CN110551804B - Detection method for chimeric rate of chimera based on donor and acceptor - Google Patents

Detection method for chimeric rate of chimera based on donor and acceptor Download PDF

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CN110551804B
CN110551804B CN201910655106.8A CN201910655106A CN110551804B CN 110551804 B CN110551804 B CN 110551804B CN 201910655106 A CN201910655106 A CN 201910655106A CN 110551804 B CN110551804 B CN 110551804B
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彭海
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Wuhan Baiken Biological Technology Co ltd
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Abstract

The invention relates to a method for detecting the chimeric rate of chimeras based on a donor and a receptor. The detection method comprises the following steps: utilizing the primer group to amplify the detection site of the genome DNA of the chimera through multiple PCR to obtain a multiple PCR amplification product; performing high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment; obtaining the allele types of the true polynucleotide polymorphisms of the donor, the acceptor and the chimera of the detection site from the sequencing fragments; determining whether the chimera is contaminated using the allelic type of the authentic polynucleotide polymorphism; if the chimera is not contaminated, calculating the chimera rate of the chimera. The detection method for the chimeric rate of the chimera provided by the invention has high sensitivity and accuracy.

Description

Detection method for chimeric rate of chimera based on donor and acceptor
Technical Field
The invention relates to the field of biological detection, in particular to a method for detecting the chimeric ratio of chimeras based on a donor and a receptor.
Background
In bone marrow transplantation experiments in both animals and humans, the chimerism rate of a chimera is an important reference index that indicates whether the donor's bone marrow is functioning. In recent years, methods for detecting the chimerism rate of a chimera include an erythrocyte antigen method, a chromosome karyotype method, a human leukocyte antigen typing method, and a Short Tandem Repeat (STR) method. Among them, the STR method is most widely used, but the STR method has a phenomenon of slippage during PCR (Polymerase Chain Reaction) amplification, which results in low detection sensitivity.
The method comprises the steps of carrying out multiple PCR amplification on genome DNAs of a donor, a receptor and a chimera by adopting a primer group to obtain an amplification product, detecting the amplification product through high-throughput sequencing, carrying out typing to obtain a plurality of (25-96) SNP (single nucleotide polymorphism) sites, respectively calculating the chimerism rate of each SNP site, and taking the average value of the chimerism rates of all the SNP sites as the chimerism rate of a sample (chimera) to be detected. The primer group comprises a label primer, and the labeled cross contamination condition of the sample to be detected can be identified through a label in the sequencing result so as to improve the detection sensitivity.
In the existing method for detecting chimera, only 1 SNP locus is detected in each detection locus, so that the number of allelic genes in each detection locus is at most 2, and more than 3 allelic genes cannot be detected, so that whether a sample is polluted before labeling is judged, and the detection result is inaccurate.
Disclosure of Invention
In order to solve the problems of the prior art, the embodiment of the invention provides a method for detecting the chimeric rate of a chimera based on a donor and a receptor. The technical scheme is as follows:
the embodiment of the invention provides a detection method of a chimeric rate of chimeras based on a donor and a receptor, which comprises the following steps:
respectively amplifying the detection sites of the genome DNA of the donor, the receptor and the chimera by utilizing a primer group through multiple PCR to obtain multiple PCR amplification products, wherein the primer group comprises a forward primer and a reverse primer;
performing high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment;
obtaining the true polynucleotide polymorphism alleles of the donor, the acceptor and the chimera of the detection site according to the sequencing fragments, wherein the true polynucleotide polymorphism allele determination method comprises the following steps:
aligning the sequencing fragments to a reference genome to obtain an allele type of a potential polynucleotide polymorphism, wherein the allele type of the potential polynucleotide polymorphism is a combination of base sequences on the sequencing fragments, which are different from the reference genome;
the number of the sequencing fragments of the allelic type of all the potential polynucleotide polymorphisms of the ith detection site is from large to arranged, wherein the number of the allelic type of the jth potential polynucleotide polymorphism and the number of the sequencing fragments of the allelic type of the jth potential polynucleotide polymorphism are respectively denoted as Ap ij And Np ij When A is ij In the absence, note Np ij ＝0；
When determining Ap ij Ap being the allele type of the true polynucleotide polymorphism ij For the allelic form of the true polynucleotide polymorphism, denoted At ij Support At ij The number of the sequenced fragments of (a) is denoted as Nt ij ；
For the donor and the acceptor:
when in use
Figure BDA0002136621660000011
When it is determined that Ap is present i1 Is the allele type of the true polynucleotide polymorphism;
when in use
Figure BDA0002136621660000012
When it is determined that Ap is present i1 And Ap i2 Are all allelic forms of the true polynucleotide polymorphism;
for the chimeras:
at of the donor and the acceptor ij (ii) is also the allele type of the true polynucleotide polymorphism in the chimera;
judging whether the chimera is contaminated or not by using the obtained true polymorphism allele type of the polynucleotide;
if the chimera is not contaminated, the chimera rate is calculated.
Specifically, the detection site comprises a plurality of base sites of single nucleotide polymorphisms.
Specifically, the allele type of the true polynucleotide polymorphism and the allele type of the potential polynucleotide polymorphism are each a combination of the allele types of a plurality of single nucleotide polymorphisms.
