactttcc tgtcatc 27 <210> 376 <211> 27 <212> DNA <213> Artificial Sequence <400> 376 agtgagggag catgactaaa aatagaa 27 <210> 377 <211> 22 <212> DNA <213> Artificial Sequence <400> 377 ccctcgcttc caatgaatct ct 22 <210> 378 <211> 22 <212> DNA <213> Artificial Sequence <400> 378 aggtcccaca cccttaagag aa 22 <210> 379 <211> 22 <212> DNA <213> Artificial Sequence <400> 379 gaagctgagt tcaacctggg aa 22 <210> 380 <211> 22 <212> DNA <213> Artificial Sequence <400> 380 gatcttgact gccactgtct ca 22 <210> 381 <211> 23 <212> DNA <213> Artificial Sequence <400> 381 gggaaacaat actggctgat cac 23 <210> 382 <211> 22 <212> DNA <213> Artificial Sequence <400> 382 aggttaggtc gttcttcacg ag 22 <210> 383 <211> 22 <212> DNA <213> Artificial Sequence <400> 383 aaatgatggg cctcactgtc tg 22 <210> 384 <211> 23 <212> DNA <213> Artificial Sequence <400> 384 cactacacaa tgatgctggt cca 23 <210> 385 <211> 22 <212> DNA <213> Artificial Sequence <400> 385 ggtgcccttt ggatctcttt cc 22 <210> 386 <211> 19 <212> DNA <213> Artificial Sequence <400> 386 tgagaggggc agcctcttt 19 <210> 387 <211> 19 <212> DNA <213> Artificial Sequence <400> 387 acaaatgcca gtcggtgct 19 <210> 388 <211> 17 <212> DNA <213> Artificial Sequence <400> 388 agagccccca gcctctg 17 <210> 389 <211> 22 <212> DNA <213> Artificial Sequence <400> 389 cagaacgagc tggacaagta cc 22 <210> 390 <211> 22 <212> DNA <213> Artificial Sequence <400> 390 gacagacaat acagagaccc gc 22 <210> 391 <211> 22 <212> DNA <213> Artificial Sequence <400> 391 gagcctaaca ctcctcttca ca 22 <210> 392 <211> 23 <212> DNA <213> Artificial Sequence <400> 392 aaggcctagg gaaaagacat tga 23 <210> 393 <211> 22 <212> DNA <213> Artificial Sequence <400> 393 tgtggttcat gtgttcagca ct 22 <210> 394 <211> 23 <212> DNA <213> Artificial Sequence <400> 394 catcacacag ctgtgaaaga agc 23 <210> 395 <211> 23 <212> DNA <213> Artificial Sequence <400> 395 cctttgtcca ttgtgggtaa gca 23 <210> 396 <211> 23 <212> DNA <213> Artificial Sequence <400> 396 aagcctattg tgctaggagc tca 23 <210> 397 <211> 22 <212> DNA <213> Artificial Sequence <400> 397 tcactgacta ggttgccatt gc 22 <210> 398 <211> 22 <212> DNA <213> Artificial Sequence <400> 398 gtgttgggat gtgtcaagtc ca 22 <210> 399 <211> 24 <212> DNA <213> Artificial Sequence <400> 399 gcactcttac aagtctatgg gctt 24 <210> 400 <211> 24 <212> DNA <213> Artificial Sequence <400> 400 ggatttgtca cttggagttg ggta 24 <210> 401 <211> 33 <212> DNA <213> Artificial Sequence <400> 401 aattttttat acaattttca ttccatttct gca 33 <210> 402 <211> 33 <212> DNA <213> Artificial Sequence <400> 402 agcaaatgaa cacagtttta taaacaaata ttt 33 <210> 403 <211> 27 <212> DNA <213> Artificial Sequence <400> 403 aagtaatatc tcttgtcgtg tctctca 27 <210> 404 <211> 32 <212> DNA <213> Artificial Sequence <400> 404 ctgatggatt gtccctaaaa ataaaataat gg 32 <210> 405 <211> 23 <212> DNA <213> Artificial Sequence <400> 405 cctaactgct tcccattagc cat 23 <210> 406 <211> 27 <212> DNA <213> Artificial Sequence <400> 406 gatcattgtt gactgacata aacacca 27 <210> 407 <211> 20 <212> DNA <213> Artificial Sequence <400> 407 ccgcccgaga aaaagtttcg 20 <210> 408 <211> 18 <212> DNA <213> Artificial Sequence <400> 408 acctgtacgt ctgcggct 18 <210> 409 <211> 19 <212> DNA <213> Artificial Sequence <400> 409 atcggagagg ggaggaagc 19 <210> 410 <211> 17 <212> DNA <213> Artificial Sequence <400> 410 tctgcttctg cgcctgc 17 <210> 411 <211> 24 <212> DNA <213> Artificial Sequence <400> 411 gccagtaaat cttccctctc ttgc 24 <210> 412 <211> 27 <212> DNA <213> Artificial Sequence <400> 412 tgtgcagctg ataagatgaa aaaagaa 27 <210> 413 <211> 23 <212> DNA <213> Artificial Sequence <400> 413 ctcctttgca ggatgttgtt cac 23 <210> 414 <211> 21 <212> DNA <213> Artificial Sequence <400> 414 tcagggtgct gcatttcaga t 21 <210> 415 <211> 22 <212> DNA <213> Artificial Sequence <400> 415 gaagaggctg agctgtttgg ta 22 <210> 416 <211> 22 <212> DNA <213> Artificial Sequence <400> 416 actctgcatg tgttcctact gg 22 <210> 417 <211> 25 <212> DNA <213> Artificial Sequence <400> 417 cctggaggtg gataaatcag aagag 25 <210> 418 <211> 23 <212> DNA <213> Artificial Sequence <400> 418 ccaagaactc atgtgtgttg ctg 23 <210> 419 <211> 22 <212> DNA <213> Artificial Sequence <400> 419 aaccctgttc cacagtcatg tc 22 <210> 420 <211> 22 <212> DNA <213> Artificial Sequence <400> 420 aggaagcaga ggcgatcaaa ag 22 <210> 421 <211> 22 <212> DNA <213> Artificial Sequence <400> 421 aactataatc tgggcccccc aa 22 <210> 422 <211> 21 <212> DNA <213> Artificial Sequence <400> 422 agaccaacat gggcaactgt t 21 <210> 423 <211> 29 <212> DNA <213> Artificial Sequence <400> 423 aggagcttaa gtatcttgtt tttctctct 29 <210> 424 <211> 22 <212> DNA <213> Artificial Sequence <400> 424 gtgtagctgc