CN108504749B - 29 micro haplotype sites, screening method, composite amplification system and application - Google Patents
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Abstract
The invention discloses 29 micro haplotype sites, a screening method, a composite amplification system and application. According to the invention, by acquiring group data, acquiring micro haplotype genotype and allele frequency from the group data, calculating forensic related parameters of candidate loci in different groups, and excluding loci influencing subsequent sequencing and typing to obtain 29 micro haplotype loci; designing primers and amplification conditions according to the 29 micro haplotype sites and establishing a composite amplification system based on a three-wheel PCR amplification system, wherein the composite amplification system is used for amplifying a DNA template extracted from a mixed biological detection material and performing large-scale parallel sequencing on an amplification result to obtain a numerical value of the sequencing depth of each micro haplotype site in the DNA template, thereby finally achieving the purpose of typing the mixed biological detection material. The composite amplification system is suitable for a large-scale parallel sequencing technology, and has the advantages of high detection flux, high detection speed, large obtained data volume, more accurate detection result and the like.
Description
Technical Field
The invention belongs to forensic physical evidence identification, and particularly relates to 29 micro haplotype sites, a screening method, a composite amplification system and application.
Background
The mixed biological material is a material in which two or more biological components are mixed together. The mixed biological material is a common difficult material for medical examination in forensic science. With the continuous progress of scientific and technical means, more and more criminal cases are added to the inspection and use of DNA in mixed biological samples. Mixed biological samples are likely to appear in various cases, and are common in cases of sexual assault and violent injury (such as killing people and corpses). Suspicious dander, skin swabs or bodily fluid evidences and the like collected by an experimenter on site are often originated from different individuals. Therefore, the DNA detection and determination of the mixed biological detection material plays an important role in the effective detection of the case.
At present, the analysis strategies for mixed biological samples are divided into two categories, i.e., separation of different individual components before DNA extraction and analysis after DNA extraction.
For a portion of the mixed sample, such as a semen/vaginal fluid mixed spot, differential lysis can be used to separate male and female DNA components prior to DNA extraction. Other methods of isolation include micromanipulation to isolate cells, laser capture microdissection techniques, etc., which can isolate single cells for analysis, and theoretically have higher sensitivity, but these methods are generally used to isolate different cell types, and the amount of DNA template isolated is low, and the amplification and typing effects are not ideal. Therefore, the strategy of isolating individual components before extracting DNA is limited by the type of the mixed sample, and it is impossible to isolate mixed spots derived from the same body fluid.
Short Tandem Repeat (STR) located in autosome is a genetic marker widely applied in the field of forensic physical evidence and is also a main method for analyzing DNA components in mixed biological samples at present. At present, the common STR typing method is a capillary electrophoresis technology, and a large-scale parallel sequencing technology is also gradually applied to STR typing. However, when the ratio of DNAs from different individuals in a mixed biological sample is too different (less than 5% to 10%), individuals with few DNA components can hardly be successfully detected and typed due to the influence of dominant amplification, shadow peak (stutter), random effect, and the like. When the Y-STR genetic marker is used, the detection ratio of male DNA components in a female DNA background can be increased, but female components cannot be detected, and it is difficult to distinguish individuals having a paternal genetic relationship, and even if a Rapid Mutation (RM) Y-STR genetic marker with a high mutation rate is used, about 50% of father-child pairs and 40% of brother pairs cannot be distinguished. Therefore, the testing and typing of the mixed biological detection materials still remains a technical problem to be solved in the field of forensic physical evidence.
A mini-haplotype refers to a DNA sequence of short length that contains 2 or more SNPs. Using the haplotypes formed by these SNPs as alleles, the minihaplotype loci theoretically have at most 2n(n is the number of SNPs included in the mini-haplotype site) alleles. Since the concept of the mini-haplotype was first proposed in 2013 by professor Kenneth k. Kidd of yale university, the mini-haplotype has the advantages of both SNPs and the unique advantages of multiple alleles and high polymorphism, and thus has potential application in forensic personal identification, genetic relationship identification, mixed biological material identification, ethnic information inference and the like.