Specifically, the detection sites are as follows:
Figure BDA0002136621660000021
Figure BDA0002136621660000031
Figure BDA0002136621660000041
the start and end points refer to base positions on the reference genome of a human with version number hg 19.
Further, the sequences of the primer groups are respectively shown as SEQ ID NO 1-SEQ ID NO 200 in the sequence table.
Specifically, a =0.2.
Specifically, the method for determining whether the chimera is contaminated comprises the following steps:
in the chimera, with all At ij All different Ap ij As the allele type called as a false negative polynucleotide polymorphism, the allele type of the b-th false negative polynucleotide polymorphism and the number of the sequenced fragments thereof are respectively denoted as Af ib And Nf ib ；
For each Af of the chimera ib Determination inequality
Figure BDA0002136621660000042
If the result is true, where k is the determination coefficient, k ≧ 1, e is the detection error rate, m bj The number of bases that differ between the allelic type of the non-true polynucleotide polymorphism of the b-th type and the allelic type of the true polynucleotide polymorphism of the j-th type;
counting Af satisfying the inequality in the chimera ib And is recorded as M;
when M >0, determining that the chimeric is contaminated;
when M =0, the chimera was judged to be uncontaminated.
Preferably, k =10.
Further, when it is determined that the chimera is not contaminated, a method of calculating the chimera ratio of the chimera is:
with all of said true polynucleotide polymorphisms of said donorThe allele type of the true polynucleotide polymorphism of the receptor having different allele types is defined as the allele type of the receptor-specific polynucleotide polymorphism, and the allele type of the c-th receptor-specific polynucleotide polymorphism is designated as Atr c In the chimera, support of Atr c The number of the sequencing fragments is recorded as Ntr c ；
Calculating out
Figure BDA0002136621660000043
Wherein N is i Is Atr c The sum of the number of the sequencing fragments of the chimera in the detection site, in the receptor, when Atr c (ii) when there are only 1 allele types of said true polynucleotide polymorphisms in said test site, then S =1, when Atr c (ii) S =2 if there are 2 true polynucleotide polymorphism alleles in the test site;
all of R c The median value R of the values is the chimerism rate of the chimerism.
In the detection method provided by the embodiment of the invention, one detection site may contain a plurality of SNPs instead of one SNP. Thus, the ability to distinguish between donor and acceptor in chimeras is greatly enhanced. Assuming that the frequency of both alleles in a SNP in a population is 50%, the probability that the recipient and donor alleles are not the same is 50%, i.e., the SNP has only a 50% probability of distinguishing the donor from the recipient and thus can be used for chimera differentiation. In the present invention, site 1 contains 3 SNP bases, and thus, the 3 SNP bases can distinguish between a donor and a receptor in a chimera as long as one of the 3 SNP bases is different between the donor and the receptor, that is, can be used for detection of a chimera with a probability of 1- (50%. Times.50%) 3 =98.44%, therefore, more detection sites are available and effective for detection of chimeras in the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below. The procedures or specifications of the procedures not shown or described in detail in the examples of the present invention are well known to those skilled in the art of molecular biology. Reagents or biological materials not mentioned in the examples of the present invention are commercially available reagents or biological materials, which are well known to those skilled in the art of molecular biology and commercially available.
Examples
The embodiment of the invention provides a method for detecting the chimeric rate of a chimera based on a donor and a receptor. The detection method specifically comprises the following steps:
counting the cells of three cell lines A, B and C with different genetic backgrounds, and mixing the three cell lines according to a certain proportion to form chimera samples 1-10. The chimeric samples 1 to 8 included only cell line a and cell line B, and the cell line a and cell line B were used as the recipient and donor, respectively, and the cell ratio of cell line a in the chimeric sample was used as the reference value of the chimeric rate. In chimera sample 9, only cell line a served as a control sample for the receptor. In chimera sample 10, which includes cell line a, cell line B and cell line C, in a ratio of 50. The reference values and the measured values of the chimera samples and the chimera rates are shown in the table I.
Table I shows the reference value and measured value of the chimera and the chimera rate
Figure BDA0002136621660000051
As can be seen from table one, the calculation methods of the chimera samples based on the SNP allelic types showed that the chimera rates of the chimera samples 5 to 9 were all 0, and it was not possible to calculate the chimera rate of less than 1% of the chimera samples. The embodiment of the invention calculates the chimeric rate of chimera which is lower than 1 percent, and the chimeric rate is not greatly different from a reference value, so that the detection result is more accurate compared with the calculation of SNP allelic gene type.
In table one, the method for detecting the measured chimeric ratio (R) of the chimeric samples 1 to 10 provided by the present invention specifically comprises the following steps:
and respectively extracting the detection sites of the genome DNA of the donor, the receptor and the chimera.
The primer set is used for amplifying the detection site of the genomic DNA of the donor, the detection site of the genomic DNA of the receptor and the detection site of the genomic DNA of the chimera respectively through multiple PCR (Polymerase Chain Reaction). Genomic DNA from cell line A, cell line B, cell line C and chimera samples 1-10 were extracted and purified according to the procedure described in Qiagen's human tissue and cell DNA extraction kit, cat # 51304 and the instructions.