atgctggtct at 22 <210> 425 <211> 26 <212> DNA <213> Artificial Sequence <400> 425 agggggattg acatgataga atttcc 26 <210> 426 <211> 23 <212> DNA <213> Artificial Sequence <400> 426 acgtgttcag aactctgcag aaa 23 <210> 427 <211> 25 <212> DNA <213> Artificial Sequence <400> 427 tgcacttaaa aattccagct tggaa 25 <210> 428 <211> 25 <212> DNA <213> Artificial Sequence <400> 428 ctgtgtctca gaaaaagctg tcttc 25 <210> 429 <211> 20 <212> DNA <213> Artificial Sequence <400> 429 ccgcccgaga aaaagtttcg 20 <210> 430 <211> 18 <212> DNA <213> Artificial Sequence <400> 430 acctgtacgt ctgcggct 18 <210> 431 <211> 19 <212> DNA <213> Artificial Sequence <400> 431 atcggagagg ggaggaagc 19 <210> 432 <211> 17 <212> DNA <213> Artificial Sequence <400> 432 tctgcttctg cgcctgc 17 <210> 433 <211> 24 <212> DNA <213> Artificial Sequence <400> 433 gccagtaaat cttccctctc ttgc 24 <210> 434 <211> 27 <212> DNA <213> Artificial Sequence <400> 434 tgtgcagctg ataagatgaa aaaagaa 27 <210> 435 <211> 23 <212> DNA <213> Artificial Sequence <400> 435 ctcctttgca ggatgttgtt cac 23 <210> 436 <211> 21 <212> DNA <213> Artificial Sequence <400> 436 tcagggtgct gcatttcaga t 21 <210> 437 <211> 22 <212> DNA <213> Artificial Sequence <400> 437 gaagaggctg agctgtttgg ta 22 <210> 438 <211> 22 <212> DNA <213> Artificial Sequence <400> 438 actctgcatg tgttcctact gg 22 <210> 439 <211> 25 <212> DNA <213> Artificial Sequence <400> 439 cctggaggtg gataaatcag aagag 25 <210> 440 <211> 23 <212> DNA <213> Artificial Sequence <400> 440 ccaagaactc atgtgtgttg ctg 23 <210> 441 <211> 22 <212> DNA <213> Artificial Sequence <400> 441 aaccctgttc cacagtcatg tc 22 <210> 442 <211> 22 <212> DNA <213> Artificial Sequence <400> 442 aggaagcaga ggcgatcaaa ag 22 <210> 443 <211> 22 <212> DNA <213> Artificial Sequence <400> 443 aactataatc tgggcccccc aa 22 <210> 444 <211> 21 <212> DNA <213> Artificial Sequence <400> 444 agaccaacat gggcaactgt t 21 <210> 445 <211> 29 <212> DNA <213> Artificial Sequence <400> 445 aggagcttaa gtatcttgtt tttctctct 29 <210> 446 <211> 22 <212> DNA <213> Artificial Sequence <400> 446 gtgtagctgc atgctggtct at 22 <210> 447 <211> 26 <212> DNA <213> Artificial Sequence <400> 447 agggggattg acatgataga atttcc 26 <210> 448 <211> 23 <212> DNA <213> Artificial Sequence <400> 448 acgtgttcag aactctgcag aaa 23 <210> 449 <211> 25 <212> DNA <213> Artificial Sequence <400> 449 tgcacttaaa aattccagct tggaa 25 <210> 450 <211> 25 <212> DNA <213> Artificial Sequence <400> 450 ctgtgtctca gaaaaagctg tcttc 25 <210> 451 <211> 23 <212> DNA <213> Artificial Sequence <400> 451 agctgctgta gtagcagaaa cag 23 <210> 452 <211> 29 <212> DNA <213> Artificial Sequence <400> 452 ggaaaaagaa atactgagga actctttgt 29 <210> 453 <211> 23 <212> DNA <213> Artificial Sequence <400> 453 gtttctctct gaggcttgag gtt 23 <210> 454 <211> 25 <212> DNA <213> Artificial Sequence <400> 454 ctctgtatgt aactgaccac catct 25 <210> 455 <211> 19 <212> DNA <213> Artificial Sequence <400> 455 cagtcagaac caggcacca 19 <210> 456 <211> 21 <212> DNA <213> Artificial Sequence <400> 456 acctagaggg gtagggagtt g 21 <210> 457 <211> 22 <212> DNA <213> Artificial Sequence <400> 457 ttgtaggggt tgtaagggtc gt 22 <210> 458 <211> 20 <212> DNA <213> Artificial Sequence <400> 458 atcctgctga tccgcgacaa 20 <210> 459 <211> 21 <212> DNA <213> Artificial Sequence <400> 459 cccgttgttc tcccattgga t 21 <210> 460 <211> 21 <212> DNA <213> Artificial Sequence <400> 460 ttccctcacg tgatttgagc c 21 <210> 461 <211> 23 <212> DNA <213> Artificial Sequence <400> 461 ccaggcactg atttatccat tgg 23 <210> 462 <211> 22 <212> DNA <213> Artificial Sequence <400> 462 catcaggcat ctctgctgtt ga 22 <210> 463 <211> 23 <212> DNA <213> Artificial Sequence <400> 463 gaagagggcc tattgatctg caa 23 <210> 464 <211> 32 <212> DNA <213> Artificial Sequence <400> 464 gcatattatt tatcatagtc tgaccacata cc 32 <210> 465 <211> 26 <212> DNA <213> Artificial Sequence <400> 465 gaaatgaaaa gcaacccaaa agtacc 26 <210> 466 <211> 32 <212> DNA <213> Artificial Sequence <400> 466 tggatgtttt atttgtagtt ttgtctttag tt 32 <210> 467 <211> 20 <212> DNA <213> Artificial Sequence <400> 467 acgtcgagaa gccacatagc 20 <210> 468 <211> 20 <212> DNA <213> Artificial Sequence <400> 468 gagaagttcc tgcgctcagt 20 <210> 469 <211> 20 <212> DNA <213> Artificial Sequence <400> 469 cagcggtgac tccagatgag 20 <210> 470 <211> 15 <212> DNA <213> Artificial Sequence <400> 470 acagcagccc ccgcg 15 <210> 471 <211> 23 <212> DNA <213> Artificial Sequence <400> 471 tcacctttca gcaaaaccta agg 23 <210> 472 <211> 19 <212> DNA <213> Artificial Sequence <400> 472 tcacagcagt tggaggctg 19 <210> 473 <211> 29 <212> DNA <213> Artificial Sequence <400> 473 ctgtgtcttg tttaacttgt ttcttttcc 29 <210> 474 <211> 26 <212> DNA <213> Artificial Sequence <400> 474 tctgttgttt ggtgccatta ttcaaa 26 <210> 475 <211> 24 <212> DNA <213> Artificial Sequence <400> 475 gatgttgcat gcacttctaa atgc 24 <210> 476 <211> 24 <212> DNA <213> Artificial Sequence <400> 476 actgaattcc tagaagagca acca 24 <210> 477 <211> 29 <212> DNA <213> Artificial Sequence <400> 477 cacaatcctg atttcttact gtcaagatg 29 <210> 478 <211> 29 <212> DNA <213> Artificial Sequence <400> 478 ggtaggttcc atatggaatt atcgactta 29 <210> 479 <211> 28 <212> DNA <213> Artificial Sequence <400> 479 tgagcaccta cgtattcttt atttctct 28 <210> 480 <211> 31 <212> DNA <213> Artificial Sequence <400> 480 tggacctttg ataccctaaa aataatgaat g 31 <210> 481 <211> 32 <212> DNA <213> Artificial Sequence <400> 481 gtgttatgaa gaccaattag aaaaatacca tg 32 <210> 482 <211> 29 <212> DNA <213> Artificial Sequence <400> 482 aggtaaattt cagaaaacaa gttaaggct 29 <210> 483 <211> 25 <212> DNA <213> Artificial Sequence <400> 483 cccaactgtt cttacacatg tcaag 25 <210> 484 <211> 25 <212> DNA <213> Artificial Sequence <400> 484 tgtcactttg ggttgtaatt gtgtg 25 <210> 485 <211> 28 <212> DNA <213> Artificial Sequence <400> 485 tcagctttca tttagttgaa aaagagct 28 <210> 486 <211> 25 <212> DNA <213> Artificial Sequence <400> 486 gcaatctaat gcttttctct tgcct 25 <210> 487 <211> 26 <212> DNA <213> Artificial Sequence <400> 487 tctctacaaa gcataacact catcga 26 <210> 488 <211> 26 <212> DNA <213> Artificial Sequence <400> 488 aagccagtaa tgtgtggatg agataa 26 <210> 489 <211> 23 <212> DNA <213> Artificial Sequence <400> 489 gaatcctccc tgtgtttcaa cca 23 <210> 490 <211> 22 <212> DNA <213> Artificial Sequence <400> 490 agctcctgga acacctggaa ta 22 <210> 491 <211> 20 <212> DNA <213> Artificial Sequence <400> 491 cttcagggtg agacagccaa 20 <210> 492 <211> 21 <212> DNA <213> Artificial Sequence <400> 492 actgcccatt tgtggtgcta g 21 <210> 493 <211> 23 <212> DNA <213> Artificial Sequence <400> 493 gatgtgcaaa tctgaggctt cac 23 <210> 494 <211> 22 <212> DNA <213> Artificial Sequence <400> 494 cacacactca aggataggcc aa 22 <210> 495 <211> 27 <212> DNA <213> Artificial Sequence <400> 495 tgaggattag taaataccga ccacttc 27 <210> 496 <211> 24 <212> DNA <213> Artificial Sequence <400> 496 agagagaagt cagaaggttg gaga 24 <210> 497 <211> 22 <212> DNA <213> Artificial Sequence <400> 497 caggtcctcc cttttcttca ca 22 <210> 498 <211> 22 <212> DNA <213> Artificial Sequence <400> 498 gacagtcaga tatgccgtga ca 22 <210> 499 <211> 22 <212> DNA <213> Artificial Sequence <400> 499 aatgcactct gatatgggcc ta 22 <210> 500 <211> 23 <212> DNA <213> Artificial Sequence <400> 500 accaaacatt tgcaatgatg ctc 23 <210> 501 <211> 30 <212> DNA <213> Artificial Sequence <400> 501 ggtttaaaaa atgcatttga tttccttctg 30 <210> 502 <211> 23 <212> DNA <213> Artificial Sequence <400> 502 cgattacttt ccaggagtgt tgc 23 <210> 503 <211> 25 <212> DNA <213> Artificial Sequence <400> 503 gagaaaagca tagcattcaa gccat 25 <210> 504 <211> 26 <212> DNA <213> Artificial Sequence <400> 504 tgcatccaaa agagagagac attttc 26 <210> 505 <211> 22 <212> DNA <213> Artificial Sequence <400> 505 cctaaaggag atgacgcaca ct 22 <210> 506 <211> 22 <212> DNA <213> Artificial Sequence <400> 506 agcaagatag agatcctggg ct 22 <210> 507 <211> 23 <212> DNA <213> Artificial Sequence <400> 507 gagttcctgg ttttcaaagg ctt 23 <210> 508 <211> 23 <212> DNA <213> Artificial Sequence <400> 508 ggaactagtg tctcaaatgc agc 23 <210> 509 <211> 34 <212> DNA <213> Artificial Sequence <400> 509 tctctagtaa gtctcaaata aaattatttg aagt 34 <210> 510 <211> 17 <212> DNA <213> Artificial Sequence <400> 510 cctgggcacc aggcgat 17 <210> 511 <211> 27 <212> DNA <213> Artificial Sequence <400> 511 cactgtcaca taaagatgag ctaagtc 27 <210> 512 <211> 25 <212> DNA <213> Artificial Sequence <400> 512 tggttaagtg atctctgaag gctac 25 <210> 513 <211> 21 <212> DNA <213> Artificial Sequence <400> 513 cctgctgaga atgcatggat g 21 <210> 514 <211> 22 <212> DNA <213> Artificial Sequence <400> 514 gagagccatt ttcaccacga tc 22 <210> 515 <211> 25 <212> DNA <213> Artificial Sequence <400> 515 aactagttcc gtgtatgtct tctca 25 <210> 516 <211> 24 <212> DNA <213> Artificial Sequence <400> 516 ttcctgaaca cgctggaaaa taaa 24 <210> 517 <211> 22 <212> DNA <213> Artificial Sequence <400> 517 cccagtgctt tttaaggcct tc 22 <210> 518 <211> 22 <212> DNA <213> Artificial Sequence <400> 518 ggatgtccac tggttccatc tt 22 <210> 519 <211> 25 <212> DNA <213> Artificial Sequence <400> 519 caatgtattc ttagagtggc gactg 25 <210> 520 <211> 26 <212> DNA <213> Artificial Sequence <400> 520 cttgagtgaa gtcataatct catcgg 26 <210> 521 <211> 33 <212> DNA <213> Artificial Sequence <400> 521 actttattac tggattttat aaccaattcc cat 33 <210> 