Disclosure of Invention
The invention aims to provide 29 micro haplotype sites which can provide a brand-new technical idea for the detection and typing of mixed biological samples.
It is still another object of the present invention to provide a method for screening 29 mini-haplotype sites as described above.
It is still another object of the present invention to provide a multiplex amplification system constructed for the 29 mini-haplotype sites and suitable for the practical application of test typing of mixed biological samples.
The present invention further provides a practical application of the multiplex amplification system in testing and typing of mixed biological assay materials, namely a library construction scheme of the multiplex amplification system, wherein the multiplex amplification system is applied to a massively parallel sequencing platform to obtain genotyping and sequencing depth.
The invention is realized in such a way that 29 micro haplotype sites are specifically:
the invention further discloses a screening method of the 29 micro haplotype sites, which comprises the following steps:
(1) population data obtained from a thousand human genome database;
(2) obtaining the micro haplotype genotype and allele frequency from the group data according to the screening rule, and calculating the detailed process of forensic related parameters of the candidate sites in different groups;
(3) the sequence of the selected mini-haplotype locus is further checked, and the locus influencing the subsequent sequencing and typing is eliminated.
Preferably, in step (2), the screening rule is that the sequence length is less than 50bp, the allele has 4 or more alleles, the frequency of at least 4 alleles is greater than 0.1, A is in Chinese Han nationality populationeThe value is greater than 3.
Preferably, in step (3), the excluding of sites that affect subsequent sequencing typing comprises: dinucleotide polymorphisms, contiguous repeats, and contiguous pluralities of identical bases.
The invention further discloses a composite amplification system of the 29 micro haplotype sites, which comprises:
(1) first round PCR amplification system
The first round of PCR amplification system takes a DNA template as a target for amplification, primers in the system are respectively designed according to the screened micro haplotype sites, and the first round of PCR amplification system also comprises a bridging sequence;
(2) second round PCR amplification system
The second round PCR amplification system takes the diluent of the first round amplification product as a template to carry out second round amplification;
(3) third round PCR amplification System
The third round of PCR amplification system uses the second round of PCR amplification product as a template to carry out third round of amplification, and the primer used in the third round of PCR amplification system is a complementary sequence of 'a tag sequence + a barcode sequence + a bridging sequence'.
Preferably, in the first round of PCR amplification system, the names and sequences of the primers respectively corresponding to 29 mini-haplotype sites are as follows:
preferably, the bridging sequence is specifically:
pre-amplification primer kpF: 5' -ACGACGTGTCGAGTTCAGG +,
post amplification primer kpB: 5' -CAGTGAGTCGCCACAGGTCA +;
in the third round of PCR amplification system, the tag sequence and the barcode sequence are individually designed according to different massively parallel sequencing platforms selected by a user.
The invention further discloses the application of the composite amplification system in the aspect of testing and parting of mixed biological test materials.
Preferably, the mixed biological detection material is a mixed biological detection material of Chinese population.
Preferably, the method of application comprises the steps of:
(1) respectively amplifying the DNA template extracted from the mixed biological detection material by a first round PCR amplification system, a second round PCR amplification system and a third round PCR amplification system in a multiple-micro-haplotype locus composite amplification system;
(2) and (3) carrying out large-scale parallel sequencing on the final amplification result of the multiple-micro-haplotype locus composite amplification system to obtain the average value of the sequencing depth of each micro-haplotype locus in the DNA template.
Professor Kidd, 2015 et al, proposed the use of effective number of alleles (A)e) To evaluate the ability of the mini-haplotype sites to detect mixed plaques. A. theeHas a calculation formula of 1
，
Frequency of the ith allele, A of one locuseHigh value, reflecting the high number of alleles and uniform frequency distribution of alleles at the locus, thus the locus appeared in the mixed spotNow, the probability of more alleles is high, namely the efficiency of identifying and distinguishing mixed plaques is strong. To verify AeValue the ability to use for mini-haplotype site screening, the present application calculates the use of different numbers of mini-haplotype sites (assuming Ae= 3), the results are shown in table 1 below:
TABLE 1 use different amounts of (A)e= 3) cumulative probability of detecting mixed plaques composed of two randomly independent individuals at a mini-haplotype site
P bCumulative probability, the calculation formula:
and p is the probability of 3 or more alleles appearing in the mixed spot at a genetic locus with 3 alleles and all alleles of equal frequency (p = 4/9).
