CN116769924A - Detection kit comprising 90 short fragment micro-haplotype sites and application thereof - Google Patents

Abstract

The invention discloses a detection kit comprising 90 short fragment micro-haplotype sites and application thereof, and relates to the technical field of medicine. The target composite amplification system comprising 90 target micro-haplotype sites is provided, and a corresponding human body detection material parting kit is prepared based on the target composite amplification system, so that the research of different groups of forensic medical individual identification, spoilage degradation detection materials, unbalanced mixed detection materials, complex genetic identification and family border inference is satisfied.

Description

Detection kit comprising 90 short fragment micro-haplotype sites and application thereof

Technical Field

The invention relates to the technical field of medicine, in particular to a detection kit comprising 90 short fragment micro-haplotype sites and application thereof.

Background

The increased number of paternity cases required to be supplemented and rechecked in forensic examination works, the forensic DNA identification often encounters some difficult problems in daily practice, such as identification of problematic relationships (half siblings, full siblings, etc.), identification typing of severely degraded biological examination materials, puzzle of population origin, etc., and the above problems have not prevented the progress of forensic science. The analysis of DNA genetic markers in on-site biological detection materials is the most valuable individual identification tool at present, various DNA genetic markers have advantages, disadvantages and application ranges, and the selection and evaluation of DNA genetic markers with high polymorphism, which are different from common DNA genetic markers, are important links for solving the problems.

The most commonly used DNA genetic markers at present are STR (short tandem repeat), which plays an important role in forensic individual identification and affinity identification, but in actual forensic practice, high-level spoilage degradation detection materials, unbalanced mixed spot detection materials, complex difficult and complicated affinity identification cases and the like are frequently encountered, and when the difficult cases are solved, STR often cannot provide enough information, and cannot make conclusions about the high-level spoilage detection materials, the individual identification of small mixed spots contributors and the elimination or identification of the complex difficult and complicated affinity. In addition, because of the higher mutation rate of STR, the efficacy of the STR is limited in the research of deducing the source of forensic genetic ancestor, and smaller-fragment DNA genetic markers with lower mutation rate are key to solving the problems. SNP genetic markers and InDel genetic markers are genetic markers which play an important role in the judicial practice of forensic science due to smaller amplified fragments and lower mutation rate, but the two genetic markers are two-level genetic markers, have poor polymorphism, and need more sites to solve the problems. MH is a DNA fragment region composed of more than 2 SNP loci, and belongs to multiallelic genetic markers. The MH has the advantages of small SNP/InDel fragment and low mutation rate, has the advantage of high STR polymorphism, and is the most potential forensic practice supplementary genetic marker at present.

The Kidd professor of the university of United states medical college, 2013, et al, proposed the concept of Microshaplotype (MH) and applied it to the court science field for the first time. MH is a polymorphic site combination comprising two or more single nucleotide polymorphisms (single nueleotide polymorphism, SNPs) within a DNA region and having linkage disequilibrium and a length of less than 200 bp. MH has unique advantages as a novel DNA genetic marker over other genetic markers, and has recently been receiving attention from scientific researchers in the fields of forensic science and human genetics research.

MH has the following advantages over the genetic markers currently in common use: (1) Compared with STR genetic markers, the method has the advantages that interference of a stutter peak does not occur after amplification, the complexity of a secondary contributor of an interpretation mixed sample can be reduced, and the accuracy is improved; (2) The polymorphism is high and the mutation rate is low, the MH can comprise a plurality of SNP loci, belongs to multi-allele genetic markers, has high polymorphism of STR genetic markers, but the mutation rate is 5-6 orders of magnitude lower than that of STR genetic markers like SNP; (3) The fragment is short, and can be perfectly combined with the NGS technology, so that the multi-site high-efficiency synchronous detection typing can be realized. In view of the characteristics of the MH, the MH is expected to be a powerful supplement for traditional genetic markers such as forensic personal identification, paternity test, and suspicion genetic relationship test.