And respectively amplifying the detection sites of the genome DNA of the donor, the receptor and the chimera by utilizing a primer group through multiple PCR to obtain multiple PCR amplification products, wherein the primer group comprises a forward primer and a reverse primer. Wherein the detection site comprises a plurality of Single Nucleotide Polymorphisms (SNPs) base sites. The detection sites and corresponding primer sets are shown in table two:
TABLE II detection sites and corresponding primer sets
Figure BDA0002136621660000061
Figure BDA0002136621660000071
Figure BDA0002136621660000081
In table two, start and end points refer to base positions on the reference genome of a human with version number hg 19.
In practice, genomic DNA from cell line A, cell line B, cell line C and chimera samples 1-10 were extracted and purified according to the procedure described in Qiagen's human tissue and cell DNA extraction kit cat # 51304 and the instructions for its operation. All of the multiplex amplification primers from site 1 to site 100 in Table I were synthesized and mixed at the United states of America, thermoelectricity, inc., to obtain a mixture of multiplex amplification primers. The extracted genomic DNA was amplified according to the amplicon library construction kit manufactured by U.S. thermal electric corporation under the cat No. 4475345 and the instructions thereof to obtain multiple amplification products, and the multiple PCR amplification products were further used to construct a high-throughput sequencing library according to the kit and the instructions thereof. And performing high-throughput sequencing on the constructed high-throughput sequencing library by using an S5 high-throughput sequencer produced by the American thermal electric company and an operation instruction thereof, wherein the sequencing depth is set to be two million sequencing fragments per sample, and the sequencing length is 300bp.
And obtaining the allele types of the true polynucleotide polymorphisms of the donor, the acceptor and the chimera of the detection site according to the sequencing fragment. The true polynucleotide polymorphism's allele-type and the potential polynucleotide polymorphism's allele-type are each a combination of the plurality of single nucleotide polymorphism's allele-types.
The method for determining the allele type of the true polynucleotide polymorphism comprises the following steps:
comparing the sequencing fragments to a reference genome to obtain the allelic gene type of the potential polynucleotide polymorphism, wherein the allelic gene type of the potential polynucleotide polymorphism is the combination of base sequences on the sequencing fragments, which are different from the reference genome;
sequencing fragments of all the allelic forms of the potential polynucleotide polymorphisms of the i-th detection site from size to arrangement, wherein the allelic forms of the j-th potential polynucleotide polymorphism and the number of the sequencing fragments of the allelic forms of the j-th potential polynucleotide polymorphism are respectively referred to as Ap ij And Np ij When A is ij In the absence, note Np ij ＝0；
When determining Ap ij Ap being the allele type of the true polynucleotide polymorphism ij For the allelic form of the true polynucleotide polymorphism, denoted At ij Support At ij The number of sequenced fragments of (2) is denoted as Nt ij ；
For the donor and acceptor:
when in use
Figure BDA0002136621660000091
When it is determined that Ap is present i1 An allelic type for an actual polynucleotide polymorphism;
when in use
Figure BDA0002136621660000092
When it is determined that Ap is present i1 And Ap i2 An allelic type for an actual polynucleotide polymorphism;
for chimeras:
at of Donor and Acceptor ij And also the true allele type of the polynucleotide polymorphism in the chimera.
The obtained sequenced fragments of cell line A were aligned to the first detection site of the human reference genome (version number hg 19) using Bowtie 2 software (version number 2.1.0) and a total of 18725 sequenced fragments resulted in a successful alignment. The combination of base sequences on the sequenced fragments that differ from the reference genome is 5551 in total, i.e., an allele in which 5551 potential polynucleotide polymorphisms are present. Sequencing the number of the allelic fragments of all the potential polynucleotide polymorphisms of the first detection site from size to arrangement to obtain Ap 11 And Ap 12 Number of fragments supporting sequencing, i.e. Np 11 =15031 and Np 12 And =12. In the present invention, 400 samples of randomly selected volunteers (human) were verified, when
Figure BDA0002136621660000093
Then, the locus is truly heterozygous, so in this example, the value a =0.2, and therefore,
Figure BDA0002136621660000094
thus, ap is determined 11 Is the allelic form of the true polynucleotide polymorphism and is designated as At 11 And Nt 11 =15031. That is, the cell line A has only one true polymorphism allele at the first detection site, and is homozygous. The allele type of the true polynucleotide polymorphism of all the test sites in the cell line A is determined in the same manner as the first test site. According to the same method as that of the cell line A,and obtaining the allele types of the real polynucleotide polymorphisms of all the detection sites in the cell line B and the cell line C.
For chimeras, at for donor and acceptor ij The true polynucleotide polymorphisms in both the chimeras are also allelic, i.e., the true polynucleotide polymorphisms in the donor and the recipient are also allelic for the chimeras.
And judging whether the chimera is polluted or not by using the obtained allele type of the true polynucleotide polymorphism.
The method for judging whether the chimera is polluted comprises the following steps: in chimera, all At will be linked ij All different Ap ij As the allele of the false polynucleotide polymorphism, the allele of the b-th false polynucleotide polymorphism and the number of the sequenced fragments thereof are designated as Af, respectively ib And Nf ib . For each Af of chimera ib Judgment inequality
Figure BDA0002136621660000095
If the detection error rate is satisfied, where k is a determination coefficient and k ≧ 1, e is the detection error rate, m bj The number of bases that differ between the allele of the b-th unreal polynucleotide polymorphism and the allele of the j-th actual polynucleotide polymorphism.
Af satisfying the inequality in statistical chimeras ib And is denoted as M. When M is>0, judging that the chimeric body is polluted; when M =0, the chimera was judged to be uncontaminated.