522 <211> 23 <212> DNA <213> Artificial Sequence <400> 522 cctttcctac ctgaaaacca cca 23 <210> 523 <211> 31 <212> DNA <213> Artificial Sequence <400> 523 gaaatgtttg atctgtttta tttgttccct a 31 <210> 524 <211> 27 <212> DNA <213> Artificial Sequence <400> 524 ctgtgtaggt gaatttgcta tttcctt 27 <210> 525 <211> 30 <212> DNA <213> Artificial Sequence <400> 525 gtaatgtcat gatcatgtac attttgtcct 30 <210> 526 <211> 30 <212> DNA <213> Artificial Sequence <400> 526 gaacaagagg aacacatatg gagttaaatt 30 <210> 527 <211> 28 <212> DNA <213> Artificial Sequence <400> 527 tttttcctca accagatcaa tgtagatc 28 <210> 528 <211> 27 <212> DNA <213> Artificial Sequence <400> 528 cctaatacag ccattactca gatgact 27 <210> 529 <211> 30 <212> DNA <213> Artificial Sequence <400> 529 tcaaaataaa catggtaaaa taagcctggt 30 <210> 530 <211> 23 <212> DNA <213> Artificial Sequence <400> 530 actgcaacat ggaaacagga gaa 23 <210> 531 <211> 21 <212> DNA <213> Artificial Sequence <400> 531 gttttgctca aatgggctgc t 21 <210> 532 <211> 22 <212> DNA <213> Artificial Sequence <400> 532 gatgaaagca tgccagatcc ac 22 <210> 533 <211> 26 <212> DNA <213> Artificial Sequence <400> 533 gcatttcttg catgtaaacc tttgtg 26 <210> 534 <211> 31 <212> DNA <213> Artificial Sequence <400> 534 cactgttaca gctccatatg taaaatagtt c 31 <210> 535 <211> 29 <212> DNA <213> Artificial Sequence <400> 535 actacagggc agaataatac ttaaaggaa 29 <210> 536 <211> 29 <212> DNA <213> Artificial Sequence <400> 536 gtagcaagta attttcatgc catatctct 29 <210> 537 <211> 22 <212> DNA <213> Artificial Sequence <400> 537 gcatatgggt gtgcaaaact gt 22 <210> 538 <211> 32 <212> DNA <213> Artificial Sequence <400> 538 catgaacata tggaatattg ttgttttaga at 32 <210> 539 <211> 26 <212> DNA <213> Artificial Sequence <400> 539 tccatgacac cagataacaa gagaag 26 <210> 540 <211> 24 <212> DNA <213> Artificial Sequence <400> 540 tagcctttat gggaactagc gttc 24 <210> 541 <211> 22 <212> DNA <213> Artificial Sequence <400> 541 tctcagttga aggtggtctg ga 22 <210> 542 <211> 22 <212> DNA <213> Artificial Sequence <400> 542 cttccatttt gccctccttg gt 22 <210> 543 <211> 22 <212> DNA <213> Artificial Sequence <400> 543 acatgaccag gaagcactag gt 22 <210> 544 <211> 22 <212> DNA <213> Artificial Sequence <400> 544 cttgagtcaa gtgccccatg tt 22 <210> 545 <211> 28 <212> DNA <213> Artificial Sequence <400> 545 ccagaatacg acttcaaaac atatgaca 28 <210> 546 <211> 27 <212> DNA <213> Artificial Sequence <400> 546 actgttcctg tttaggataa tgtgtga 27 <210> 547 <211> 23 <212> DNA <213> Artificial Sequence <400> 547 gaaagagcct gctattcagg act 23 <210> 548 <211> 24 <212> DNA <213> Artificial Sequence <400> 548 gctaagcatg aagatttctg tgca 24 <210> 549 <211> 26 <212> DNA <213> Artificial Sequence <400> 549 gaatgactag tgactcagga tggtag 26 <210> 550 <211> 26 <212> DNA <213> Artificial Sequence <400> 550 ttggtatggt cacttaatgt tggttc 26 <210> 551 <211> 22 <212> DNA <213> Artificial Sequence <400> 551 actgaagcca gtgggaaaca ac 22 <210> 552 <211> 22 <212> DNA <213> Artificial Sequence <400> 552 gcttatgcag ccttctaatg gc 22 <210> 553 <211> 24 <212> DNA <213> Artificial Sequence <400> 553 tggaagatga aatggaaaac cgaa 24 <210> 554 <211> 24 <212> DNA <213> Artificial Sequence <400> 554 ctagtggttc acgtagagag gaag 24 <210> 555 <211> 23 <212> DNA <213> Artificial Sequence <400> 555 catcttttcc aggagctccc tag 23 <210> 556 <211> 21 <212> DNA <213> Artificial Sequence <400> 556 ttggccacct catcatgatc c 21 <210> 557 <211> 27 <212> DNA <213> Artificial Sequence <400> 557 gcttccagaa gttggtacaa atatgtc 27 <210> 558 <211> 25 <212> DNA <213> Artificial Sequence <400> 558 caactgcata tcagcaacaa actga 25 <210> 559 <211> 22 <212> DNA <213> Artificial Sequence <400> 559 atgcaggcca tagctagatc ac 22 <210> 560 <211> 22 <212> DNA <213> Artificial Sequence <400> 560 gttggcttga tgatggctcc ta 22 <210> 561 <211> 23 <212> DNA <213> Artificial Sequence <400> 561 cccttgagca ttttgagctg tac 23 <210> 562 <211> 23 <212> DNA <213> Artificial Sequence <400> 562 ctactgatgc aaaaccgact cag 23 <210> 563 <211> 25 <212> DNA <213> Artificial Sequence <400> 563 gtacctggag agccctatta aacag 25 <210> 564 <211> 25 <212> DNA <213> Artificial Sequence <400> 564 gatggctgga catctgtata ctagc 25 <210> 565 <211> 24 <212> DNA <213> Artificial Sequence <400> 565 tgtcattctt cactttggga ctca 24 <210> 566 <211> 25 <212> DNA <213> Artificial Sequence <400> 566 tgacgccaaa ctctaaaaaa tgacc 25 <210> 567 <211> 27 <212> DNA <213> Artificial Sequence <400> 567 cacatctcag cagtataagt aaaagca 27 <210> 568 <211> 24 <212> DNA <213> Artificial Sequence <400> 568 gagtggggta gtcagactac tcta 24 <210> 569 <211> 24 <212> DNA <213> Artificial Sequence <400> 569 catttgtttg tagccctctc tcct 