Mixed spots are biological specimens frequently encountered in forensic practice, and commonly used short tandem repeat (short tandem repeat, STR) genetic markers are poorly effective in unbalanced mixed spots, with minor contributors being difficult to detect in mixed spots below 1:20. Research shows that a Micro Haplotype (MH) detection system can realize the qualitative of main contributors in balanced and unbalanced mixed spots, and simultaneously can quantitatively analyze main components and secondary components in the unbalanced mixed spots, and can realize the qualitative of secondary contributors with the proportion higher than 1:99 in the mixed spots if enough sites can be captured. Forensic medical practice often encounters forensic examination materials damaged by environmental factors such as high temperature, microbial degradation and the like, and conventional genetic markers STR and the like often cannot obtain enough genetic information so that forensic identification work is in trouble. The characteristic of small MH genetic mark fragments can be well applied to the identification of putrefactive degradation detection materials, more genetic information can be obtained aiming at the simulated degradation detection materials compared with the traditional STR genetic mark, and the parting success rate is far higher than that of the STR genetic mark. Meanwhile, researchers evaluate the identification efficiency of the MH detection system in complex affinities, and the results show that the full sibling, half sibling and irrelevant individuals can be well distinguished by different likelihood distributions in different distant affinities. Furthermore, MH achieves noninvasive prenatal paternity testing even with extremely fragmented fetal free DNA obtained in maternal peripheral serum. The low mutation rate of MH and the characteristic that the genetic stability is higher than that of STR make it have unique advantage in tumor examination material or tumor cell line identification.

In summary, MH has excellent application value and prospect in forensic judicial practice and forensic genetics, but population genetic data of MH is still insufficient at present, and on the other hand, it is necessary to develop a sufficient number of MH sites with multiple alleles, small fragments and high polymorphism to realize the application value.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a detection kit comprising 90 short-segment micro-haplotype sites and application thereof, and provides a targeting composite amplification system comprising 90 target micro-haplotype sites, and a corresponding human body detection material parting kit is prepared by using the targeting composite amplification system as a basis, so that the detection kit can meet the requirements of different groups of forensic individual identification, putrefactive degradation detection materials, unbalanced mixed detection materials, complex genetic identification and family border inference research.

In order to achieve the above object, the present invention is realized by the following technical scheme: the method for obtaining 90 micro-haplotype sites is characterized by comprising the following steps: the method comprises the following steps:

step one: based on polymorphic SNP loci (minimum allele frequency > 0.1) recorded in a thousand-person genome database, a Perl program is compiled, and loci with polymorphic SNP in the flanking region of 100bp of the SNP loci are screened out. Next, these sites were further selected according to the following criteria:

a. the selected SNP locus is required to be positioned in a pseudogene, a gene intron or a non-coding region, so that the influence of potential pathogenic causes of individuals is avoided;

b. constructing MH haplotypes in each region by adopting PHASE v2.1 software, wherein SNP loci in a single MH region have different allele frequency distribution, and the linkage disequilibrium between two SNPs is checked that r2 is more than 0.6;

c. the screened MH locus accords with Hardy-Weinberg equilibrium (HWE) rule in east Asia population, and the MH locus on the same chromosome is not deviated from Linkage Disequilibrium (LD) test;

d. the MH site screened was higher polymorphic in the east asia population and was not contained in ALFRED and MicroHapDB databases.

Step two: the genetic polymorphism and forensic application value of the candidate sites in 26 individuals 2504 groups in 5 different areas of Africa, south Asia, east Asia, europe and America are evaluated, STRAF software is adopted to calculate parameters including He, PIC, CMP and CPE, and the site with the highest forensic parameters in the east Asia groups is selected.

Step three: optimizing a system site: the system site amplification compatibility is good, and the GC content is between 45% and 55%; and the system site number is controlled within 100 by considering the test efficiency and economy.

