CN108300790B - Forensic medicine next-generation sequencing kit based on 165Y-SNPs - Google Patents
Forensic medicine next-generation sequencing kit based on 165Y-SNPs Download PDFInfo
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Abstract
The invention belongs to the field of forensic genetics, and particularly relates to a secondary forensic sequencing kit based on 165Y-SNPs. The invention aims to solve the technical problem that a second-generation sequencing technology is used, and the position of a Chinese male biological test material with unknown source on a Y chromosome evolutionary tree is divided at high resolution by utilizing a Y-SNP genetic marker so as to deduce the male ancestry source. The technical scheme of the invention is that the forensic second-generation sequencing kit based on 165Y-SNP genetic markers comprises a mixture of 165 pairs of primers for composite amplification, so that the simultaneous detection of 165Y-SNPs becomes practical. The kit of the invention applies the single-tube composite amplification and second-generation sequencing technology, can obtain the genotyping of 165Y-SNP genetic markers of a plurality of biological detection materials at one time, and deduces the geography, ethnicity and even family source of male samples from Chinese population sources.
Description
Technical Field
The invention belongs to the field of forensic genetics, and particularly relates to a 165Y-SNP-based forensic second-generation sequencing kit for high-resolution division of positions of biological test materials of unknown Chinese males on a Y chromosome evolutionary tree.
Background
The Y chromosome is a chromosome only existing in male individuals, 95% of the regions of the chromosome are non-recombination regions (non-recombination regions of Y chromosome, NRY, the Y chromosomes which are not specially explained below all refer to non-recombination regions), and the chromosome has the characteristics of paternal inheritance, non-recombination, small effective population, specificity in biophysiology and the like, so that the chromosome plays a role in the aspect of forensic medicine, such as sexual invasion of males as suspects, identification of the source of victims of the group disaster event and investigation of missing personsPlays an important role. The use of Y chromosome short tandem repeats (Y-STR) for the search of male suspects for criminal cases has been highly successful. However, since the mutation rate of Y-STR is very high (about 10)-3) Individuals of the same paternal line may mutate to present different Y-STR genotypes, which hinders the inference of sample origin. Therefore, the method solves the potential confusion caused by the mutation of the Y-STR, correctly judges the geography, the ethnicity and even the family source of the crime scene inspection material, and is a scientific problem to be solved by the forensic medicine.
The mutation rate of Y chromosome single nucleotide polymorphism (Y-SNP) is much lower than that of Y-STR (about 10)-9) The probability of mutation of the same parent individual is very low, and thus a Y chromosome evolutionary tree has been established. In the study of human origin, evolution, migration, geneticists can estimate the time when a nearest common ancestor of a population or race exists by using a Y chromosome evolutionary tree, thereby providing clues for predicting potential geographic or ancestral origin. The detection technology which is continuously developed provides verification and optimization for the construction of the Y chromosome evolutionary tree, more and more Y-SNP sites are discovered, and the Y chromosome evolutionary tree is expanded to the great extent, so that the division of the single group can be more refined. By 18/5/2016, 58198Y-SNP sites provided online by the International Society of Genetic genetics (ISOGG) for the construction of Y chromosome evolutionary trees have been reached. The Y chromosome evolutionary tree under the subdivision can trace back to a nearest common ancestor in a shorter generation number, so that the application of the Y chromosome evolutionary tree to geographic, ethnic and even family traceability of male samples becomes possible. This inspires that we apply Y-SNP haplotype group to overcome the adverse effect caused by the mutation of Y-STR in the forensic medicine tracing process. Individuals from the same paternal line, including more distantly related individuals, all have identical Y-SNP haplotypes, which may correspond to the same branch on the Y chromosome evolutionary tree. Thus, if the unknown male test material is not identical to the reference paternal haplotype group, indicating that co-ancestral relationships occur at a greater time, it may be excluded from the same paternal line. If the haplotype groups are identical, it cannot be excluded that they are derived from the same father line. Obviously, forThe identity of the paternal line is believed to depend on the degree of subdivision of the haplotype group and the density of the genetic markers used. However, it is cumbersome and time consuming to use the currently existing highest resolution Y chromosome evolutionary tree (http:// www.isogg.org/tree/index. html) only to reconstruct the parents within the local region. vanOven et al constructs a minimum reference tree with 759 SNP sites, providing distribution profiles of major haplotype groups in continents (wherein the major haplotype groups in asia including china are 12), but the tree has low resolution, which greatly limits the specific source speculation for unknown male material in a certain region in practical application. The territorial area of China is third in the world, and the population is first in the world, including 56 nationalities with population structure differences. Therefore, we need to select the most representative and most reasonable Y-SNP to construct a non-redundant and high-resolution Y chromosome evolutionary tree for chinese population, and at the same time, the tree must have a stable and complete topology. The method provides convenience for deducing the geography, the ethnicity and even the family source of the male sample from the Chinese population source, and provides a reliable prediction range for the police investigation work.
The integrity of the Chinese specific Y chromosome evolutionary tree depends on the non-missing inclusion of representative SNP sites (including InDel sites) of all levels of branches, but the continuous detection of a plurality of samples and a large number of SNP sites cannot be carried out by using the traditional detection methods of forensic science, such as SNaPshot, pyrosequencing and the like. The second-generation sequencing technology has great advantages in accuracy and high throughput, and is suitable for detection of multiple samples and multiple Y-SNPs. The method is used as a technical means, so that when a high-resolution Y chromosome evolutionary tree is constructed, the Y-SNP and the number of branches are not forced to be reduced due to detection limitation, the integrity of a system is ensured in each detection, and a typing error is avoided.
In conclusion, a group of suitable Y-SNP genetic markers are urgently needed to be screened, a second-generation sequencing system based on a non-redundant high-resolution complete Y chromosome evolutionary tree suitable for Chinese people is constructed by applying a second-generation sequencing technology, and a new technical means is provided for forensic traceability.
Disclosure of Invention
The invention aims to solve the technical problem of carrying out high-resolution division on the position of a biological test material of Chinese males with unknown sources on a Y chromosome evolutionary tree by utilizing a Y-SNP genetic marker so as to deduce the origin of male descent.
The technical scheme for solving the technical problems comprises the following steps: a forensic second generation sequencing kit based on 165Y-SNPs is provided, the kit comprising a composite amplification primer mixture. The composite amplification primer mixture in the forensic medicine next-generation sequencing kit based on 165Y-SNP genetic markers comprises 330 amplification primers in total of 165Y-SNP genetic markers. Further, the nucleotide sequences of the amplification primers are shown as SEQ ID No.1 to SEQ ID No.330 in Table 2, respectively.