In Table 1, the cumulative probability of detecting 3 or more alleles in a mixed spot consisting of two randomly unrelated individuals has reached 0.9999999 when 28 mini-haplotype sites are used.
On the basis, the invention overcomes the defects of the prior art and provides 29 completely new micro haplotype sites, a screening method, a composite amplification system and application. The invention firstly utilizes the medical parameters of the law, such as heterozygosity, personal identification capability and the like and A based on Chinese Han group data in a thousand-person genome databaseeAnd 29 micro haplotype sites suitable for Chinese population are screened by the indexes, the micro haplotype is the latest genetic marker, and compared with the short tandem repeat Sequence (STR) commonly used by the current public security physical evidence, the micro haplotype has the advantages of short amplification length, no shadow peak, balanced allele amplification and the like, and meanwhile, the composite amplification of a plurality of sites can reduce the DNA dosage and reduce the difficulty of extracting evidence at a crime scene. Therefore, the 29 micro haplotype sites of the invention can provide a brand new technical idea for the detection and typing of the mixed biological detection materials.
Furthermore, the invention further establishes a composite amplification system suitable for large-scale parallel sequencing, so that 29 micro haplotype sites can be completed in one amplification system, and the composite amplification system is used for a large-scale parallel sequencing platform to obtain genotyping and sequencing depth and achieve the purpose of mixed biological detection material identification.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: the 29 completely new micro haplotype sites, the multiplex amplification system and the library construction scheme (application) are suitable for the large-scale parallel sequencing technology, and compared with the commonly used capillary electrophoresis technology, the method has the advantages of high detection flux, high detection speed, large obtained data volume, more accurate detection result and the like, and can effectively solve the identification problem of common mixed biological detection materials in the public security material evidence identification work.
Drawings
FIG. 1 is a partial population data display of a screening in the thousand human genome database in an example of the present invention;
FIG. 2 shows that the SNPs in chromosome I are screened for mini-haplotype sites within 200bp according to physical distance in the example of the present invention;
FIG. 3 is a representation of the relevant parameters of a portion of candidate mini-haplotype sites in an embodiment of the present invention;
FIG. 4 is a forensic parameter display of a portion of candidate mini-haplotype sites in a portion of a population of a region in an embodiment of the present invention;
FIG. 5 is A of a portion of candidate mini-haplotype sites in a portion of a population of a geographic region according to an embodiment of the present inventioneDisplaying the value distribution condition;
FIG. 6 is a mean value of the sequencing depths of 29 mini-haplotypes in example of the present invention; wherein, FIG. 6A is the mean value of the sequencing depth of 29 micro-haplotypes in M1 and M2 samples, and FIG. 6B is the mean value of the sequencing depth of an error sequence (drop in) in 14 error sequence sites;
FIG. 7 shows the correlation between the sequencing depth of 29 mini-haplotype sites and the amount of DNA templates in the library in the example of the present invention;
FIG. 8 shows the results of the ratio of the sequencing depth of two alleles of each individual in the examples of the present invention;
FIG. 9 is a comparison of the relative differences in sequencing depth of two alleles for each individual in the examples of the invention;
FIG. 10 shows the results of the ratio of the sequencing depths of two alleles of heterozygous individuals according to the example of the present invention;
FIG. 11 is a comparison of the allelic balance of heterozygotes of individuals according to the present embodiment with the change in the DNA mixing ratio.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The invention relates to a method for screening 29 completely new micro haplotype sites, which comprises the following steps:
(1) population data obtained from a thousand human genome database;
(2) obtaining the micro haplotype genotype and allele frequency from the group data according to the screening rule, and calculating the detailed process of forensic related parameters of the candidate sites in different groups;
(3) the sequence of the selected mini-haplotype locus is further checked, and the locus influencing the subsequent sequencing and typing is eliminated.