Specific information for the 90 microsloid markers selected is shown in table 1:

TABLE 1 composition and distribution information of 90 micro-haplotype loci screened according to the invention

Genomic DNA extraction and quantification of the present invention

a. Genomic DNA extraction: and extracting the whole genome DNA from the whole blood sample according to the instruction of the kit by using the Ezup column type blood genome DNA extraction kit. The invention is also applicable to genomic DNA extracted from other human subjects. In particular, it may be serum, saliva stain, saliva, sperm stain, semen or other tissue, etc.

b. Quantification of genomic DNA: sample genomic DNA was quantified using Qubit 4.0 and QubitTM dsDNAHS Assay Kit.

Multiplex PCR-targeted amplification of the present invention

a. The multiplex PCR targeted amplification system comprises 90 pairs of primers and is characterized by being simultaneously applicable to Ion S5 ^TM Platform and Illumina platform. The primer sequences used in the 90 micro-haplotype targeted amplification systems provided by the invention are shown in Table 2:

table 2: primer pair of 90 micro-haplotype sites screened by the invention

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b. The target amplification system comprises the following components: template DNA10ng,5 Xion AmpliSeq ^TM HiFi Mix buffer 4. Mu.L, primer Mix 10. Mu.L, make up ddH ₂ O to 20. Mu.L. The multiplex PCR reaction conditions were: 99 ℃ for 2min;99 ℃,15s,60 ℃,4min,16 cycles; 60 ℃ for 10min;4 ℃, and preserving.

c. Digestion of amplification products: 2. Mu.L FuPa was added to the amplification sample reaction tube, vortexed and mixed well, and centrifuged instantaneously to ensure no bubbles in the tube. Placing a PCR instrument, wherein the PCR reaction conditions are as follows: 50 ℃,10min,55 ℃,10min,60 ℃ and 20min.

Library construction, quantification and sequencing of the invention: linker and purification according to the instructions, ionLibrary was usedThe library was qPCR quantified using the Quantitation Kit. The quantitative result was multiplied by the dilution factor, the library concentration was calculated, and the library was diluted to 40pM based on the calculated library concentration. Absorbing 25 mu L of diluted library in Ion S5 ^TM And (5) sequencing on a platform machine.

The invention has the beneficial effects that:

1. the invention provides a kit for detecting more than 90 short fragment micro-haplotype sites. Compared with the STR which is most commonly used at present, the method has the advantages of low mutation rate, no interference of stutter peak typing, more balanced allele amplification, shorter fragments and the like, and the typing can be perfectly combined with an NGS technology, so that multi-site synchronous typing can be realized, and the component proportion of different alleles from different samples can be accurately estimated. The gene is not coincident with the reported micro haplotype locus, is not contained in ALFRED and MicroHapDB databases, is a set of brand-new haplotype locus, and has the advantage of shorter amplified fragments. Experiments prove that the synchronous parting detection kit based on the NGS technology can be applied to forensic putrefaction degradation detection materials, low-proportion unbalanced mixed spot detection materials and complex and difficult genetic relationship identification.

2. The micro-haplotype site reported by the invention has the following advantages:

a. the polymorphism is higher, and the fragment is small. The length of the inserted fragment of the constructed 90MH locus sequencing system is less than 100bp, wherein the Ae value of 80 MH loci is more than 2, the system is more suitable for individual identification of putrefactive degradation detection materials and mixed detection materials and other forensic medical practices, and the research result also supports the conclusion.

b. The system efficiency is better. From the detection result of 224 Henan Han nationality samples, the average allele sequencing depth (depth of coverage, doC) value of 90MH loci is 279-567, and the overall distribution is more average; average allele coverage ratio (allele coverage ratio, ACR) for 90MH sites was 0.8611-0.9674, with higher ACR for 90 sites, indicating more balanced allele amplification for each site. These values demonstrate that the 90MH site amplification system has better sequencing performance.

c. The population genetics has better application value. The screened 90MH loci show larger genetic variation in different intercontinental populations, and the degree of distinction of the intercontinental populations is better; wherein the In value of 20 sites is more than 0.1, and the AIM can be used as a candidate AIM for the ancestor identification study of the intercontinental groups.