Based on the obtained results, the allele type of the authentic polynucleotide polymorphism At the first detection site in chimera sample 1 is the At of the donor 11 And At of the receptor 11 Number of sequencing fragments it supports, i.e. Nt ij 9561 and 10023 strips, respectively. The alleles of all 5620 potential polynucleotide polymorphisms, other than the true polynucleotide polymorphisms, are non-true polynucleotide polymorphisms, wherein the allele of the first non-true polynucleotide polymorphism is assignedAs Af 11 Number of fragments Nf that support sequencing 11 And (5) =2. In the present embodiment, k =10,e =1% is taken in order to make the conclusion of the contamination determination more accurate. Allelic forms of two authentic Polynucleotide polymorphisms of chimeras with Af 11 The number of the different bases of (1) is 1, i.e., m bj =1. Substituting the above parameters into
Figure BDA0002136621660000096
Figure BDA0002136621660000097
In (1), the determination inequality does not hold. Sequentially determining the Af of all chimeric samples 1 by the same method as the allele type of the first unreal polynucleotide polymorphism ib That is, it is determined whether the inequality holds, and Af for holding the inequality is counted ib And is denoted as M. In the present example, M =0 of the chimera sample 1, and therefore, it was determined that the chimera sample 1 was not contaminated.
According to the method of the chimeric sample 1, whether the chimeric samples 2 to 10 are polluted or not is sequentially judged, and the result shows that: when M of all of chimeric samples 2 to 9 is equal to 0, it is judged that none of the samples is contaminated, and when M =12 of chimeric sample 10, it is judged that the sample is contaminated. The result is consistent with the real situation, which shows that the method for judging pollution in the embodiment of the invention is accurate.
If the chimera is not contaminated, the chimera ratio is calculated. Specifically, the actual polynucleotide polymorphism allele type of the acceptor which is different from all the actual polynucleotide polymorphism alleles of the donor is regarded as the acceptor-specific polynucleotide polymorphism allele type, and the c-th acceptor-specific MNP allele type is designated as Atr c Support of Atr in chimeras c The number of sequencing fragments was recorded as Ntr c . Computing
Figure BDA0002136621660000098
Wherein N is i Is Atr c In the detection site, the sum of the number of chimera sequencing fragments, in the receptor, when the Atr c Therein is arrangedWhen only 1 true polynucleotide polymorphism allele type exists in the detection site, S =1, when Atr c And S =2 when there are 2 true polynucleotide polymorphism alleles in the detected site. All of R c The median R of the values is the chimerism rate of the chimerism.
In this example, none of the chimera samples 1 through 9 was contaminated, and thus, it was possible to calculate the chimera ratio. For the first detection site of the chimera sample 1, both the donor and the acceptor have only one true polymorphism genotype of polynucleotides, which are At of the donor 11 And At of the receptor 11 . Due to the At of the donor 11 And At of the receptor 11 Is not identical, therefore, the At of the receptor 11 Referred to as receptor-specific polynucleotide polymorphism allele type and designated as Atr 1 . Support for Atr in chimera sample 1 1 Number of sequencing fragments of (1) Ntr 1 =10023, number of sequenced fragments N of first detection site in chimera sample 1 1 =19233. Due to the fact that in the receptor, atr 1 The first test site at which only 1 true polynucleotide polymorphism allele was present, so S =1. Substituting the values of the above parameters
Figure BDA0002136621660000101
In (1), is obtained by calculation
Figure BDA0002136621660000102
Figure BDA0002136621660000103
The R's of the respective receptor-specific polynucleotide polymorphism alleles of the chimeric sample 1 were sequentially calculated in the same manner as for the first detection site c Value, obtain all R c The median value R =51.71%, i.e., the chimerism rate of the chimeric sample 1 was 51.71%. The chimeric ratios of chimeric samples 2 to 9 were obtained in the same manner as in the calculation of chimeric sample 1, and the results are shown in Table II. As can be seen from the second table, the reference values of the fitting ratios of the measured values of the fitting ratios are relatively close, which indicates that the detection method provided by the embodiment of the invention isIs accurate.
In the detection method provided by the embodiment of the invention, one detection site may contain a plurality of SNPs instead of one SNP. Thus, the ability to distinguish between donor and acceptor in chimeras is greatly enhanced. Assuming that the frequency of both alleles in a SNP in a population is 50%, the probability that the recipient and donor alleles are not the same is 50%, i.e., the SNP has only a 50% probability of distinguishing the donor from the recipient and thus can be used for chimera differentiation. In the present invention, site 1 contains 3 SNP bases, and thus, the 3 SNP bases can distinguish between a donor and a receptor in a chimera as long as one of the 3 SNP bases is different between the donor and the receptor, that is, can be used for detection of a chimera with a probability of 1- (50%. Times.50%) 3 =98.44%, therefore, more detection sites are available and efficient for detection of chimeras in the present invention.