TABLE 2 composite amplification primers
Further, the kit also comprises a composite amplification reaction mixed solution.
Further, the kit may further comprise a standard DNA.
Preferably, the standard DNA is 007 standard DNA.
The invention also provides the application of the kit in the division of the single group of Chinese male samples with unknown sources.
The invention has the beneficial effects that: the kit comprises 165Y-SNP genetic markers, gives consideration to the integrity and high resolution of the Y chromosome evolutionary tree, achieves higher system resolution capability by using as few sites as possible, and can correctly divide any Chinese male individual test material into the currently recognized branches of the Y chromosome evolutionary tree. The kit adopts single-tube composite amplification and a higher-flux second-generation sequencing technology, can obtain the genotyping of 165Y-SNP genetic markers of a plurality of male-source biological detection materials at one time, and quickly realizes the source tracing of the biological detection materials, the affiliated crowds and even the family tracing. The kit comprises a typing result of standard DNA, and can ensure accurate typing; the length of the composite amplification product of the kit is only 125bp at the shortest and not more than 224bp at the longest, so that the kit has advantages for detecting degradation detection materials common in forensic medicine, and has good application prospect and popularization value.
Drawings
FIG. 1 is a Y chromosome evolutionary tree consisting of 165Y-SNP sites detected by the present invention, wherein the end of each horizontal line points to a haplotype group, and the upper part of the horizontal line is the decisive Y-SNP site of the haplotype group.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments.
The invention relates to a forensic medicine second-generation sequencing kit based on Y-SNP genetic markers, which is constructed by utilizing a second-generation sequencing system which is rapidly developed at present based on 165Y-SNP genetic markers obtained by screening. The kit consists of a composite amplification primer mixture, standard DNA and a composite amplification reaction mixed solution.
The working principle of the kit is that 165 DNA fragments of all 165Y-SNP of a single sample are obtained by simultaneously amplifying a composite amplification primer mixture and a composite amplification reaction mixed solution in a single tube at one time (a sequencing library is constructed).
Then, the 165 DNA fragments are used as templates, and adapters are added respectively (the adapters are consistent in a single sample, if simultaneous sequencing of a plurality of samples is required, different labels are added to different samples respectively on the basis of the adapters for distinguishing the samples. Mixing a plurality of samples including typing standard DNA samples, and then carrying out emulsion PCR amplification and library enrichment.
Finally, the amplification product of the 007 standard DNA is used as a positive control to perform a sequencing reaction with the samples at the same time, so as to obtain a sequencing result of each sample; and then, firstly analyzing whether the sequencing result of the typing standard DNA is accurate or not, and if the sequencing result of the typing standard DNA is correct and shows that the sequencing result is reliable, further analyzing and determining the typing results of 165Y-SNP genetic markers of the sample to be detected.
In the invention, the selection of the Y-SNP is extremely key to the construction of the traceability complex detection kit for the Chinese male population. As described above, Y chromosome inheritance represents paternal inheritance, and is of great significance in human evolution research. In the Y chromosome evolutionary tree constructed by using Y-SNP, a group of similar haplotypes (haplotypes) is represented by a haplotype group (Hg). The existing research results show that the distribution of the DNA fragments has obvious population and geographical specificity, and any reasonable Y chromosome DNA detection system cannot ignore the division of the haplotype group. The Y chromosome phylogenetic tree provided by ISOGG (website www.isogg.org) on line gives all sites which are considered to be available for dividing haplotype groups at present, which is undoubtedly the most complete and highest-resolution Y chromosome evolutionary tree known at present, but because the number of the contained sites is extremely large, the stability of partial sites is to be examined, and the SNP sites on each node have redundancy, the Y chromosome phylogenetic tree is not suitable for daily detection. van Oven et al screened 759Y-SNPs including major haplotype group nodes of each continent on the basis of phylogenetic trees published by ISOGG and YCC to construct a minimum reference tree, but due to its low resolution, in practical application, it has limited ability to further geographically, nationally and even family traceability on unknown male biological samples. Therefore, we need to select the most representative and reasonable Y-SNP to construct a non-redundant and high-resolution Y chromosome evolutionary tree for Chinese population to infer the geography, ethnicity and even family source of the male sample from the Chinese population.
The selection of the Y-SNP sites in the kit aims to improve the resolution of the Y chromosome evolutionary tree as much as possible while using fewer Y-SNP sites. Therefore, the invention obtains the adoption standard of the Y-SNP locus in the kit through a large amount of researches: 1) the number of sites contained in lower branches of different main single groups is different according to the percentage of the single groups in the population, and the larger the proportion of the sites in the population is, the more Y-SNP sites contained in the lower branches are; 2) the important intermediate node Y-SNP of the main single group to which the Chinese group belongs is used for ensuring the integrity of the topological structure of the tree; 3) Y-SNP sites with good polymorphism in Chinese population; 4) can design the amplification product within 250 bp. Wherein the last one is a requirement for each site. According to the above criteria, the present invention designs the combinatorial sequencing of 165 loci for the first time, and the key to the selection of these loci is whether the haplotypes they decided appear in the Chinese population, so long as the haplotypes are observed in the Chinese population, the decision loci will be included in the kit. At present, the haplotype group determined by the 165 loci is only observed in the Chinese population, so that the invention finally confirms that 165Y-SNP genetic markers are used for establishing the composite detection system based on the second-generation sequencing technology.
The 165Y-SNP genetic markers confirmed by the research of the invention can cover the main single-fold groups and the subgroup thereof of all Chinese people, and the total number is 161. Group investigation experiments (namely detection experiments of the embodiment) prove that the Y-SNP composite detection system based on the second-generation sequencing technology can correctly attribute any Chinese male sample to the currently accepted Y chromosome evolutionary tree branch.
The 165Y-SNP sites referred to in the kit are shown in Table 3.
TABLE 3165Y-SNP sites
The kit of the invention applies a composite amplification technology in the detection of Y-SNP. The technology can amplify a plurality of target DNA fragments in a single reaction system simultaneously, and has the advantages of convenience, rapidness, sample saving, cost saving and the like. However, the design of the pair of primers has higher requirements, the design of the primers is finished by the 'white glove' plan of the Saimei Fei company, and 165 pairs of primers after the design is finished are mixed in the same primer pool.
The kit of the invention utilizes the composite amplification primer to obtain an amplification product (namely, a library) containing 165Y-SNP genetic markers, and then takes the amplification product as a template to carry out next generation sequencing reaction.