Drawings

The invention is described in detail below with reference to the drawings and the detailed description;

FIG. 1 is a graph of CMP and CPE at 90 sites in the Henan Han population;

fig. 2 is a graph of He and PIC value distributions for 90MHs in 5 different intercontinental populations; (a) a distribution of He values for 90MHs in different intercontinental populations; (b) He value distribution of different intercontinental populations 90 MHs; (c) a distribution of PIC values for 90MHs in different intercontinental populations; (d) PIC value distributions of different intercontinental populations 90 MHs;

FIG. 3 is a graph of the In value distribution of 90MHs In 5 different intercontinental populations and Henan Han nationality, where (a) 90MHs different intercontinental populations may be sourced and potencies In differentiating intercontinental populations; (b) cumulative In value distribution for 90 different intercontinental populations of MHs; (c) 90MHs are arranged In the In value of Henan Han nationality;

FIG. 4 shows the allele sequencing depth (a), allele coverage (b) and sequencing noise level (c) for 90MH sites;

FIG. 5 shows the typing results of 20 STRs after ultrasonic disruption at different times for positive standards;

FIG. 6 shows allele sequencing depth (a), allele coverage (b), and sequencing noise level (c) for 90 microsloid sites;

FIG. 7 shows the results of STR typing at 5 representative sites with different mixing ratios for 9947A and 2800M standards;

FIG. 8 is a box plot of noise, dilution 200-fold, 100-fold, 40-fold, and 20-fold NL;

FIG. 9 is an isotactic/independent individual Log10LR distribution density map and graph (a) and a hemi-sibling/independent individual Log10LR distribution density map and graph (b) simulated using the 90MH locus of this study.

Detailed Description

The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.

Based on a public database and an NGS technology, the specific embodiment screens more micro-haplotype sites with high polymorphism and insert less than 100bp, and evaluates the forensic application value of the sites. The forensic efficacy evaluation is carried out on 90 selected short segment MH sites in Henan Han population, the forensic application value of the system in simulated putrefactive degradation detection materials and unbalanced mixed detection materials is evaluated, and the full sibling/half sibling genetic identification efficacy test is carried out on the system. Based on this, a targeted multiplex amplification system comprising 90 short-fragment MH sites was constructed.

Example 1

This example is an evaluation of the system efficacy of 90MH genetic markers.

1. Sample collection and reference populations

1.224 Henan Han nationality samples were collected in Henan area of China, 224 volunteers were healthy unrelated individuals, and peripheral blood samples were collected with EDTA anticoagulant tubes at 2ml. All participants were informed of the purpose and content of the study and signed informed consent.

2. Reference population information

The reference population was from five intercontinental populations (Africa, american, south Asia, east Asia and European populations) and the reference population genetics data was from the thousand genome project ((http:// www.1000genomes.org)). Basic information for 26 populations of 5 different intercontinents is shown in table 3.

Table 3: basic information of 26 groups between 5 different intercontinents

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2. Experimental method

DNA extraction and quantification

Extracting DNA by using Ezup column type blood genome DNA extraction kit (Shanghai bioengineering Co., ltd.), adding 200ul of buffer solution GB and 20ul of premixed solution of proteinase K into 200ul of peripheral blood sample, fully and reversely mixing, standing at 56 ℃ for 10min, and reversely mixing for several times; standing at room temperature for 5min, adding 350ul buffer BD, and fully and reversely mixing; rinsing the DNA, the rinsing being repeated twice; the DNA was collected by elution into a centrifuge tube. The tissue sample can be subjected to DNA extraction by using other commercial kits, and no special requirement is made. The DNA concentration of each sample was determined using a Qubit 4.0 assay.

2. Multiplex PCR targeting amplification

The configuration of the amplification system was as shown in Table 4, and the PCR reaction was carried out as shown in Table 5.