The embodiment of the invention provides a method for detecting the chimeric rate of a chimera based on a donor and a receptor, which is to calculate the chimeric rate of the chimera based on a polynucleotide polymorphism (MNP) marker in order to compare the accuracy rate of the chimera based on the polynucleotide polymorphism marker and an SNP marker. We split the polynucleotide polymorphic markers in cell line a, cell line B and chimeric samples 1 to 9 into SNP markers, e.g., the polynucleotide polymorphic allele at the second detection site of chimeric sample 1 is "AC" which consists of two SNP alleles, "a" and "C", thus splitting the MNP allele into SNP allele "a" and SNP allele "C". The chimerism rates of the chimerism samples 1 to 9 based on the SNP alleles were calculated in the same manner and in the same manner as in this example, except that the MNP alleles were divided into SNP alleles, and the results are shown in Table II. As can be seen from Table two, the reference value of the chimerism rate from the chimerism sample 5 to the chimerism sample 9 was less than 1%, and the measured value of the chimerism rate based on the SNP allelic type was 0%, which indicates the calculation method of the chimerism rate based on the SNP allelic type, and the chimerism rate of the chimerism having the chimerism rate of less than 1% could not be calculated due to the experimental error. The embodiment of the present invention is a calculation method based on the chimerism rate of the MNP allele, and therefore, a chimerism rate of less than 1% of the chimerism can be detected because experimental errors are reduced.
Also, M =0 in the chimera sample 10 was counted as above based on the SNP allele type method, because the principle of "there are more than 4 alleles" cannot be utilized to determine whether the chimera sample 10 has been contaminated before the PCR reaction.
Since the PCR amplification and sequencing are erroneous, the probability of erroneously detecting the SNP allele of the donor in the chimera as the SNP allele of the acceptor is e, and in the present invention, the polynucleotide polymorphism allele of the donor is erroneously judged as the acceptor allele only when the alleles of m SNPs different between the polynucleotide polymorphism alleles of the donor and the acceptor are simultaneously erroneous, or conversely, the polynucleotide polymorphism allele of the acceptor is erroneously judged as the donor allele, and in the present invention, the probability of erroneous allele of the donor or acceptor polynucleotide in the chimera is reduced to e, based on the statistical principle that the probability of simultaneous occurrence of independent events is equal to the product of the probabilities of the respective occurrences m It can be seen that the error rate decreases exponentially, enabling more sensitive detection of the chimerism rate. For example, assuming that e =1%, in the present invention, if the allele types of the polynucleotide polymorphisms of the donor and the recipient are AAA and CCC, respectively, the probability of the AAA being caused by the error of CCC is e = (1%) 3 As for =0.0001% < 1%, the chimera could be detected at a chimera rate of 1% or less (the chimera was composed of 1% AAAA and 99.5% CCCC). Therefore, the embodiment calculates the chimeric rate of the chimera less than 1%, and has little difference with the reference value, and the result is more accurate.
In addition, when any contamination type exists in the chimera sample, the number of allelic forms of the polynucleotide polymorphism of the chimera may exceed the maximum value of no contamination, i.e., 4, so that the presence of contamination is determined and the overall quality control of contamination is realized. In addition, the primer group of the embodiment of the invention does not need a sequence tag, and the number of the forward primers and the reverse primers and the primer cost can be greatly reduced.
Figure BDA0002136621660000111
Figure BDA0002136621660000121
Figure BDA0002136621660000131
Figure BDA0002136621660000141
Figure BDA0002136621660000151
Figure BDA0002136621660000161
Figure BDA0002136621660000171
Figure BDA0002136621660000181
Figure BDA0002136621660000191
Figure BDA0002136621660000201
Figure BDA0002136621660000211
Figure BDA0002136621660000221
Figure BDA0002136621660000231
Figure BDA0002136621660000241
Figure BDA0002136621660000251
Figure BDA0002136621660000261
Figure BDA0002136621660000271
Figure BDA0002136621660000281
Figure BDA0002136621660000291
Figure BDA0002136621660000301
Sequence listing
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tcaaacagat ggcaagaatt aggtctc 27
<210> 9
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tcagaaaaat aactagaaat gaaggaaagt gga 33
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ccgtgcccat ctcttaccaa g 21
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gggaagtgga ctctccttcc ta 22
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggtcaaacct cttttatcag gaaacctta 29
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ttccagtgga gacacctgac taa 23
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtggaaggaa gaccggagag gtaaa 25
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agcaagaatg agtgcagcaa ga 22
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cctggtggaa ggtatttagg tcact 25
<210> 17
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tgatttagca aaagattgga caggcta 27
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
caacctcctt tggaaacact gac 23
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ccagcacagt ctctacctag tcc 23
<210> 20
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
agattagact agatgtagag tggatattga c 31
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
aagagggttg gcaagccata aa 22
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acatgatggc ccagcttgaa aa 22
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cccaccctca agaacaggat tt 22
<210> 24
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtgtctggtg aaggttcact ttctaa 26
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cttgaccctc aggccatcaa ta 22
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gggttgtgtg ggaggaaaag tag 23
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
taatgggagg cctagggcag c 21
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tggcctgagg tgacaaatct g 21
<210> 29
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gttctcatgt atgagtttgg gactgt 26
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aatgacgttc ccatgaatgt gaaga 25
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
caagtggagt gaggaagatg ct 22
<210> 32
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cttccttgat tattcaccct gtatggaa 28
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ctcatctaat tgggttggct ctcat 25
<210> 34
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
agccggtata ctatttgatg aagaaacg 28
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
agtgaccatt aagacctgcg g 21
<210> 36