Sequencing used with the kit can be performed on the S5XL (Ion S5XL sequence, from seimei). The second generation sequencing technology of the platform is a detection technology based on the principle that hydrogen ions are released during base extension, so that the pH value of a microenvironment is changed. The chip contains a large number of micropores (different according to models), each micropore theoretically only contains a single microbead, and after emulsion PCR reaction, only a plurality of clones of one amplification fragment theoretically exist on one microbead, namely, only a plurality of clones of one amplification primer theoretically exist in a single micropore. The actually extended base type can be reflected by simultaneously detecting whether the pH value of the reaction solution in each micropore changes after different bases are added until the detection of all libraries is finished. The method is characterized in that a plurality of SNP sites of a plurality of samples can be analyzed simultaneously, and the obtained result is the whole nucleotide sequence including the flanking sequence, so that the base distribution condition of a target position can be known, the base arrangement of the flanking sequence can be seen, and the reliability of the typing result can be evaluated more intuitively by comparing standard sequences.
Under the condition that the number of SNP sites detected by each sample is certain, in order to realize the detection of more samples in a single time, the balance of the quantity among the sequencing templates needs to be noticed, and the main influence factor of the balance is the library construction result, namely the difference of the quantity among the fragments of the amplification products in the library, and the main reason of the difference is the difference of the amplification efficiencies of different primers, so that the special attention needs to be paid when the primer design and the mixed primer pool are carried out.
The sequences of all the finally designed composite amplification primers for 165Y-SNP sites are shown in Table 2.
The positive control is introduced into the kit of the invention to be used as quality control for evaluating whether a single sequencing result is reliable or not. The positive control provided in the present invention included the standard typing results of all 165Y-SNP genetic markers of the 007 standard DNA sample. When an unknown sample is detected, standard DNA amplification and sequencing are performed in parallel, and the reliability of the current detection result is evaluated by analyzing the 007 standard DNA typing result to determine whether to perform subsequent analysis. If the result is reliable, the unknown sample can be typed, and the single group to which the unknown sample belongs can be divided.
More specifically, the kit of the invention specifically comprises the following components:
a) complex amplification reaction mixture: PCR buffer solution containing DNA polymerase and enucleated enzyme water;
b) composite amplification primer mixture: a composite amplification primer pool consisting of amplification primer pairs of 165Y chromosome SNP genetic markers as shown in Table 1;
c) partial digestion primer sequence reagents: FuPa reaction solution;
d) linker ligation reaction reagents: comprises a joint, a label, a conversion solution and a ligase;
e) library purification reagents: comprises that
XP reaction solution, 70% ethanol and low-concentration TE buffer solution;
f) library quantification of reagents: comprises E.coli standard substance,
MasterMix and
assay; quantifying each library respectively, diluting each library according to a corresponding proportion according to a quantification result, wherein the total volume after mixing is 20 mu L, and the final concentration is within the range of 30-60pM so as to ensure the template content balance from each sample in a template mixture for emulsion PCR;
g) emulsion PCR reaction reagent: ion 520 containing buffer, enzyme, ISP (Ion Sphere particle), reaction oil and demulsifying solutionTM&Ion 530TMA Chef reagent compartment;
h) library enrichment reagents: an enrichment bin v2 containing eluent, library enrichment Beads, NaOH solution, Beads eluent, Tween solution, neutralization solution and enucleated enzyme water;
i) sequencing the reaction mixture: ion S5 containing polymerase, sequencing primer and internal reference ISPTMA Chef solution bin;
j) sequencing reaction other reagents: ion S5 containing NaOH solution and dNTPsTMSequencing reagent cartridge, Ion S5TMWashing solution, Ion S5TMA cleaning solution;
k)007 standard DNA: as a quality control index, the library was constructed and processed in parallel with other samples.
The reagents a, c, d, f to j in the above-mentioned examples are usually pure water and NaOH, butNeeds to use a matched commercial product, and the reagent used in this time is Hi-QTMSeries, purchased from zeimer airlines. In library purification reagent e
XP reaction was purchased from Beckman. Reagent k was purchased from Saimer Fei as a male sample standard.
As the template for extracting DNA from a sample to be tested, various conventional reagents currently used in the art can be used, and the extraction of the DNA template can be carried out by referring to the conventional methods or using commercially available reagents according to the guidelines.
By using the kit provided by the invention, forensic DNA samples can be analyzed. The analysis method comprises the following steps;
1) extracting DNA of a sample to be detected as an amplification template;
2) performing single-tube composite amplification on the DNA extracted in the step 1 and the 007 standard DNA by using the composite amplification reaction mixed solution and the composite amplification primer mixture;
the cycle parameters of the reaction of the multiplex amplification PCR are as follows: 99 ℃ for 2 minutes; storing at 10 ℃ after 21 cycles of 99 ℃, 15 seconds, 60 ℃ and 4 minutes;
3) partially digesting the primers of the library constructed in the step 2) by using the partially digested primer sequence reagent;
the cycle parameters of the partial digestion library primers were: 50 ℃, 10 minutes, 55 ℃, 10 minutes, 60 ℃, 20 minutes;
4) adding a linker to the product in step 3 of the method using the linker ligation reaction reagent, and purifying the product using a library purification reagent;
the circulation parameters of the adding joint are as follows: 22 ℃, 30 minutes, 72 ℃, 10 minutes;
5) quantifying the product in the step 4 of the method by using the library quantitative reaction reagent so as to ensure the balance of the amount of each library during subsequent library mixing;
the cycle parameters of the quantitative reaction are as follows: 50 ℃, 2 minutes, 95 ℃, 20 seconds; 40 cycles of 95 ℃, 3 seconds, 60 ℃, 32 seconds;
6) performing emulsion PCR reaction on an Ion Chef System by using the emulsion PCR reaction reagent and the library enrichment reagent, and further enriching the product to ensure that the quality and the quantity during sequencing reach higher levels; automatically mixing the enriched product with a reagent required by sequencing in an Ion ChefSystems by using the sequencing reaction mixture, and loading the reagent into a sequencing chip;
the cycling parameters of the emulsion PCR reaction depend on the kit used: the instrument presets parameters for providing all available kits, and the used kits are selected when in use;
7) sequencing the chip in the step 6 by using other reagents of sequencing reaction to obtain standard DNA and genotypes of all sites of the sample;
the cycle parameters of the sequencing reaction depend on the type of genetic marker and the fragment length to be sequenced, the chip used, etc.: the instrument is preset to provide all available parameters, and corresponding settings are selected when the instrument is used.
Further, the sequencing result analysis in step 7 of the method comprises the following steps: performing sequencing reaction on an Ion S5XL sequence, obtaining an excel table containing the site sequencing result after the reaction is finished, and a sequencing original file (comprising a sequence information file bam and a corresponding positioning information file bai), and after 007 standard DNA typing result and sequence analysis are considered as credible sequencing result, analyzing other sequencing samples to obtain the genotype of the sample to be tested.