Table 4: proportion of amplification System

Table 5: multiplex PCR reaction conditions

3. Library preparation, digestion of amplified products, ligation junctions and purification

2. Mu.L FuPa was added to the amplification sample reaction tube, vortexed and mixed well, and centrifuged instantaneously to ensure no bubbles in the tube. The PCR instrument was placed and the setup procedure was as follows: 50 ℃,10min,55 ℃,10min,60 ℃ and 20min. To the resulting digested product of the previous step, 4. Mu.L of Switch solution, barcodeadapter mix. Mu.L of diluted DAN Ligase 2. Mu.L, and a final volume of 30. Mu.L were sequentially added. After vortex mixing, the PCR instrument was placed and set up as follows: 22 ℃,30min,68 ℃,5min,72 ℃ and 5min. The liquid in the reaction well is collected by instantaneous centrifugation at the bottom of the tube and transferred to 54. Mu.L of Agenemy added in advance ^TM AMPure ^TM In a 1.5mL non-stick tube of XP Reagent, vortex mix well. The next purification step was performed according to the instructions. The concentration was determined for Qubit after elution.

4. Library quantification, template preparation and sequencing

a. Using Ion LibraryThe library was qPCR quantified using the Quantitation Kit. The reaction system was 10uL, according to 5.0uL 2×/>Master Mix，0.5uL 20×Ion/>Assay and 4.5uL diluted library to configure the reaction system, standard library dilutions and negative control settings were repeated twice. The setup procedure was as follows: 95 ℃,1s,60 ℃,20s,40 cycles. The quantitative result was multiplied by the dilution factor, the library concentration was calculated, and the library was diluted to 40pM based on the calculated library concentration.

b. Creating an experiment plan: logging in to connect Ion Chef using Torrent Browser ^TM The Server of the instrument (Torrent Server) browses pages; selecting a Plan tab page, clicking on Templates, selecting an application type to be Run, and selecting Plan New Run; in the Kit tab page, select Ion Chef, and select Ion 540 in the Template Kit drop-down menu ^TM Kit Chef option; selecting Ion S5 in Sequencing Kit drop-down menu ^TM A Sequencing Kit option; a Ion Chef Library Sample Tube bar code (Barcode) is then entered in the Sample Tube Label field in the Plan tab page, saving the Plan.

c. Diluting the sample library: library stock solutions were diluted with nuclease-free water.

d. Preparing libraries and consumables: ion 540 was turned on 45 minutes before use ^TM Chef Reagent Cartridge, allowing to equilibrate to room temperature; mu.L of diluted library was pipetted into Ion Chef ^TM Library Sample Tube bottom; and (3) unsealing all consumable materials and placing beside the instrument for standby, installing a suction head box and a PCR plate according to the illustration of an instrument instruction book, and installing a reagent and solution module.

e. Start Ion Chef ^TM And (3) running: determining that all reagents and consumables are installed correctly, clicking a set up run option, selecting a Step by Step option, and then setting according to the content of a guide page of the instrument.

f. Sequencing: from Ion Chef ^TM Taking out the adapter/chip centrifugal basket combination from the instrument, carefully taking out the chip, and putting the chip into Ion S5 ^TM Sequencing instrument, initiallyThe instrument was initialized and the sequencing reaction was started as prompted.

g. Cleaning an instrument: according to the operation instructions.

3. Experimental results

Firstly, the forensic application value of 90 micro-haplotype genetic markers in Henan Han population is evaluated. The STRAF software was used to calculate forensic parameters for 90 micro-haplotype genetic markers in Henan Han population. The Ae value distribution of the 90 micro-haplotype sites was obtained as shown in table 6. The Ae values for 90 micro-haplotype sites were distributed as 1.7649 (MH 45) -3.9792 (MH 50), and the Ae values for 80 MH sites were all greater than 2. In the Henan Han population, the average He, ho, PIC, MP, DP and PE values for 90MH sites were 0.5788, 0.5851, 0.5039, 0.2608, 0.7392 and 0.2806, respectively. The cumulative match probability (cumulative match probability, CMP) and cumulative non-father exclusion probability (cumulative probability of exclusion, CPE) for 90MH sites in the study population were calculated, as shown in fig. 1, and it was seen that the CMP values for 90MH sites in the study population were less than 10-54, and the CPE values for 90MH sites reached 0.999999999999923.