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttgaagatga tgagctcatt ggtaatttaa ga 32
<210> 37
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
catcatgagc ataattccta acatttattg agc 33
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gcctcagttt cctctacacc at 22
<210> 39
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tcaaaaagtt ctattggaca gtgctgat 28
<210> 40
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ttgggaagca gaggtttaat tgagt 25
<210> 41
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttgggaagca gaggtttaat tgagt 25
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gcttgctctg tgaatccagc aa 22
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcaccaacca gtttgtaagg c 21
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
aaaaagtggg tccttgtgtc c 21
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gggactacat gactttccac tagtt 25
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
aagccagagc atctggaata tgg 23
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cgtttgaatt cagagccact caga 24
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gctctgtctc tgttctgggt ttc 23
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gctttacgtt tagccacagg aaa 23
<210> 50
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cctggcttta caaatgagga cact 24
<210> 51
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
catctggtgc ggagcagta 19
<210> 52
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ataaccgagg tccggtcct 19
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gtgatggctg gttcccttac aa 22
<210> 54
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
catctctctg cagattgcct cat 23
<210> 55
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
aaacacctcg actagacaag ttcg 24
<210> 56
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gcttctttgt ggttctgttt ctcagt 26
<210> 57
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cctatcggca gattaaatcc ttctagc 27
<210> 58
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gagggcaata gtgatgataa acctcag 27
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
acttgttctc agggtcccag aa 22
<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
atcacctgaa tgttctgcca ttct 24
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
cggcactacc aatccacaaa ct 22
<210> 62
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tcagagaaag acaaggcatc cttaatg 27
<210> 63
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
tggcatcttt aagtgaactc agaatttct 29
<210> 64
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ggctttcatg gtgatccctg tt 22
<210> 65
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cccactgagc tcttttgacc c 21
<210> 66
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
aatgagtggc cagaggtaga ttaattg 27
<210> 67
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
atagattagg cacaatgact tcaattcagt 30
<210> 68
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ccctgtggta gactgatcta ttttacctt 29
<210> 69
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
cctgttacca ctgctgcaga g 21
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tggagcccaa aagagagtgt g 21
<210> 71
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
acactcctct atcagtaatt gacagaca 28
<210> 72
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ttatgctctt cttgtatgaa gtcaacaact 30
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ctagtacaca tgcatgcacg tg 22
<210> 74
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
tttgtctctt agcttaatga aaactgcaaa tt 32
<210> 75
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ctggtgaatg gataagcact ctgt 24
<210> 76
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gtatgtcact tttgaatctg gcttctttc 29
<210> 77
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tctccctggt acatgcatta aacc 24
<210> 78
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
cacaactaca gagcagcaga ttct 24
<210> 79
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
tctataggtt tgaagcctag gctgta 26
<210> 80
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tctataggtt tgaagcctag gctgta 26
<210> 81
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gcttccctgg atgggagtta gt 22
<210> 82
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gtcctggacc gagacaatga tc 22
<210> 83
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
actaaaggca tattgatctt gtgattggt 29
<210> 84
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ttcttcctct gttgtttgac agttaaca 28
<210> 85
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ggacaaggca caatatgtga tttttct 27
<210> 86
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
aaagatgatg ctgtatttat tgagcacttg 30
<210> 87
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
tggtgcaagg gatgaaaact cc 22
<210> 88
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gttcttccca tttggactta tgggt 25
<210> 89
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gcctgatgaa gcttcagtcc t 21
<210> 90
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
cccttctctg tacagccaac t 21
<210> 91
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
tgcattcagc aaatatttat gaagagccta 30
<210> 92
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ggtttatttg cttcactaag aggtttcc 28
<210> 93
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
cccaaattga ttctctcagg aaagg 25
<210> 94
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gcaagctggc tcacaaggaa t 21
<210> 95
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ccctgtatac acgaggttgc tt 22
<210> 96
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
aaatctcgtg ctctcttgct gt 22
<210> 97
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
actttgtaaa catttactat gtacctgct 29
<210> 98
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
atttcaatga aggagatgac ttgactatcc 30
<210> 99
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
ctgcttctca gttcagtccc aa 22
<210> 100
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
gttgtgagtt gttttcaggt taccatg 27
<210> 101
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
caaagacagg gccacctttt g 21
<210> 102
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
ccaaagtcag cagcttgatc tatg 24
<210> 103
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
gggatcttaa aaagacccag cag 23
<210> 104
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
cctgaaatgt catgttgctg cac 23
<210> 105
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
gctgtgcaag gtagggagtt ag 22
<210> 106
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ttgacaaacc cagcgaccta ag 22
<210> 107
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ttccttatag ggagctctaa agagact 27
<210> 108
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
cccttcctct ctttgttttt atccttctg 29
<210> 109
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
tggtgactga atgtcctttt tctgt 25
<210> 110
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
agctttcctc cacactgtca ag 22
<210> 111
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
ggcagaagaa ttacttcaac atgggat 27
<210> 112
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tttttattat ggttagctgt cacatacaac ttt 33