The present invention is further illustrated by the following specific examples, in which the following reagents and instruments are used without specific indication:
1) nanodrop-1000 micro uv-vis spectrophotometer, semer fly;
2) ProFlexPCR amplification apparatus, Saimer Millipore;
3) pure water unit, Millipore corporation;
4) desktop high speed centrifuge, semer fly;
5) pipette EPPENDORF;
6) magnetic frame, Saimei Fei corporation;
7) model 7500, Saimei Fei, real-time quantitative PCR Instrument;
8) s5XL second generation sequencing system, seimer airlines;
9) ion 530 chip, Saimei Fei;
10) vortex oscillator, zemer airlines.
EXAMPLE 1 preparation of the kit of the invention
The Y-SNP composite detection kit for detection can comprise the following reagents packaged respectively:
a) and (3) compounding the amplification primer mixture. The amplification primers shown in Table 3 were mixed, synthesized by Saimer Miller and mixed into one primer pool.
b) Composite amplification reaction mixed liquor and partial digestion primer sequence reagent. In this example, a PCR reaction mixture supplied from Ion AmpliSeq Library Kit 2.0, Saimer fly corporation, was used.
c) The linker connects the reagents. In this embodiment, an Ion Xpress Barcode Adapter of the zemer airlines is used to connect and tag the interface as 1: mix 1 to tag-linker mix and dilute 2-fold for use. It should be noted that, since the main role of the tag is to distinguish different samples, the tag may not be used in the case where the number of single sequencing samples is only 1.
d) Library purification reagents. In this example use
XP reaction solution, 70% ethanol solution according to the weight ratio of ethanol to water 230: 100, the solution needs to be prepared fresh.
e) The reaction reagents were quantified from the library. In this example, Ion Library TaqMan quantification kit was used, in which
MasterMix and
assay reagent 10: mixing at a ratio of 1 for use.
f) Emulsion PCR reaction reagent and library enrichment reagent. Ion 520& Ion 530Kit-Chef Kit was used in this example.
g) Reagents for sequencing reactions. In this example, Ion S5Sequencing Kit was used.
Packaging the reagents according to respective conventional requirements to prepare the forensic medicine second-generation sequencing kit based on the Y-SNP genetic marker for subsequent experiments.
Example 2 detection of 51 unrelated Male test specimens Using the kit of the present invention
51 unrelated Chinese male individuals were tested by using the forensic second-generation sequencing kit based on 165Y-SNPs. The specific detection process is carried out according to the following steps:
a. extracting DNA from blood sample of unrelated Chinese male by kit or salting-out method as composite amplification template (the DNA extracted by salting-out method comes from existing DNA in laboratory);
b. and c, carrying out single-tube composite amplification on the DNA template in the step a and the standard DNA 007 by using the composite amplification reaction mixed solution and the composite amplification primer mixture to construct each sample library. The system is 20 mu L: amplifying primer mixture 10 mu L, compound amplifying reaction mixed liquor 4 mu L, template DNA1 mu L, adding enucleation enzyme water to complement 20 mu L; circulation conditions are as follows: 2 minutes at 99 ℃, 15 seconds at 99 ℃ and 4 minutes at 60 ℃ and 21 times of circulation.
c. Partial digestion of primers: and (c) adding 2 mu L of FuPa reaction solution into the product in the step (b) respectively to make the total volume be 22 mu L, and then carrying out reaction at 50 ℃ for 10 minutes, at 55 ℃ for 10 minutes and at 60 ℃ for 20 minutes.
d. Adding a joint: mu.L of the conversion solution and 2. mu.L of the diluted tag-linker mixture were added to each product in step c, and after mixing well, 2. mu.L of DNA ligase was added to make the volume 30. mu.L, followed by 30 minutes at 22 ℃ and 10 minutes at 72 ℃.
e. Library purification: transferring the product obtained in step d to a medium containing 45 μ L of the product
Incubation in EP tubes with XP reaction solution at room temperatureFor 5 minutes. Then placing the mixture on a magnetic frame for about 2 minutes, removing supernatant, respectively rinsing with 150 mu L70% ethanol solution for three times, respectively removing the mixture from the magnetic frame after the last washing is finished, airing, adding 50 mu L low-concentration TE solution for full resuspension, and then standing and sucking the supernatant for later use.
f. Library quantification: respectively taking 5 mu L of the product in the step e, diluting by 100 times, respectively taking 9 mu L, and adding
MasterMix、
The Assay mixture (11. mu.L) was added to a total volume of 20. mu.L for quantification. The cycle parameters are: 2 minutes at 50 ℃ and 20 seconds at 95 ℃; cycling 40 times at 95 ℃ for 3 seconds and 60 ℃ for 32 seconds.
g. Emulsion PCR reaction, library enrichment, chip loading: preparation of the library mixture: and f, diluting the library quantified in the step f (not less than 30pM) according to the lowest concentration, mixing to make the content of each library uniform, taking the diluted library mixture, adding the enucleation enzyme water to 25 mu L, and ensuring that the final concentration is in the range of 30-60 pM. 20 mu L of the library is added into the corresponding positions of the Ion 520& Ion 530Chef reagent bins according to the instruction of an operation manual, the library is installed on an Ion Chef System, an enrichment bin v2, an Ion S5Chef solution bin, a chip and corresponding accessories are installed at the same time, and the apparatus automatically completes the emulsion PCR reaction and library enrichment according to the set program. The enriched product is automatically mixed with the reagents required for sequencing in an Ion Chef System and loaded into a sequencing chip.
h. Sequencing was performed using Ion S5XL Sequencer second generation sequencing system from semefree: according to the operation manual, accessories such as Ion S5sequencing reagent bins, Ion S5 washing solution, Ion S5 cleaning solution and the like are used for the installation reaction, an S5XL sequencing system is cleaned and initialized, and chips loaded with the library are installed for the sequencing reaction.
Sequencing conditions are as follows: the kit used was selected as indicated, the flow number was set to 500, Torrent _ Suite Server (v5.2.1) was used for raw file collection in this example, and VariantCaller (v5.2.1.38) and CoverageAnalyzis (v5.2.1.2) were used for data analysis. Subsequent analyses used IGV (Integrated Genomics viewer) v 2.3.72.