In step (1), candidate mini-haplotype sites were screened from the thousand human genome database, as shown in FIG. 1, which is a partial population data obtained from the thousand human genome database.
In the step (2), the screening rule is that the sequence length is less than 50bp, 4 or more alleles are provided, the frequency of at least 4 alleles is greater than 0.1, and the Ae value in Chinese Han nationality population is greater than 3.
In the step (2), each screening result is more intuitive as shown in fig. 2-5.
FIG. 2 shows that the SNPs in chromosome I are screened for mini-haplotype sites within 200bp according to physical distance; the initial SNP of one mini-haplotype site is shown in black, and the remaining SNPs within the same site are in gray.
FIG. 3 shows the sequence length, number of SNPs, number of alleles, standard deviation of allele frequencies, individual alleles and frequencies of candidate mini-haplotype sites. The four allele frequencies of the yellow-labeled mini-haplotype sites in the figure are all > 0.1.
FIG. 4 is a forensic parameter of candidate mini-haplotype sites in a partial population: allele number, allele frequency, heterozygosity, random match probability, personal identification ability, non-paternal exclusion rate, number of valid alleles, ancestor information content, etc. (some data not shown).
FIG. 5 is A of candidate mini-haplotype sites in a population of a fraction of the populationeThe distribution of values.
In step (3), the sequence of the selected site of the mini-haplotype is further examined to exclude sites that may affect the subsequent sequencing and typing, such as dinucleotide polymorphisms, consecutive repeated sequences, consecutive bases, etc.
The 29 completely new mini-haplotype loci of the present invention are finally obtained by the screening method, which are specifically shown in the following table 2:
TABLE 229 names of Brand New mini-haplotype sites and Intra-site SNPs
Example 2
The invention relates to a composite amplification system of 29 micro haplotype sites, which comprises the following components:
(1) first round PCR amplification system
The first round of PCR amplification system takes a DNA template as a target for amplification, primers in the system are respectively designed according to the screened micro haplotype sites, and the first round of PCR amplification system also comprises a bridging sequence;
(2) second round PCR amplification system
The second round PCR amplification system takes the diluent of the first round amplification product as a template to carry out second round amplification;
(3) third round PCR amplification System
The third round of PCR amplification system uses the second round of PCR amplification product as a template to carry out third round of amplification, and the primer used in the third round of PCR amplification system is a complementary sequence of 'a tag sequence + a barcode sequence + a bridging sequence'.
According to the above 29 micro-haplotype sites of the present invention, in this embodiment, the multiplex amplification system for the 29 micro-haplotype sites is more specifically:
1. sample preparation
DNA is extracted according to a conventional method, the concentration of the DNA is measured by an ultraviolet spectrophotometer and the like, the dosage of the DNA template in the composite system is recommended to be 60-90 ng, and the concentration is more than 10-15 ng/ul.
2. First round PCR amplification system
The first round of PCR amplification system proposed by the present invention is shown in the following Table 3:
TABLE 3 first round PCR amplification System
The reaction conditions of the first round PCR amplification system proposed by the present invention are shown in the following Table 4:
TABLE 4 PCR procedure for the first round PCR amplification System
The composite system comprises 29 micro haplotype sites in total, and the sequences and the concentrations of all primers are shown in the following table 5:
TABLE 5 first round PCR amplification System primer sequences and concentrations
The 5' ends of the front and back primers need to be added with a bridging sequence (which can be adjusted according to different sequencing platforms), and the recommended bridging sequence of the system is as follows:
pre-amplification primer kpF: 5' -ACGACGTGTCGAGTTCAGG +,
post amplification primer kpB: 5' -CAGTGAGTCGCCACAGGTCA +.