24 <210> 570 <211> 25 <212> DNA <213> Artificial Sequence <400> 570 tggttcttat gcctttgagg attga 25 <210> 571 <211> 20 <212> DNA <213> Artificial Sequence <400> 571 actctggcaa aacgtcctgt 20 <210> 572 <211> 22 <212> DNA <213> Artificial Sequence <400> 572 cacttttgcc ttcccaatct cc 22 <210> 573 <211> 26 <212> DNA <213> Artificial Sequence <400> 573 ggaggacctg ggaagataaa taactc 26 <210> 574 <211> 23 <212> DNA <213> Artificial Sequence <400> 574 atggcagagc caactgtcta aac 23 <210> 575 <211> 26 <212> DNA <213> Artificial Sequence <400> 575 gtacgactgt gtttaagaag atgcat 26 <210> 576 <211> 27 <212> DNA <213> Artificial Sequence <400> 576 aggacgttga tgtatgtctt ttactgt 27 <210> 577 <211> 23 <212> DNA <213> Artificial Sequence <400> 577 gcttcttgct gcattcagag aac 23 <210> 578 <211> 28 <212> DNA <213> Artificial Sequence <400> 578 gtcttcaact gaactgaatt aagtgtgg 28 <210> 579 <211> 25 <212> DNA <213> Artificial Sequence <400> 579 ctaatctgtt ctggtttgct tgtgt 25 <210> 580 <211> 22 <212> DNA <213> Artificial Sequence <400> 580 tgacatgttt ttgcccagca ag 22 <210> 581 <211> 26 <212> DNA <213> Artificial Sequence <400> 581 ggatcaatta tgagttggcc ctaaac 26 <210> 582 <211> 26 <212> DNA <213> Artificial Sequence <400> 582 gaaggatatg gtgaagaaat agcctg 26 <210> 583 <211> 21 <212> DNA <213> Artificial Sequence <400> 583 tcctcttcac gctcttcctg a 21 <210> 584 <211> 22 <212> DNA <213> Artificial Sequence <400> 584 tctcaggaga acccacatgt ct 22 <210> 585 <211> 23 <212> DNA <213> Artificial Sequence <400> 585 cagggaacaa cttgatggtg cta 23 <210> 586 <211> 23 <212> DNA <213> Artificial Sequence <400> 586 actgattttc ccctttacac ccc 23 <210> 587 <211> 22 <212> DNA <213> Artificial Sequence <400> 587 gatgtctgga agccagaacc at 22 <210> 588 <211> 24 <212> DNA <213> Artificial Sequence <400> 588 gactttcatt cccttttagg ctcc 24 <210> 589 <211> 27 <212> DNA <213> Artificial Sequence <400> 589 tctgataatg attgtgaatc accagga 27 <210> 590 <211> 30 <212> DNA <213> Artificial Sequence <400> 590 gtgagacgta atatcgagta aagaaagaag 30 <210> 591 <211> 22 <212> DNA <213> Artificial Sequence <400> 591 gcgagtgtca ttgctttgaa gc 22 <210> 592 <211> 22 <212> DNA <213> Artificial Sequence <400> 592 ggaagaaaac accaccaatg gc 22 <210> 593 <211> 25 <212> DNA <213> Artificial Sequence <400> 593 ggttccttcc aggaatacat acact 25 <210> 594 <211> 25 <212> DNA <213> Artificial Sequence <400> 594 cttctgggat tctgacataa agcat 25 <210> 595 <211> 22 <212> DNA <213> Artificial Sequence <400> 595 acctccagtg agtcttcagc at 22 <210> 596 <211> 29 <212> DNA <213> Artificial Sequence <400> 596 cagtggaata ccttgtaaac aacttagaa 29 <210> 597 <211> 29 <212> DNA <213> Artificial Sequence <400> 597 tctttttcta ggctctataa acttttcca 29 <210> 598 <211> 25 <212> DNA <213> Artificial Sequence <400> 598 cttgacatgt gtaagaacag ttggg 25 <210> 599 <211> 21 <212> DNA <213> Artificial Sequence <400> 599 actcatgacc agccattcag a 21 <210> 600 <211> 23 <212> DNA <213> Artificial Sequence <400> 600 tggtcaacag agatgctaat gtg 23 <210> 601 <211> 22 <212> DNA <213> Artificial Sequence <400> 601 gaagggtgaa gtggctaagt ga 22 <210> 602 <211> 22 <212> DNA <213> Artificial Sequence <400> 602 gaagcacatg ctttcatgga gt 22 <210> 603 <211> 24 <212> DNA <213> Artificial Sequence <400> 603 ctggcttctt ttgcattttg catg 24 <210> 604 <211> 25 <212> DNA <213> Artificial Sequence <400> 604 cagtcaacag agaaaaaaca aggca 25 <210> 605 <211> 29 <212> DNA <213> Artificial Sequence <400> 605 cagatctggt tatttcttga aaaaatgca 29 <210> 606 <211> 23 <212> DNA <213> Artificial Sequence <400> 606 tgctgctttt cttttagatg ccc 23 <210> 607 <211> 26 <212> DNA <213> Artificial Sequence <400> 607 gttgctatct aggttagtga aggcta 26 <210> 608 <211> 25 <212> DNA <213> Artificial Sequence <400> 608 ctgtcttcat ttgtcttgcc tttct 25 <210> 609 <211> 29 <212> DNA <213> Artificial Sequence <400> 609 gcatgcttta atcttctctt tatcaaacc 29 <210> 610 <211> 24 <212> DNA <213> Artificial Sequence <400> 610 gaagaggaga ctgagttttg acca 24 <210> 611 <211> 22 <212> DNA <213> Artificial Sequence <400> 611 gcgttcatct ccaaccttct ga 22 <210> 612 <211> 20 <212> DNA <213> Artificial Sequence <400> 612 aagcattctt cctgccccac 20 <210> 613 <211> 26 <212> DNA <213> Artificial Sequence <400> 613 gcttttctat aagccatgtt tgaggt 26 <210> 614 <211> 28 <212> DNA <213> Artificial Sequence <400> 614 acagtgtgta ttctttttgt ttctgagt 28 <210> 615 <211> 23 <212> DNA <213> Artificial Sequence <400> 615 gtggcctgat tcaaaatgat gca 23 <210> 616 <211> 23 <212> DNA <213> Artificial Sequence <400> 616 ctaggtgaat ggaatgctgt gga 23 <210> 617 <211> 24 <212> DNA <213> Artificial Sequence <400> 617 cctgagagtt actcctcttc ttgg 24 <210> 618 <211> 22 <212> DNA <213> Artificial Sequence <400> 618 gaagtgcagg actgtcccat at 22 <210> 619 <211> 22 <212> DNA <213> Artificial Sequence <400> 619 tggatgcaag acagtgacat gg 22 <210> 620 <211> 22 <212> DNA <213> Artificial Sequence <400> 620 ccaaattgag gctcagggaa ct 22 <210> 621 <211> 23 <212> DNA <213> Artificial Sequence <400> 621 cagctgagag attgctgttg ttg 23 <210> 622 <211> 27 <212> DNA <213> Artificial Sequence <400> 622 gagagagagg gagagataga cagataa 27 <210> 623 <211> 22 <212> DNA <213> Artificial Sequence <400> 623 tccaagggga aacacaaacc at 22 <210> 624 <211> 27 <212> DNA <213> Artificial Sequence <400> 624 catgaatgct ctttctgaag tgtatga 27 <210> 625 <211> 21 <212> DNA <213> Artificial Sequence <400> 625 caaaaggtga tgctggccaa c 21 <210> 626 <211> 21 <212> DNA <213> Artificial Sequence <400> 626 cagtgatccc agcaatccca a 21 <210> 627 <211> 30 <212> DNA <213> Artificial Sequence <400> 627 acctgaaaat aaagatatct gataacacca 30 <210> 628 <211> 25 <212> DNA <213> Artificial Sequence <400> 628 ctacctggct ggttgaataa aaact 25 <210> 629 <211> 25 <212> DNA <213> Artificial Sequence <400> 629 cctggtggca aggtataata aacac 25 <210> 630 <211> 22 <212> DNA <213> Artificial Sequence <400> 630 agacagatgc tgcttttggg aa 22 <210> 631 <211> 26 <212> DNA <213> Artificial Sequence <400> 631 gggggatttt ttagctgata gaaagc 26 <210> 632 <211> 27 <212> DNA <213> Artificial Sequence <400> 632 gaagggattt tccaacaaaa tgaatgc 27 <210> 633 <211> 24 <212> DNA <213> Artificial Sequence <400> 633 caggtccaca tgtttcagat gtct 24 <210> 634 <211> 24 <212> DNA <213> Artificial Sequence <400> 634 atgcgctgtg tttctgaaaa ttgg 24 <210> 635 <211> 23 <212> DNA <213> Artificial Sequence <400> 635 tgccccgtat tatggagatg aac 23 <210> 636 <211> 24 <212> DNA <213> Artificial Sequence <400> 636 cgttcccatt ctaatctaag cagc 24 <210> 637 <211> 22 <212> DNA <213> Artificial Sequence <400> 637 cacgtttggt ttggagagtc ca 22 <210> 638 <211> 22 <212> DNA <213> Artificial Sequence <400> 638 gcatgccatc actttcagta gc 22 <210> 639 <211> 22 <212> DNA <213> Artificial Sequence <400> 639 aaagaaggcc ctgaagctga tg 22 <210> 640 <211> 25 <212> DNA <213> Artificial Sequence <400> 640 ttccctgcaa tgaatcagta gatgc 25 <210> 641 <211> 22 <212> DNA <213> Artificial Sequence <400> 641 ggtgctcatt gtcgatgtgt ct 22 <210> 642 <211> 22 <212> DNA <213> Artificial Sequence <400> 642 tgtctgggaa ggttgctgtc ta 22 <210> 643 <211> 22 <212> DNA <213> Artificial Sequence <400> 643 gtcatttgcc cctttgagaa cc 22 <210> 644 <211> 24 <212> DNA <213> Artificial Sequence <400> 644 aacccttcag tcttgtgtat gaca 24 <210> 645 <211> 23 <212> DNA <213> Artificial Sequence <400> 645 caggttttac gtggtacact gga 23 <210> 646 <211> 23 <212> DNA <213> Artificial Sequence <400> 646 cgtaaggggc actctcactt aaa 23 <210> 647 <211> 23 <212> DNA <213> Artificial Sequence <400> 647 ccgcctgatc ttcagtaaac tca 23 <210> 648 <211> 25 <212> DNA <213> Artificial Sequence <400> 648 gagtgtctca ttggaactcc ctatg 25 <210> 649 <211> 22 <212> DNA <213> Artificial Sequence <400> 649 gaagatgagc caactccagc tt 22 <210> 650 <211> 22 <212> DNA <213> Artificial Sequence <400> 650 gagaatggca caatgcagga tc 22 <210> 651 <211> 31 <212> DNA <213> Artificial Sequence <400> 651 gttcaagctt gaggaatttc atatttttca g 31 <210> 652 <211> 30 <212> DNA <213> Artificial Sequence <400> 652 acgtattgtt gacatcctgt atctatgtaa 30 <210> 653 <211> 27 <212> DNA <213> Artificial Sequence <400> 653 tcagaagtgt attggaacta tcaggaa 27 <210> 654 <211> 24 <212> DNA <213> Artificial Sequence <400> 654 cacaggtgta aaacatgctg tcat 24 <210> 655 <211> 27 <212> DNA <213> Artificial Sequence <400> 655 gagataaaat attgaccgat gcagcta 27 <210> 656 <211> 25 <212> DNA <213> Artificial Sequence <400> 656 actgaaaaac ctgtaaagct gtacc 25 <210> 657 <211> 26 <212> DNA <213> Artificial Sequence <400> 657 tcagtgactt gtctcactaa gttgac 26 <210> 658 <211> 24 <212> DNA <213> Artificial Sequence <400> 658 aaggcttcag tcttcctgtg aaaa 24 <210> 659 <211> 24 <212> DNA <213> Artificial Sequence <400> 659 agcaaaagag gctgaatgta cact 24 <210> 660 <211> 34 <212> DNA <213> Artificial Sequence <400> 660 tgtctataag tcttataatc aattgcttgt ttgt 34 <210> 661 <211> 26 <212> DNA <213> Artificial Sequence <400> 661 gctcaatctc acactaaaga acacca 26 <210> 662 <211> 23 <212> DNA <213> Artificial Sequence <400> 662 tctcctccta cacagtgacc aat 23 <210> 663 <211> 22 <212> DNA <213> Artificial Sequence <400> 663 tcctagccca gctagaaaag ga 22 <210> 664 <211> 22 <212> DNA <213> Artificial Sequence <400> 664 gagaaaatgc acagtgtgag gc 22 <210> 665 <211> 22 <212> DNA <213> Artificial Sequence <400> 665 cattctcaca ccatcatgcg tt 22 <210> 666 <211> 25 <212> DNA <213> Artificial Sequence <400> 666 