The results of typing of all the male samples and 007 standard DNA are shown in Table 4 (T: Tibetan sample; H: Hui family sample; U: Vigorean family sample), and as can be seen from the table, they were divided into 28 haplotype groups. The specific division method comprises the following steps: as shown in FIG. 1, since the evolutionary tree is distributed in a tree, different haploids are branches of the tree, and SNPs are distributed at respective nodes on the tree. When the haplotype group is divided, if the SNP of the node is a mutant type, namely positive, the SNP can go up along the branch when looking up from the root of the tree, if a certain node is negative, the node where one SNP is located on the node is an end point, and the suggested haplotype group is the haplotype group where the sample is located. Thus, a single haplotype group is often determined by typing multiple SNPs.
TABLE 4 test results
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 14
acactagcta taagcaaaag aaatctaacg a 31
<210> 15
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tacctccttg aacttggaaa gttttaaa 28
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caagtggtca gttatgcaat tgcaa 25
<210> 17
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
acttgtatta aaattggact cttgtgct 28
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atcctgtggt tctgctgtta ttgcta 26
<210> 19
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
caaccaacaa caataaacaa tcttaattac ca 32
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
atggagagag gaaagatata gaaaattgc 29
<210> 21
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cccatgtact tacctcaaat tcagctac 28
<210> 22
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
catccactcc tctccagatt gtattt 26
<210> 23
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cttccctttc tctctttctc tcaacaga 28
<210> 24
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttctagagca ttttatatcc aacacctg 28
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gaggctgagg ccaggagaat gat 23
<210> 26
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gggagaatag aagatgtagt cagcatgt 28
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ttatggaggg caatctgctg cac 23
<210> 28
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cttcttagct tgaaagaaag ggatctgc 28
<210> 29
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tacatacatt cctcaaaatg acactcca 28
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cacctgctga gcaagactaa caa 23
<210> 31
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cagagttata aatcgtccat ctgacaat 28
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gctgaacttt ttatttcata tgcctgttga 30
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
catgctcagc atcactggca ag 22
<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gcactcttgc tgttgttatg atgc 24
<210> 35
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cacccacttt gttgctttgt aaagg 25
<210> 36
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttgaagctag gagaactgtt ccttctg 27
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
cacaagtgaa cagatcccaa cttc 24
<210> 38
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gtctttgatt ttgaggcaaa ttttcgtg 28
<210> 39
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ctggctatta atgtgaagga aggtatga 28
<210> 40
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tctctcttca gcaacagtaa gtcgaa 26
<210> 41
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
caaaatcctc tcgtacagat ctgtttc 27
<210> 42
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
atttgataaa gctgctgtgg ctttc 25
<210> 43
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gtctttttga gtggagttaa aaatgctt 28
<210> 44
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
ttgctaagtt tgcccttaat cattttct 28
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
catgaaaaat acacgtgcgc aaact 25
<210> 46
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
attcacgcat acatgcagtc actc 24
<210> 47
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
aacagagggt ttcagtgttt aaacagaa 28
<210> 48
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ttttgccagt aaaaagatga aggcttag 28
<210> 49
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tttgttttat cccttccact cttagctt 28
<210> 50
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tccaagtttc agtgtcacat ataagtca 28
<210> 51
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ttacctgtgg gcatttgtaa gagaat 26
<210> 52
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
cctgttttag tgaaattcct ggagagag 28
<210> 53
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tagcaatgga aaagctgtat gaaatcct 28
<210> 54
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
ttcccttctc aactgtcctt ccaatga 27
<210> 55
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
tacaggagaa ggatttttaa tcccaact 28
<210> 56
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tacacgttag atttgtgata atcgcttg 28
<210> 57
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gtatatttgc catgagttag gccaatgt 28
<210> 58
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gggactggtg attattggtt ctaag 25
<210> 59
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
acactctttt tggaataaag ccactgt 27
<210> 60
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gctttaagca gtctctttat ttggttgg 28
<210> 61
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
acatctgcta gagctcagtc taatc 25
<210> 62
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
agtgaaactg tcatttcaaa tggaacat 28
<210> 63
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
cctggattca gctctcttcc taaggt 26
<210> 64
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
cacacagagt atcaggagag gctg 24
<210> 65
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
gcatggatca caaggacaag agatt 25
<210> 66
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gatggagtct ctgactgtca cca 23
<210> 67
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ccaagatata aactgcttgt gcgat 25
<210> 68
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
gacattgagt gtgagctgca ataatg 26
<210> 69
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
gagtatatgt caaattgtga cactgcaa 28
<210> 70
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ctatcagctt catccaacac taagtacc 28
<210> 71
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
tattaacctt tgcaacccag ggca 24
<210> 72
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
actatagatt tggaccccct cctgt 25
<210> 73
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gagagttttc taacagggcg taacaaa 27
<210> 74
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
atatgggaat cacttttgca actttcat 28
<210> 75
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
aagctgagag gtgaataagt ttcctg 26
<210> 76
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
tgtagctaaa gcaggttggg agac 24
<210> 77
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
taaaacagca acaactacca cactactg 28
<210> 78
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
aggaaccata aggttggaaa tttaggc 27
<210> 79
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
caggacactg accccaaaac attag 25
<210> 80
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ccaggccaag aactaatgaa gagaa 25
<210> 81
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
agtatccaag atatgagtgt tcttcctg 28
<210> 82
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
aaaatctcaa atagtcagtc cctggatg 28
<210> 83
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
agagtagttc atattgaaac cttgtggt 28
<210> 84
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
agggagatat ctattagaga taggatgga 29
<210> 85
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
aaatttcatg gaagcaatct tctacgtt 28
<210> 86
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gccttttctg taagtgagat ttccaag 27
<210> 87
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gtagtcggac ttgaaggaat cagc 24
<210> 88
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
ttatctcccc ttagctctcc tgtttt 26
<210> 89
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tgttacattc aatatctcat gcaataattc tg 32
<210> 90
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tctagaatga tagagacttg actgaacc 28
<210> 91
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gccttagatg aggatgtcct tcg 23
<210> 92
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ggaggaagta cagtgcagaa aatcac 26
<210> 93
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
catgtttgtt caaataattg cagctctt 28
<210> 94
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
accacacaaa aacaggtcct catttta 27
<210> 95
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
aaccacttcc taattattca gactcaaga 29
<210> 96