3. Second round PCR amplification system
100ul of double distilled water is added into each hole of the first round PCR amplification product, and the mixture is instantaneously centrifuged and kept stand for 10 minutes at room temperature. Taking the diluent as a second round amplification template, the recommended amplification system and reaction conditions are shown in the following tables 6 and 7:
TABLE 6 second round PCR amplification System
TABLE 7 second round PCR amplification System PCR program
4. Third round PCR amplification System
Taking the second round of PCR amplification product as a third round of PCR amplification template, and taking the primer as a tag sequence + barcode sequence + bridging sequence complementary sequence, wherein the tag sequence and the barcode sequence are designed according to the selected massively parallel sequencing platform, and the amplification system and the reaction conditions are shown in the following tables 8 and 9:
TABLE 8 third round PCR amplification System
TABLE 9 third round PCR amplification System PCR program
Example 3
The invention discloses an application of the composite amplification system in the aspect of testing and parting of mixed biological test materials, in particular to an application of the mixed biological test materials in the aspect of testing and parting of Chinese population, and the application method comprises the following steps:
(1) respectively amplifying the DNA template extracted from the mixed biological detection material by a first round PCR amplification system, a second round PCR amplification system and a third round PCR amplification system in a multiple-micro-haplotype locus composite amplification system;
(2) and (3) carrying out large-scale parallel sequencing on the final amplification result of the multiple-micro-haplotype locus composite amplification system to obtain the average value of the sequencing depth of each micro-haplotype locus in the DNA template.
In step (2), fastq sequence files obtained after massively parallel sequencing are analyzed by using FLfinder software to obtain amplified micro-haplotype site sequences and corresponding sequencing depths (reference of FLfinder software: Zhu J, Zhou N, Jiang Y, et al. FLfinder: A novel software for the microhaplotypmaker [ J ]. Forensic Science International Genetics Supplement 2015, 5: e622-e 624.).
To verify the accuracy of the multiplex amplification system of the present invention, the present invention first identified 29 mini-haplotypes in A from African, European, Asian populationseThe results of analysis of the distribution of values show the distribution of Ae values in 6 populations in africa, europe and asia (chinese beijing chinese, chinese southern chinese, japan tokyo, utah, northern european afterfilin, babassu african caribbean, southern african american), for the site of the mini-haplotype in the composite system as a heat map (the figure is omitted here).
The multiplex amplification system constructed by the invention is applied to two random unrelated individuals (M1, M2) according to the total amount of 600ug, respectively9 mixed samples mixed for 1:1, 1:3, 1:5, 1:9, 1:19, 19:1, 9:1, 5:1, 3:1 were tested. FIG. 6 is the average of the sequencing depth (sequencing depth) of 29 sites in M1 and M2 samples, wherein FIG. 6A is the average of the sequencing depth of 29 mini-haplotypes in M1 and M2 samples, and FIG. 6B is the average of the sequencing depth of error sequences (drop in) in 14 error sequence sites. The sequencing depth range of 29 sites was 91.5
-2044.5
Average of 488
(FIG. 6A). Allele insertions (wrong sequences) were detected at 14 sites, with sequencing depths ranging from 2
-26.7
Average value of 7.5
(FIG. 6B).
After the composite amplification is finished, performing single qPCR on the amplification product, and adjusting the primer concentration in the first round of PCR according to the CT value. In the present example, template amounts of 29 sites were calculated using 2-CT, and correlation analysis was performed with the final sequencing depth of these sites, with a pearson correlation coefficient of 0.4208, p = 0.023, which were significantly correlated (as shown in fig. 7). Suggesting that the strategy of adjusting the primer concentration in the composite system according to qPCR is completely feasible.