Table 6: forensic parameters of 90 micro-haplotype genetic markers in Henan Han population

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The genetic diversity and forensic application value of 90 microscales in different intercontinental populations were then assessed. By analyzing the thousand person genome data (http:// www.1000genomes.org), 90 candidate MHs genotypes from 26 populations (2504 persons) of 5 different regions (AFR, south asia AMR, eastern asia EAS, european EUR and american SAS) were used to evaluate the polymorphisms and forensic application value of these genetic loci. He, PIC, DP, PE, CMP and CPE were calculated based on the genotyping data for 90MHs of the 5 regions described above. He and PIC values were first calculated (fig. 2). As can be seen from the results, the He values of 82 other MHs except for MH18, MH29, MH40, MH44, MH45, MH53, MH79 and MH90 were all greater than 0.5 in the EAS population (fig. 2 a). The highest and lowest He values are distributed over MH50 and MH79. Some MHs, such as MH3, MH10, MH18, MH21, MH24, MH37, MH46, MH83 and MH88, were also observed for which He values were widely different between the populations. The average He value of 90MHs was greater than 0.5 (fig. 2 b). For PIC values, the distribution pattern in the population is similar to He values. The PIC values of 83 MHs in the EAS population were greater than 0.4 (fig. 2 c) and the average PIC value of 90 micro-haplotypes was greater than 0.5 (fig. 2 d). CPE and CMP values for 90MHs sites in the 5 intercontinental populations were calculated (see Table 7), and for 90MHs in the east Asia population were 1.1688X 10-54 and 0.999999999998954, respectively. Overall, these sites have higher polymorphisms in the east asia population.

Table 7: CPE and CMP values for 90 micro-haplotype sites in these 5 intercontinental populations

To assess the genetic distribution of 90MHs sites In different intercontinental populations, the In values of these sites In africa, south asia, east asia, european and american populations and the hewanan han family were calculated using INFOCALC software, see fig. 3. The possible sources of the ancestor population for each site are shown, see fig. 3 (a), which shows the higher efficacy of these sites in distinguishing africa from other intercontinental populations; further showing the cumulative In values for 5 different intercontinental populations and the differences between each other, see fig. 3 (b), where it can be seen that african, eastern and european populations can be effectively distinguished; finally, the In value arrangement of Henan Han is shown, and as can be seen from FIG. 3 (c), the In value of 20 sites is greater than 0.1. Wherein, the In value of MH3 and MH71 is greater than 0.2. In summary, the present system can be used for progenitor studies.

Finally, the sequencing performance of the 90 micro-haplotype genetic marker system was evaluated, and the detection performance of the development system was evaluated using allele sequencing depth (depth of coverage, doC), allele coverage ratio (allele coverageratio, ACR) and sequencing Noise Level (NL) (fig. 4). From the detection results of 224 Henan Han nationality samples, the average DoC value of 90MH loci is 279-567, and the overall distribution is more average (FIG. 4 a); the average ACR values for 90MH sites ranged from 0.8611 to 0.9674, with higher ACR values for 90 sites (fig. 4 b), indicating a more balanced allele amplification for each site; the average NL value for 90MH sites was 0.0386-0.1036, with the exception of MH33 sites, the NL for the remaining sites were below 10% (fig. 4 c). These values demonstrate that the 90MH site amplification system has better sequencing performance.

4. Conclusion of the experiment

The constructed NGS sequencing system with 90 micro-haplotype sites shows higher polymorphism in Henan Han population and east Asia population, and the system can be used for forensic medical genetic relationship identification and individual identification application and research of the east Asia population.