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
actttcaaaa cgtcttatac caaaatatca cc 32
<210> 97
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
cctgtgtttc catttctctt ttcctcat 28
<210> 98
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tcagctagat tgtgttcttc cacac 25
<210> 99
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
agatgcccca ttatatcctc attcac 26
<210> 100
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
agttttaaat atcagccagg tacagaga 28
<210> 101
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ctgaaccagt taattcatct ttggga 26
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gggaaacatc gtgatggtta ctt 23
<210> 103
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
acttatttta tctccttctc aattacaggg t 31
<210> 104
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
aataagccac tactgtaatt tagccact 28
<210> 105
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
aattgaagaa atttgtacag ctggagga 28
<210> 106
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
agaaggctga aatcaatcca atctgt 26
<210> 107
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
gctgtaacag atgtaggaag agtgga 26
<210> 108
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
tagaagcaaa gagtcttttc acttgttc 28
<210> 109
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
caaggaattc gctgcagcat ataa 24
<210> 110
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
aatgcataat gaagtaagcg ctacc 25
<210> 111
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
gaggtcaaaa tttttagggt ccctca 26
<210> 112
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
cctgtgtaag cacaaagtag gttct 25
<210> 113
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
catttgatca aggaaatttg tgttttcca 29
<210> 114
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
gatcctctga gcccaaaagt ctgg 24
<210> 115
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
tcaaaaacta ctggttactt tcgttcgt 28
<210> 116
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
tgtcctatag gcccaggtac gc 22
<210> 117
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
tggaaaatgt gggctcgttt taattat 27
<210> 118
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
acagcaagtt tattcgttgt gtttga 26
<210> 119
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
tgaaaaacag tttggcagtg ct 22
<210> 120
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
actgttttct ttataagtta cctagcctc 29
<210> 121
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
cagctaatgc tgaactaagg caagt 25
<210> 122
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
tacttgagtc tattcctgag gaagacta 28
<210> 123
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
aaacccagca atcagtcttg tatagat 27
<210> 124
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
tgcttagtta tgattcttac agcaagaata 30
<210> 125
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
ggtcctgcaa cttcacctga aaaata 26
<210> 126
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gccagcatgg tctcaattta cattc 25
<210> 127
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
aattatatgg actctgagtg tagacttgtg 30
<210> 128
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
caagaagaac ttcagcattg ggtatg 26
<210> 129
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
actccgaaag tctgcatgga ttag 24
<210> 130
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
agtactgaat gcagcaatac tgtct 25
<210> 131
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
agtatccaag atatgagtgt tcttcctg 28
<210> 132
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
aaaatctcaa atagtcagtc cctggatg 28
<210> 133
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
caagaggtgt tggagggaca tta 23
<210> 134
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
gccaatgact gctgatacaa gatgat 26
<210> 135
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
aacttaattt tattttaagt cccaagatgt gca 33
<210> 136
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
gggagagcat caggacaaat agctattg 28
<210> 137
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
gttcagaaac tacacttttg cctagaaa 28
<210> 138
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
tcttttccat ctgatgtggg tttataca 28
<210> 139
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gatttaaact ctctgaatca ggcacatg 28
<210> 140
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
cttgattgac tgtaatccat gtactttgtc 30
<210> 141
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
aaatgttatg ggttattcca attcagca 28
<210> 142
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gccaaggtaa atgactcacc ctaag 25
<210> 143
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
agttgttaac ctgtgctcaa atcct 25
<210> 144
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
cgaatgtgct gtattgtgct gta 23
<210> 145
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
gtgaggtatc tcgctcaggg atta 24
<210> 146
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
gggcggtaaa gtcttgttgc ata 23
<210> 147
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
ggatcatgaa tctgcagcga aca 23
<210> 148
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
caggacaatg aaacattgag agatgttg 28
<210> 149
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
cacaggcata gaaaagaaag agtacaat 28
<210> 150
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
cattgtacac actttttcaa gtgatgtt 28
<210> 151
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
aatcacagtg gttaagtata ttgcctgta 29
<210> 152
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
tgatttttct tgaggattct acctttagct g 31
<210> 153
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
ttgtggctgg ttattaatga gaattttcag 30
<210> 154
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
cacaataaag gatgcatagg ccact 25
<210> 155
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gctatcccaa agttagtgat cactcat 27
<210> 156
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
gaaaagatag aatcatgccc atgaaact 28
<210> 157
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
cccctgtgag ggcaggatga a 21
<210> 158
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
atgggaattg gaatatagtc tggtgagttt ct 32
<210> 159
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
ctgccaggtc tgtcagaatg aaataa 26
<210> 160
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
ccatcaattt ctcccttgga aaacct 26
<210> 161
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
gctttggaga aagttgcttg agttt 25
<210> 162
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
caatcggaag cctcaatcta tacagac 27
<210> 163
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
aatattaatt atgcctgcaa cagtcaca 28
<210> 164
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
attcttaaca gtataaagca tagggaacag 30
<210> 165
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
ttggaggaca cagattatgt aatttctg 28
<210> 166
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
gggattttgt ttgtctaagg gccaa 25
<210> 167
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
actggaatgg tagatccaat aacagcct 28
<210> 168
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
tcttaaactg gatcatgctg atggaa 26
<210> 169
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
gggaacaggt aggtggtatt tgg 23
<210> 170
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gctgatacac gagaatcgct tggg 24
<210> 171
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
gaaaactata acttcacagt ctcagcga 28
<210> 172
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
ccaacaaaag gcacagttaa taaaggg 27
<210> 173
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
gatttccagt cagctgccag caata 25
<210> 174
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
cacctgtttt gtgtaagagg atccc 25
<210> 175
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
taagtattgg cgccactgat gga 23
<210> 176
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
ctgatacaac cccagtgaga agcat 25
<210> 177
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
ctaacgacaa gtccctgcat gttt 24
<210> 178
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
ctttgttcct gaagctctgc ttgatat 27
<210> 179
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
taaaaacaca aaaattaact aggcatggtg gaa 33
<210> 180
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
tttttaggtg gaatgtcctc tgttg 25
<210> 181
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
cgtgcgtcac cacacccc 18
<210> 182
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
ggtacaatat gaatatacag gccgggt 27
<210> 183
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
aatataggct atggcctagg tgct 24
<210> 184
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
ttccatgtga tggtcagtag gtgtg 25
<210> 185
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
ttcactcttg aagagggaat ctgtga 26