The detection results of 29 micro-haplotype sites in the mixed sample are divided into the following types: (1) the genotypes of the 12 sites M1 and M2 are the same, and the sites cannot distinguish mixed samples, so that the results are not shown; (2) the 2 sites M1 and M2 are homozygotes and have different alleles, in this case, although the mixed spots can only detect two alleles, the mixed spots can be suggested to exist according to the difference of the sequencing depth ratio between the alleles, the mixed ratio can be predicted (figure 8), MPS can effectively detect two alleles of 1:19 or 19:1, and the allele sequencing depth ratio of different individuals is increased along with the increase of the difference of the mixed ratio. (3) Two heterozygotes with the same allele have the same allele due to different individual components, the allele has higher sequencing depth than the other allele due to the additive effect, and the relative difference of the sequencing depths of the two alleles changes along with the change of the mixing ratio, and the result is shown in FIG. 9. (4) One heterozygote is homozygote which is different from one allele (FIG. 10), i.e., the site of the mini-haplotype where 3 alleles are different. As can be seen from FIG. 10, even in the case of 19:1 or 1:19, the allelic balance of heterozygote individuals with a low DNA proportion is not affected by the extremely high background DNA. (5) One heterozygote and one homozygote with the same allele (FIG. 11). As can be seen in FIG. 11, since one allele of the heterozygous individual is identical to that of the homozygous individual, the allele balance of the heterozygous varies with the DNA mixing ratio.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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<211> 30
<212> DNA
<213> Artificial Sequence
<400> 16
ctcttaacat atatttcaat tctgagaaac 30
<210> 17
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 17
tttctcttaa aagagctgaa tgctata 27
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 18
gaagtaccag agtttatatg catgc 25
<210> 19
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 19
atacatgata gagtactatt cagccataa 29
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 20
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 21
ggcaggagta ttccttgagt cc 22
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 22
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 23
tcttgaatgg cagtttaaca agg 23
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 24
aatccaaacc aaaatgcatc a 21
<210> 25
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 25
gtgactaatg aaaagcagaa cttctc 26
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 26
ggcagaattt gatagagatg gag 23
<210> 27
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 27
ctcagtaaga gtttacactc tctgctag 28
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 28
ggcctagaag catttctcct agg 23
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 29
cttggagaca caaggtccct c 21
<210> 30
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 30
ggtcacagat cccctagaag c 21
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 31
tgggaggtaa gactctcctc ct 22
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 32
ggtggcagct tgtagtaagt ctc 23
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 33
ccatcaccac catcactatc acc 23
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 34
ggtggtgagt ggtgacagta gc 22
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 35
agctgctaag actacagctg tgtg 24
<210> 36
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 36
gtcaagtgaa aacataagat atagttggt 29
<210> 37
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 37
aactatggaa tcattttgtc aaatg 25
<210> 38
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 38
taattaaatt aacaagaaat ataaggcc 28
<210> 39
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 39
aagaatgtat aggtaaagac ttgaaaaaa 29
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 40
aatgttcaag aaggctggac g 21
<210> 41
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 41
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 42
tacatcctaa tccctgaaac ctg 23
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 43
<210> 44
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 44
tagagggttg ggattttgaa gc 22
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 45
gctaagcatc tactatgtgg caaac 25
<210> 46
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 46
tatgtatgta ttctgtttta cagtgcc 27
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 47
tgcataaggt tttatctgag cataa 25
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 48
tcactttctc agaaggaacc aac 23
<210> 49
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 49
agatggaaca tatttttctg tgtttattg 29
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 50
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 51
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 52
tgatcacaga atatggcact tgag 24
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 53
ccatggctct aactgctttt cc 22
<210> 54
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 54
agaaagagca ggtattaaag gtgtc 25
<210> 55
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 55
ccttctgcat ccaaggatac c 21
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 56
tttattaaag aacaaatcat gctgg 25
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 57
ctgtcctgag ctgcctacca g 21
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 58
tttctgagcc tttggcaaat c 21
<210> 59
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 59
acgacgtgtc gagttcagg 19
<210> 60
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 60
Claims (4)
1. A composite amplification system of 29 micro-haplotype sites, which is characterized by comprising:
(1) first round PCR amplification system
The first round of PCR amplification system takes a DNA template as a target for amplification, primers in the system are respectively designed according to the screened micro haplotype sites, and the first round of PCR amplification system also comprises a bridging sequence;
(2) second round PCR amplification system
The second round PCR amplification system takes the diluent of the first round amplification product as a template to carry out second round amplification;
(3) third round PCR amplification System
The third round of PCR amplification system uses the second round of PCR amplification product as a template to carry out third round of amplification, and the primer used in the third round of PCR amplification system is a complementary sequence of 'a tag sequence + a barcode sequence + a bridging sequence';
the bridging sequence is specifically as follows:
pre-amplification primer: 5' -ACGACGTGTCGAGTTCAGG +,
post amplification primer: 5' -CAGTGAGTCGCCACAGGTCA +;
in the third round of PCR amplification system, the tag sequence and the barcode sequence are individually designed according to different massively parallel sequencing platforms selected by a user;
the 29 mini-haplotype sites are specifically:
2. the multiplex amplification system of claim 1, wherein in the first round of PCR amplification system, the primers corresponding to 29 mini-haplotype sites are specifically:
3. the use of the multiplex amplification system according to claim 1 for testing and typing of a mixed bioassay material for Han people in China.