<210> 113
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
cctggccaca ctctttcaat gt 22
<210> 114
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
agatgattat ttgagcagtt agtggaaatg a 31
<210> 115
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
attctaagtg ttaatgggca gcatct 26
<210> 116
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
gggcattcta aaattaccag caga 24
<210> 117
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gggcattcta aaattaccag caga 24
<210> 118
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
gcaaatctga taaaggatgt tggtgg 26
<210> 119
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cagtgttgtc aggagttctt ggt 23
<210> 120
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
tttaccacgc tgtggctatg taa 23
<210> 121
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
ttccttgttt ttggcattta gaagaagtc 29
<210> 122
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
cattaacctg agccttaatg ttaggaaaga 30
<210> 123
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
tgtctagtga caagttcaat gacaagtac 29
<210> 124
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ggacttagca tgcttcttaa ggcat 25
<210> 125
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
gccctactag cccttactca ca 22
<210> 126
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
aaagagttcc ttgagtatag gaacgaaac 29
<210> 127
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
ttggagctca tagtggacca gta 23
<210> 128
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
cagtactgct ggtctcagtt attcc 25
<210> 129
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
gctccataga gctccaggat tc 22
<210> 130
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
aattgtgaac aaaataagac attggcttga 30
<210> 131
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
agaacttgtg tgaaagaatt ttgacaagg 29
<210> 132
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
cacgagaacc cagtggaaat gt 22
<210> 133
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
gaaatattgt gctacctttc taaatcggc 29
<210> 134
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
aatcttgcct gcatcagatt tgc 23
<210> 135
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
cagcaggagg tcatttgtgg ta 22
<210> 136
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
agagttactg caagggattc atgg 24
<210> 137
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
gatcatcatc atgtagtcaa cacatagaca 30
<210> 138
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
gactgtgatg acgtccatta tctatgt 27
<210> 139
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gactgtgatg acgtccatta tctatgt 27
<210> 140
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
ggctggatgg ataggcttct atttg 25
<210> 141
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
gcaccaggct ttaagggaca 20
<210> 142
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
tgctctgatt aggagacaac acattatttt 30
<210> 143
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
caaaatgccc taatcattgc agga 24
<210> 144
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
ctctggtctc taacacctca caga 24
<210> 145
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
ggattcagag agcttcctgg ga 22
<210> 146
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cacaggtcaa gggctcaatg t 21
<210> 147
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
tgacatatct aacaggagtt tggaaagc 28
<210> 148
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
gcagtgtctg agctacattt ctcataag 28
<210> 149
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
gtgatgttac atagccacag gga 23
<210> 150
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gtagctcctt gttcccttct cttt 24
<210> 151
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
tgacgctttg aaaagaagga aattctg 27
<210> 152
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
tggatatttt agatcccaca cgtaaatgag 30
<210> 153
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
ccttatgtac agccatgcat ggt 23
<210> 154
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
tatgatggaa gtcagatata ccatgcaga 29
<210> 155
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
acagttccgc aggaaactta caa 23
<210> 156
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
tctcagagat ctgatggttt ttaaagtgg 29
<210> 157
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
gacccaaagg caaagtgcta aac 23
<210> 158
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
gctgttgttt tgcaggtgaa aaga 24
<210> 159
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
agtgtttgtg gaacatctca gtttga 26
<210> 160
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
cctgtccatc tctccagagg t 21
<210> 161
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
tgcactaagt atagtatctg ccaagtact 29
<210> 162
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
ccttctctaa aattgctccc tcatct 26
<210> 163
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
tggataaagc accatgtatc ttcagg 26
<210> 164
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
cctttaatcc aaagcaaata gattccca 28
<210> 165
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
cctttaatcc aaagcaaata gattccca 28
<210> 166
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
aggcctcaga aatctggaaa acc 23
<210> 167
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
aggcctcaga aatctggaaa acc 23
<210> 168
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
tacacagaat gactaaggaa accaatgac 29
<210> 169
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
gtgcacactt acctggtgat ct 22
<210> 170
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
agtcatgaca accaaaactg tctttaga 28
<210> 171
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
ggagccttag ttccagctca tat 23
<210> 172
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
gacaatctcc aaaggaaaaa cctagagat 29
<210> 173
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
cttgcgtgtg gctctttcaa g 21
<210> 174
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
caaatcacat actttactca caagcaagtc 30
<210> 175
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
gcagttatgg cacaggactg tt 22
<210> 176
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tgcaagatac atagatgggc taaagtattg 30
<210> 177
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
ggtgggttaa gcatttcctt agc 23
<210> 178
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
cagtctagga cacaactgca gt 22
<210> 179
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
tctaaggtct aacatctaac ttctacctgt c 31
<210> 180
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
ctgttcatta gaccatcaga catgagac 28
<210> 181
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
tctgcttctc acatcctcag gaa 23
<210> 182
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
tctgcttctc acatcctcag gaa 23
<210> 183
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
ccaccctcac taatgatggg a 21
<210> 184
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
gcgtggagaa gataaagccc t 21
<210> 185
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
ggttaaataa agacagtgtt ggtctccaa 29
<210> 186
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
ggttaaataa agacagtgtt ggtctccaa 29