<210> 186
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
ctatcttccc actctgctgg tcttaa 26
<210> 187
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
gcttagtgtt tgacccacat cca 23
<210> 188
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
acaggctact acagcactta caga 24
<210> 189
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
tggctaattt ttatattttc agtagagatg cga 33
<210> 190
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
gcacccggcc atgtcattt 19
<210> 191
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
taaccttttc cacagtccct ccataa 26
<210> 192
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
cactaagata gcacaagatg atgcaaat 28
<210> 193
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
ggcaagctgg acaaaactat aatgatc 27
<210> 194
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
agacttagct gtgtttgatt ctgttttc 28
<210> 195
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
aaacttcctg ttactgtgct gtacat 26
<210> 196
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
acaggaaatg tgtctgtctt ttgagaat 28
<210> 197
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
cattcttgag tgtgtggctt tcgta 25
<210> 198
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
catgcacaga gagaaatacc cgaatt 26
<210> 199
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
agtcacttgc tctgtgttag aaaaga 26
<210> 200
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
taagtgactg caaatggtat gcaact 26
<210> 201
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 201
acagaaacac tgtcaatctg atttgatc 28
<210> 202
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 202
atgtcatgat attcacattt ctgaaatgct 30
<210> 203
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 203
cagtaaatat tttcttgcag gcttggg 27
<210> 204
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 204
tttgacttca gagcactgaa aattcagc 28
<210> 205
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 205
ccaacatact cgccaatcca atg 23
<210> 206
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 206
cagataccca gcagtattag ctgtg 25
<210> 207
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 207
atgctgctgg ataggcaatt tga 23
<210> 208
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 208
ttcctatgat gaggccacaa aagataat 28
<210> 209
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 209
tctccttcag tttcactctt atcttgatta c 31
<210> 210
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 210
gggtttatgg caaaattttt gaatggc 27
<210> 211
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 211
acggtgcaaa gtagcttgag atttta 26
<210> 212
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 212
tactgtacta tcacacaatt ttgtcagc 28
<210> 213
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 213
tatagaactt gcttggacct gggtt 25
<210> 214
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 214
ctgaatgcat tcatgtcagc caa 23
<210> 215
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 215
ggtttctccc tattagcgtt catt 24
<210> 216
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 216
ctatgggaaa tgaggctact aagtcaac 28
<210> 217
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 217
ttgcctgtca aagtttatta atattagatc ct 32
<210> 218
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 218
gttggagagg tggaatttct tattgt 26
<210> 219
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 219
ggcagagctc tcactgagaa cctat 25
<210> 220
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 220
acagctttta cagggagcaa gatatt 26
<210> 221
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 221
aaggatctga tttcaggact gtagt 25
<210> 222
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 222
tttcccacct aaactatgga ctgtag 26
<210> 223
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 223
ttacaaagat gcgaatgagc atgaa 25
<210> 224
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 224
ggaaataaga atttgtcccc cagct 25
<210> 225
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 225
tcaagggctt tgtcagtttt ctaaga 26
<210> 226
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 226
aatcacagtc atggccattg gctta 25
<210> 227
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 227
agagtagttc atattgaaac cttgtggt 28
<210> 228
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 228
agggagatat ctattagaga taggatgga 29
<210> 229
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 229
gcaaagtgca aacaacctca gcca 24
<210> 230
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 230
cactttcact gggctggtgc tg 22
<210> 231
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 231
aatatttact gagcatgatg tgctgtg 27
<210> 232
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 232
cactaacccc aaatcccaag gtag 24
<210> 233
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 233
cttccagata ttaccagcat gcagatt 27
<210> 234
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 234
atatctcctt tctgtcagct ttagctt 27
<210> 235
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 235
actctttttg agaattctca tttaccact 29
<210> 236
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 236
cacaaccaga caatcagaat catca 25
<210> 237
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 237
tcactaaaga gcttattaga tgatagaaaa aca 33
<210> 238
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 238
aatcaaaaat accagcaaaa cttttcct 28
<210> 239
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 239
tcctcccact ttggaagaac tgga 24
<210> 240
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 240
ctgcgaggct gattcttccc tct 23
<210> 241
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 241
caaaagggaa gaggctctgg aaaaa 25
<210> 242
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 242
gcaccttagc taatgagagt cacag 25
<210> 243
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 243
tatacacaca ctttaagtca gggtcttg 28
<210> 244
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 244
ctctcaaaat atttagctaa aaatggtggc 30
<210> 245
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 245
aaaaactact ttaaagttta cttggagcca 30
<210> 246
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 246
tggttgtaga catgtggcat aattttga 28
<210> 247
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 247
agtcatcaca cgatacaagc caatat 26
<210> 248
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 248
ggcttaaccg aatgtctcaa atggac 26
<210> 249
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 249
ggccagtatc ttcaactgaa atatcc 26
<210> 250
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 250
tcaagtcagc cactttagcc t 21
<210> 251
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 251
gtcacatgtc ttttggatac aaaccttc 28
<210> 252
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 252
ggcattcgct cttaataaaa catgct 26
<210> 253
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 253
acctgctcca cttgtacata ctactc 26
<210> 254
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 254
aaaaatctgt ttgctggtgc acaa 24
<210> 255
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 255
attaggattt cagtataccc cttggga 27
<210> 256
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 256
ctccttttca ctgcaatgac cgt 23
<210> 257
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 257
ttcattgcag gacgctagga aac 23
<210> 258
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 258
ctccctgagt aaataggtag catcc 25
<210> 259
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 259
ggtcctaaga tatgtcacaa gtccccat 28
<210> 260
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 260
gactttgtaa cctatctctt gggcct 26
<210> 261
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 261
aagctaggag ttctttcagg ca 22
<210> 262
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 262
aaaaactcat tgtttcacca aggct 25
<210> 263
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 263
agtttacctg cagtatttaa gaagtgga 28
<210> 264
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 264
gcccatgtaa tacatttcag tcaaaca 27
<210> 265
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 265
ggatagcatg atttgagagc ataaactg 28
<210> 266
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 266
gtactttttg gaggagatga ggtct 25
<210> 267
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 267
ggtcatatgg tatttctggc tgtaagg 27
<210> 268
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 268
attaaatgtc aagatcaaaa gatccggt 28
<210> 269
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 269
caaaagtaca ggaaatcagt ttgagagg 28
<210> 270
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 270
ttctctgaaa ctactgcatc taggac 26
<210> 271
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 271
agccttcttc tggtactttt taaaatctt 29
<210> 272
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 272