cagtagaagc catttctcag aaaca 25 <210> 667 <211> 23 <212> DNA <213> Artificial Sequence <400> 667 gagccagcaa tgtatagtgt tgc 23 <210> 668 <211> 23 <212> DNA <213> Artificial Sequence <400> 668 agtcttggag accccatgaa ttg 23 <210> 669 <211> 23 <212> DNA <213> Artificial Sequence <400> 669 acctcctcca aatgaggatc atg 23 <210> 670 <211> 23 <212> DNA <213> Artificial Sequence <400> 670 gtgacacttt gggtgatgct atg 23 <210> 671 <211> 20 <212> DNA <213> Artificial Sequence <400> 671 tcaggagcac ttctcccctt 20 <210> 672 <211> 20 <212> DNA <213> Artificial Sequence <400> 672 catgcatctg ctttgcctcg 20 <210> 673 <211> 22 <212> DNA <213> Artificial Sequence <400> 673 agggaacctg caagcatttc tc 22 <210> 674 <211> 24 <212> DNA <213> Artificial Sequence <400> 674 gatgggtctt tctggtaact gtcc 24 <210> 675 <211> 24 <212> DNA <213> Artificial Sequence <400> 675 catgttatta ctcttggccc tcgt 24 <210> 676 <211> 24 <212> DNA <213> Artificial Sequence <400> 676 agactgctac ctgtgtctct actt 24 <210> 677 <211> 24 <212> DNA <213> Artificial Sequence <400> 677 tctgggctgg ttcttaaata tgct 24 <210> 678 <211> 23 <212> DNA <213> Artificial Sequence <400> 678 ttggcctctt tcagtatttg ggt 23 <210> 679 <211> 28 <212> DNA <213> Artificial Sequence <400> 679 gtccatcaac tttttattcc aagtgtct 28 <210> 680 <211> 32 <212> DNA <213> Artificial Sequence <400> 680 gtctaaaatg taatcttatc ctgtcttgtc tc 32 <210> 681 <211> 22 <212> DNA <213> Artificial Sequence <400> 681 ctgtgcttgt ctatcagctg gt 22 <210> 682 <211> 24 <212> DNA <213> Artificial Sequence <400> 682 gtgatgtatc ggttgtaggc ttct 24 <210> 683 <211> 23 <212> DNA <213> Artificial Sequence <400> 683 agagcctgtt cactaaacca cag 23 <210> 684 <211> 24 <212> DNA <213> Artificial Sequence <400> 684 gttgctggaa aactgtgtac tagc 24 <210> 685 <211> 26 <212> DNA <213> Artificial Sequence <400> 685 acctgaatgg aaaatgagga aaggta 26 <210> 686 <211> 32 <212> DNA <213> Artificial Sequence <400> 686 gtctcaataa tgttttgaat tgtattactg gc 32 <210> 687 <211> 22 <212> DNA <213> Artificial Sequence <400> 687 gcagtcatgt cacagagacc tt 22 <210> 688 <211> 22 <212> DNA <213> Artificial Sequence <400> 688 tggagaatgt catcatcccc ct 22 <210> 689 <211> 24 <212> DNA <213> Artificial Sequence <400> 689 cctgtacgtg acatcaaaca atgc 24 <210> 690 <211> 24 <212> DNA <213> Artificial Sequence <400> 690 acactccctt gccattaaga agtc 24 <210> 691 <211> 24 <212> DNA <213> Artificial Sequence <400> 691 tgctgcaaag caatgtctaa cttg 24 <210> 692 <211> 26 <212> DNA <213> Artificial Sequence <400> 692 tgacctctct gaatatctgc agaaac 26 <210> 693 <211> 24 <212> DNA <213> Artificial Sequence <400> 693 ctttcaaacg tttcagagca cagt 24 <210> 694 <211> 25 <212> DNA <213> Artificial Sequence <400> 694 ccttttgaat tctgcttact gctga 25 <210> 695 <211> 22 <212> DNA <213> Artificial Sequence <400> 695 gcaggtttcc aaatgtccct gt 22 <210> 696 <211> 22 <212> DNA <213> Artificial Sequence <400> 696 gtgtttggct ccttggaagg tt 22 <210> 697 <211> 27 <212> DNA <213> Artificial Sequence <400> 697 cctgaaactt acccataagt cttgaac 27 <210> 698 <211> 25 <212> DNA <213> Artificial Sequence <400> 698 actcacctca caaatatctg tgtgt 25 <210> 699 <211> 20 <212> DNA <213> Artificial Sequence <400> 699 atggaccagt tcatgcgcaa 20 <210> 700 <211> 22 <212> DNA <213> Artificial Sequence <400> 700 gaagaagggc ggcatgtcta tt 22 <210> 701 <211> 22 <212> DNA <213> Artificial Sequence <400> 701 atagttctgt gccaggcatc tc 22 <210> 702 <211> 22 <212> DNA <213> Artificial Sequence <400> 702 caagttcccc acaggagaca aa 22 <210> 703 <211> 23 <212> DNA <213> Artificial Sequence <400> 703 gaatgccaac tcagcctttt ctc 23 <210> 704 <211> 25 <212> DNA <213> Artificial Sequence <400> 704 catcttggga ggaaaagaga gagtg 25 <210> 705 <211> 29 <212> DNA <213> Artificial Sequence <400> 705 gtgaagctaa atgtttatta cccaaatgc 29 <210> 706 <211> 29 <212> DNA <213> Artificial Sequence <400> 706 tgcaattact tggttttact tttctttcc 29 <210> 707 <211> 22 <212> DNA <213> Artificial Sequence <400> 707 tgttgtctct cctctcctgt gt 22 <210> 708 <211> 22 <212> DNA <213> Artificial Sequence <400> 708 catcgttgtc gtcgtcatca tc 22 <210> 709 <211> 30 <212> DNA <213> Artificial Sequence <400> 709 caaacaactc tccttgatct atactttgag 30 <210> 710 <211> 20 <212> DNA <213> Artificial Sequence <400> 710 actggtgagc ttcagcttgc 20 <210> 711 <211> 22 <212> DNA <213> Artificial Sequence <400> 711 aagagtacta cgccaaggag gt 22 <210> 712 <211> 22 <212> DNA <213> Artificial Sequence <400> 712 aaggacagga gaacgggaaa ag 22 <210> 713 <211> 24 <212> DNA <213> Artificial Sequence <400> 713 agtcttctct gaaagcacaa tgga 24 <210> 714 <211> 27 <212> DNA <213> Artificial Sequence <400> 714 caacaacaac aacaacaaca acaaatg 27 <210> 715 <211> 25 <212> DNA <213> Artificial Sequence <400> 715 cctcccaaga tgttcagtat cccta 25 <210> 716 <211> 21 <212> DNA <213> Artificial Sequence <400> 716 ttccctcccc cacctcatat g 21 <210> 717 <211> 21 <212> DNA <213> Artificial Sequence <400> 717 gtgagggtgg tgaatcagct t 21 <210> 718 <211> 22 <212> DNA <213> Artificial Sequence <400> 718 atggtaaccc aagaacagag gc 22 <210> 719 <211> 19 <212> DNA <213> Artificial Sequence <400> 719 ccagtgcctc agatggcat 19 <210> 720 <211> 29 <212> DNA <213> Artificial Sequence <400> 720 gagagcattc atgaagtttc ttttattgg 29 <210> 721 <211> 22 <212> DNA <213> Artificial Sequence <400> 721 tcttgaggtc tggtaagggt ct 22 <210> 722 <211> 22 <212> DNA <213> Artificial Sequence <400> 722 ccatcacaca gaggtgcttt tc 22 <210> 723 <211> 22 <212> DNA <213> Artificial Sequence <400> 723 tggtttctgc tctgagagag ga 22 <210> 724 <211> 20 <212> DNA <213> Artificial Sequence <400> 724 agcacactgt tcctgcatgt 20 <210> 725 <211> 21 <212> DNA <213> Artificial Sequence <400> 725 tgatagggga cgtgggtcat c 21 <210> 726 <211> 21 <212> DNA <213> Artificial Sequence <400> 726 tctcttcctc tccaggcctt g 21 <210> 727 <211> 20 <212> DNA <213> Artificial Sequence <400> 727 aggcagtggt ggttctctct 20 <210> 728 <211> 22 <212> DNA <213> Artificial Sequence <400> 728 ccaacctctc actgacatgt cc 22 <210> 729 <211> 22 <212> DNA <213> Artificial Sequence <400> 729 agtaggcagg cagtagatgt tg 22 <210> 730 <211> 23 <212> DNA <213> Artificial Sequence <400> 730 aggagattgt cactttcctt ccc 23 <210> 731 <211> 22 <212> DNA <213> Artificial Sequence <400> 731 agggacctga ggttcattct ga 22 <210> 732 <211> 22 <212> DNA <213> Artificial Sequence <400> 732 ctgtgctctg cattgtgaca tc 22 <210> 733 <211> 20 <212> DNA <213> Artificial Sequence <400> 733 acaccccagc gagaatttgg 20 <210> 734 <211> 21 <212> DNA <213> Artificial Sequence <400> 734 tggaagccat taggggacag a 21

Claims

1. A library construction method for detecting familial thoracic aortic aneurysm and dissection related mutant genes based on high-throughput sequencing, characterized in that, It includes the following steps: (1) Sample collection: Select 12 pathogenic genes related to familial thoracic aortic aneurysm and dissection, namely ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, and TGFB3; (2) Perform the first-round multiplex PCR reaction using the kit to amplify the samples in step (1) to obtain the first-round multiplex PCR amplification products; (3) Perform the first-round magnetic bead purification treatment on the first-round multiplex PCR amplification products; (4) Perform the second-round adapter sequence PCR reaction on the purified first-round multiplex PCR amplification products using adapter sequences to obtain the second-round PCR amplification products, and perform the second-round magnetic bead purification on the second-round PCR amplification products to obtain the sequencing library; (5) Then perform high-throughput sequencing to obtain the detection results; The kit includes 1 tube of Primer pool reagent. The Primer pool reagent contains a total of 367 amplicons. Each amplicon contains a forward primer and a reverse primer. The sequences of the forward primer and the reverse primer are SEQ ID No.1 - SEQ ID No.734; The kit also includes 1 tube of IGT-I7 Index 10uM reagent stored at -20°C, 1 tube of IGT-I5 Index10uM reagent, 1 tube of Primer pool reagent, 1 tube of IGT-EM808 polymerase mixture reagent, and 1 tube of Enhancerbuffer NB 1N reagent, as well as 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent stored at 4°C; The reaction solution for the first-round multiplex PCR reaction includes: Enhancer buffer NB 1N: 3.5 ul, Nuclease-free water: 9 - x ul, Primer pool: 5 ul, gDNA: x ul, Enhancer buffer M: 2.5 ul, IGT-EM808 polymerase mixture: 10 ul, with a total of 30 ul; The reaction solution for the second-round adapter sequence PCR reaction includes: PCR product mixture: 13.5 ul, IGT-I5Index 10uM: 1 ul, IGT-I7 Index 10uM: 1 ul, IGT-EM 808 polymerase mixture: 10 ul, Enhancer buffer M: 2.5 ul, Nuclease-free water: 2 ul, with a total of 30 ul; Among them, PCR product mixture is the product obtained in step (3); The reaction procedure of the first-round multiplex PCR reaction is as follows: first, 95°C for 210 s, and then perform the cycling program: 98°C for 10 s, 60°C for 5 min, for a total of 18 cycles, and finally extend at 72°C for 5 minutes; The reaction procedure of the second-round adapter sequence PCR reaction is as follows: first, 95°C for 210 s, and then perform the cycling program: 98°C for 20 s, 68°C for 1 min, 72°C for 30 s, for 9 cycles, and finally extend at 72°C for 5 min.

Images