4. The application of claim 3, wherein the method of applying comprises the steps of:
(1) respectively amplifying the DNA template extracted from the mixed biological detection material by a first round PCR amplification system, a second round PCR amplification system and a third round PCR amplification system in a multiple-micro-haplotype locus composite amplification system;
(2) and (3) carrying out large-scale parallel sequencing on the final amplification result of the multiple-micro-haplotype locus composite amplification system to obtain the average value of the sequencing depth of each micro-haplotype locus in the DNA template.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157786A (en) * | 2018-09-06 | 2019-08-23 | 山西医科大学 | A kind of analysis method of mixing sample DNA |
| CN109346130B (en) * | 2018-10-24 | 2021-10-08 | 中国科学院水生生物研究所 | Method for directly obtaining micro-haplotype from whole genome re-sequencing data and typing micro-haplotype |
| CN110218781B (en) * | 2019-04-23 | 2023-03-31 | 河北医科大学 | Composite amplification system of 21 micro haplotype sites, next generation sequencing and typing kit and typing method |
| CN110305968A (en) * | 2019-07-14 | 2019-10-08 | 西安交通大学口腔医院 | A kind of composite amplification system in the micro- haplotype domain SNP-DIP based on NGS parting for medical jurisprudence individual identification |
| CN111667883B (en) * | 2020-06-03 | 2021-01-22 | 四川大学 | Forensic medicine mixed DNA analysis method based on composite micro-haplotype pyrophosphate sequencing atlas analysis |
| CN112063723B (en) * | 2020-08-12 | 2021-07-09 | 山西医科大学 | Degradation test material analysis micro haplotype composite amplification system and construction method thereof |
| CN111893192B (en) * | 2020-08-12 | 2021-04-09 | 山西医科大学 | Mixed detection material analysis micro haplotype composite amplification system and construction and haplotype frequency |
| CN112233724A (en) * | 2020-10-16 | 2021-01-15 | 深圳市盛景基因生物科技有限公司 | Ancestral polymorphism prediction method based on big data artificial intelligence algorithm |
| CN113981048B (en) * | 2021-08-30 | 2024-04-30 | 司法鉴定科学研究院 | Primer composition, kit and method for detecting micro-haplotype locus based on second-generation sequencing technology and application of primer composition, kit and method |
| CN115273972A (en) * | 2022-06-24 | 2022-11-01 | 厦门万基生物科技有限公司 | Method for determining noninvasive prenatal paternity relationship by using micro-haplotypes |
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| CN102115788A (en) * | 2010-12-02 | 2011-07-06 | 公安部物证鉴定中心 | SNP composite detection system and detection method |
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| CN102115788A (en) * | 2010-12-02 | 2011-07-06 | 公安部物证鉴定中心 | SNP composite detection system and detection method |
| CN102337345A (en) * | 2011-11-04 | 2012-02-01 | 四川大学 | Medicolegal composite assay kit based on twenty triallelic SNP (single nucleotide polymorphism) genetic markers |
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