ttttattttc tgtgttcctt gctccatt 28
<210> 273
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 273
gaagtgtgat actattgtaa atgtgtattt gag 33
<210> 274
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 274
gggaaataaa aatagtccac tatgttttat gtt 33
<210> 275
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 275
tgtcacaggc aggatagtaa catc 24
<210> 276
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 276
ttagagtttc acttatgtga ccgtact 27
<210> 277
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 277
tctaggagag aggatatcaa aaattggc 28
<210> 278
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 278
tacaactctc ctactctgat gagca 25
<210> 279
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 279
tacggcatag aaagtttgtg caaaa 25
<210> 280
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 280
ggttatttcg ctttaagggc tttca 25
<210> 281
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 281
gaattctctc actgctagtg ggaaag 26
<210> 282
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 282
attgggctga catcatttaa atatccct 28
<210> 283
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 283
ggaagagtaa atctgtccct ctatcct 27
<210> 284
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 284
tgtaggagga tattcttcca ctcactc 27
<210> 285
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 285
aaggaaaaat cagaagtatc cctgaaga 28
<210> 286
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 286
gtcacttcaa cctcttgttg gaagattatt 30
<210> 287
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 287
cacagtactc actttaggtt tgccatat 28
<210> 288
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 288
tttcccagat cctgaaaaca aaacact 27
<210> 289
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 289
gcctggcaaa cttttcattg ctg 23
<210> 290
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 290
atcccaaggt ttatgaaaga atgtgtc 27
<210> 291
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 291
gccttgtctt gaagtggtat tggg 24
<210> 292
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 292
catctgaaac ccacatacaa cagtc 25
<210> 293
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 293
tttacagtcc cacagaagtc ct 22
<210> 294
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 294
aattcagcat acctgtagtg ctttg 25
<210> 295
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 295
aagggcagag agaacatgta aataataatt c 31
<210> 296
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 296
catgtcaggg attcaggaaa agacaat 27
<210> 297
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 297
tttcaacttg cttacattgg taaaacaaac 30
<210> 298
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 298
tagaagatcc acttccagat tatagggt 28
<210> 299
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 299
atgtagaagc attgatttta agcaacca 28
<210> 300
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 300
gctcagttcc atgcaggtct tat 23
<210> 301
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 301
aaaagaaaga aagaaagaat gaaagaggga ata 33
<210> 302
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 302
ttctccttgc atgagctttg atg 23
<210> 303
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 303
catagaagtg gctctgcagt tgagt 25
<210> 304
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 304
ctttcagaat agaatccatg aaactgcg 28
<210> 305
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 305
tacatatgta acaaacctgc actttgcac 29
<210> 306
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 306
tcaatagtca ttttataatt gaacttcatg cc 32
<210> 307
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 307
aaaaagcaaa aggcaactac agattgaa 28
<210> 308
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 308
ttattctctc tcctgcagat tgtgt 25
<210> 309
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 309
tattctttgg tgtcagtcca tactagtg 28
<210> 310
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 310
cttgtaagga agccatccca caaatc 26
<210> 311
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 311
aaaagcctcc tacaatgtgc aa 22
<210> 312
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 312
gaagggctct tccattggg 19
<210> 313
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 313
cagaaagaag agatttctag ccagagtca 29
<210> 314
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 314
acctcttcct tgagtaaaat ttattctctc act 33
<210> 315
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 315
taaaaataca aaattagctg gatgtagtgg tgg 33
<210> 316
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 316
tttaaatttt gaggtagagt ttcactcttg tta 33
<210> 317
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 317
aactggccac cctagccttt taaatat 27
<210> 318
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 318
tatgcaaatg ccagcgttag aaac 24
<210> 319
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 319
aacaacagtt cgccatgttc ttc 23
<210> 320
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 320
ttctccttgc atgagctttg atg 23
<210> 321
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 321
atccagacaa gagtgctaaa aaccaag 27
<210> 322
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 322
caatgctgcc ccatccatat actg 24
<210> 323
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 323
caggtgtctg cactcaatct cttttt 26
<210> 324
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 324
cagggaaaac caaactcgca atg 23
<210> 325
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 325
tgtatgatca tgcctagcct cattc 25
<210> 326
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 326
cccggccact atacttcttt tgtg 24
<210> 327
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 327
ttttattcct ttgctcattt tccccttc 28
<210> 328
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 328
tggtaaacta agtaaggagt gtaaaggg 28
<210> 329
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 329
aaggatctgc aggatttggt aagtc 25
<210> 330
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 330
aacccatagc taggcagatc ctaa 24
Claims (5)
1. The forensic medicine next-generation sequencing kit based on 165Y-SNPs is characterized in that: comprises a composite amplification primer mixture; the composite amplification primer mixture comprises 330 amplification primers in total of 165Y-SNP genetic markers;
the nucleotide sequences of the composite amplification primers are respectively shown as SEQ ID No.1 to SEQ ID No. 330.
2. The forensic next-generation sequencing kit based on 165Y-SNPs according to claim 1, wherein: also comprises a composite amplification reaction mixed solution; in particular to a PCR reaction mixture provided by Ion AmpliSeq Library Kit 2.0 of Saimer Federation.
3. The forensic next-generation sequencing kit based on 165Y-SNPs according to claim 1, wherein: standard DNA is also included.
4. The forensic second-generation sequencing kit based on 165Y-SNPs according to claim 3, wherein: the standard DNA is 007 standard DNA.
5. Use of the 165Y-SNP based forensic next-generation sequencing kit of any one of claims 1 to 4 for haplotyping of a Chinese male sample of unknown origin.
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| CN112342303A (en) * | 2020-12-04 | 2021-02-09 | 郑州高新生物技术有限公司 | NGS-based human Y chromosome STR and SNP genetic marker combined detection system and detection method |
| CN112695100A (en) * | 2021-01-12 | 2021-04-23 | 郑州高新生物技术有限公司 | STR and SNP genetic marker combined detection system and detection method based on NGS |
| CN113981102A (en) * | 2021-08-30 | 2022-01-28 | 司法鉴定科学研究院 | Primer composition, kit and method for detecting Y-SNP (Y-single nucleotide polymorphism) haplotype group based on next-generation sequencing technology and application of primer composition, kit and method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614361A (en) * | 2013-08-20 | 2014-03-05 | 无锡中德美联生物技术有限公司 | Kit for multiplex amplification of 24 loci of human genome DNA |
| CN106399543A (en) * | 2016-10-26 | 2017-02-15 | 四川大学 | Forensic medicine II sequence testing kit based on 74 gama chromosome SNP genetic markers |
| CN107012226A (en) * | 2017-04-20 | 2017-08-04 | 司法部司法鉴定科学技术研究所 | A kind of detection kit and its detection method of the SNP site based on high-flux sequence |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614361A (en) * | 2013-08-20 | 2014-03-05 | 无锡中德美联生物技术有限公司 | Kit for multiplex amplification of 24 loci of human genome DNA |
| CN106399543A (en) * | 2016-10-26 | 2017-02-15 | 四川大学 | Forensic medicine II sequence testing kit based on 74 gama chromosome SNP genetic markers |
| CN107012226A (en) * | 2017-04-20 | 2017-08-04 | 司法部司法鉴定科学技术研究所 | A kind of detection kit and its detection method of the SNP site based on high-flux sequence |
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