<210> 187
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
taagtcccaa agtgttcatc aaagtgt 27
<210> 188
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
gaacaatctt ggtgactaga cttcatgaa 29
<210> 189
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
caatagcata aagacactcc cacct 25
<210> 190
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
gggtgggtta tttagaattt tctggaaaag 30
<210> 191
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
agcactgacc ctgtagagat gt 22
<210> 192
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
tatgtcagac tgaaagatac acctctgaa 29
<210> 193
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
tgagggctgt catctgactt ga 22
<210> 194
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
caacccactt gagagtgact atctg 25
<210> 195
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
acagaagtga tgaggtgtcc tttc 24
<210> 196
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
gcaaagggat agagaataca atggagag 28
<210> 197
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
agaggtcttg catttgctaa taaatcct 28
<210> 198
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
gaagggactt ttaaactgac caagtaaga 29
<210> 199
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
ggctgtcagt atgacatctg ca 22
<210> 200
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
gtgaaggcct caatggacaa ag 22

Claims (5)

1. A method for detecting the chimerism rate of a donor-recipient based chimera for non-diagnostic purposes, comprising:
respectively amplifying detection sites of genome DNA of a donor, a receptor and a chimera by utilizing a primer group through multiplex PCR, wherein the detection sites comprise a plurality of base sites with single nucleotide polymorphism to obtain a multiplex PCR amplification product, and the primer group comprises a forward primer and a reverse primer;
performing high-throughput sequencing on the multiple PCR amplification product to obtain a sequencing fragment;
obtaining the true polynucleotide polymorphism alleles of the donor, the acceptor and the chimera of the detection site according to the sequencing fragments, wherein the true polynucleotide polymorphism allele determination method comprises the following steps:
comparing the sequencing fragments to a reference genome to obtain an allelic form of a potential polynucleotide polymorphism, wherein the allelic form of the potential polynucleotide polymorphism is a combination of base sequences on the sequencing fragments, which are different from the reference genome, and the allelic form of the real polynucleotide polymorphism and the allelic form of the potential polynucleotide polymorphism are both a combination of allelic forms of a plurality of single nucleotide polymorphisms;
sequencing fragments of all the allelic forms of the potential polynucleotide polymorphism of the ith detection site are arranged from large to small, wherein the allelic form of the jth potential polynucleotide polymorphism and the number of the sequencing fragments of the allelic form of the jth potential polynucleotide polymorphism are respectively marked as Ap ij And Np ij ；
When determining Ap ij Ap as the allele type of the true polynucleotide polymorphism ij For the allelic form of the true polynucleotide polymorphism, denoted At ij Support At ij The number of the sequenced fragments of (a) is denoted as Nt ij ；
For the donor and the acceptor:
when the temperature is higher than the set temperature
Figure FDA0003895092650000011
When it is determined that Ap is present i1 Is the allele type of the true polynucleotide polymorphism;
when the temperature is higher than the set temperature
Figure FDA0003895092650000012
When it is determined that Ap is present i1 And Ap i2 Are all to(ii) the allelic type of the true polynucleotide polymorphism;
for the chimeras:
at of the donor and the acceptor ij (ii) is also the allele type of the true polynucleotide polymorphism in the chimera;
determining whether the chimera is contaminated using the obtained allelic type of the true polynucleotide polymorphism;
if the chimera is not contaminated, calculating the chimera rate of the chimera by the following method:
the allele type of the true polynucleotide polymorphism of the acceptor which is different from the allele types of all the true polynucleotide polymorphisms of the donor is regarded as the allele type of the acceptor-specific polynucleotide polymorphism, and the allele type of the c-th acceptor-specific polynucleotide polymorphism is designated as Atr c Support of Atr in the chimera c The number of the sequencing fragments of (1) is recorded as Ntr c ；
Computing
Figure FDA0003895092650000013
Wherein N is i Is Atr c The sum of the number of the sequencing fragments of the chimera in the detection site, in the receptor, when Atr c (ii) when only 1 allele of said true polynucleotide polymorphism is present in said test site, then S =1, when Atr c (ii) S =2 if there are 2 true polynucleotide polymorphism alleles in the test site;
all R c The median value R of values is the chimerism rate of the chimerism.
2. The detection method according to claim 1, wherein the detection sites are as follows:
Figure FDA0003895092650000014
Figure FDA0003895092650000021
Figure FDA0003895092650000031
Figure FDA0003895092650000041
the start and end points refer to base positions on the reference genome of a human with version number hg 19.
3. The detection method according to claim 2, wherein the sequences of the primer sets are respectively shown as SEQ ID NO 1-SEQ ID NO 200 in the sequence Listing.
4. The detection method according to claim 1, wherein the method for determining whether the chimera is contaminated is:
in the chimera, with all At ij All different Ap ij An allelic form called a false negative polynucleotide polymorphism, wherein the allelic form of the false negative polynucleotide polymorphism at the b-th and the number of the sequenced fragments thereof are denoted as Af, respectively ib And Nf ib ；
For each Af of the chimera ib Judgment inequality
Figure FDA0003895092650000042
If the result is true, where k is the determination coefficient, k ≧ 1, e is the detection error rate, m bj The number of bases that differ between the allelic type of the non-true polynucleotide polymorphism of the b-th type and the allelic type of the true polynucleotide polymorphism of the j-th type;
make statistics ofAf satisfying the inequality in chimeras ib And is denoted as M;
when M >0, determining that the chimeric is contaminated;
when M =0, the chimera was judged to be uncontaminated.
5. The detection method according to claim 4, wherein k =10.
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CN107674920A (en) * 2016-12-19 2018-02-09 苏州大学附属第医院 Chimera multiple PCR primer composition and detection method
CN109554443A (en) * 2018-12-29 2019-04-02 杭州迪安医学检验中心有限公司 A kind of primer and its application based on high throughput sequencing technologies detection DMD genetic mutation

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