The screened 90 micro haplotype sites show larger genetic variation in different intercontinental populations, and the degree of distinction of the intercontinental populations is better; wherein the In value of 20 sites is more than 0.1, and the AIM can be used as a candidate AIM for the ancestor identification study of the intercontinental groups.

Example 2

The present example is to evaluate the application value of 90MH genetic markers in spoilage degradation, low-proportion mixed samples and complex genetic relationship identification.

1. Experimental method

1. Preparation of putrefactive degradation sample

The spoilage samples were simulated by subjecting 2800M (concentration 10 ng/. Mu.L) to various cycles of ultrasonic fragmentation using a non-contact ultrasonic breaker (Diagenode Corp.) with the following cycle numbers: 15 cycles (15 min), 30 cycles (30 min), 45 cycles (45 min), 60 cycles (60 min).

2. Mixed sample preparation

The 9947A and 2800M standards were mixed in different proportions to prepare mixed samples, the mixing proportions being: 1:4, 1:9, 1:19, 1:40, 1:49, 1:99.

3. Full sibling/half sibling paternity test

1000 pairs of isotactic (H1), 1000 pairs of hemi (H1) and 1000 pairs of irrelevant individuals (H2) were simulated using family 3 software, the Likelihood (LR) in the relationship was calculated based on the frequency data of 90MH sites of the study population, the LR distribution map in the relationship was plotted using R software (https:// www.r-project. Org /), and the identification efficacy of the system on the relationship was presented.

2. Experimental results

Application value of 1.90 micro-haplotype genetic markers in degradation sample

First applyThe 21 kit evaluates the simulated degradation detection materials one by one, evaluates the fragment range of the simulated degradation detection materials on one hand, and presents the identification capability of the conventional most commonly used STR amplification system on the market on the degradation detection materials on the other hand, and compares the identification capability with the 90 micro-haplotype genetic markers. The positive standard 2800M was used for typing in this section, and the results are shown in FIG. 5. As a result, 15min of ultrasonic disruption can affect the typing of STR sites with the size of more than 400bp, 30min of ultrasonic disruption can affect the typing of STR sites with the size of more than 300bp, 45min of ultrasonic disruption can affect the typing of STR sites with the size of more than 150-200bp, and 60min of ultrasonic disruption can affect the typing of STR sites with the size of more than 100-150 bp. Moreover, it can be seen that the longer the sonication time, the poorer the equalization of the allele amplification at the same site, for example the D1S1656 site and the D12S391 site of the short fragment, which increases the risk of misinterpretation and loss of the allele. The statistics of the number of the amplification lost and unbalanced sites after ultrasonic crushing of the standard products at different times is shown in Table 8, and the result shows that the standard product detection materials subjected to ultrasonic crushing treatment for 30min, 45min and 60min by using the system can not be identified by individuals.

Table 8: number of lost and unbalanced sites amplified after different times of ultrasonic disruption of standard

The simulated degradation samples (30 min, 45min and 60min ultrasonic crushing treatment) are simultaneously detected by using the constructed 90 small-fragment micro-haplotype locus system, and as a result, the samples subjected to ultrasonic treatment at different times can be completely typed. Good sequencing performance, see FIG. 6, doC in FIG. 6 (a), ACR in FIG. 6 (b), and NL in FIG. 6 (c).

Application value of 2.90 micro-haplotype genetic markers in mixed samples

First applyThe kit 21 detects the 5 groups of mixed samples, and results show that when the mixing ratio is 1:4 and 1:9, the low-ratio samples in the mixed samples can be basically subjected to result interpretation. At a mixing ratio of 1:19, the amplified low-ratio sample peak is very susceptible to interference from the stutter peak, which is close in height to the main peak, resulting in difficult allele resolution. At mixing ratios of 1:40, 1:49, and 1:99, the pp21 system was barely able to amplify low proportions of sample peaks. Here, 5 representative site amplification peak plots are shown, the results are shown in FIG. 7.

The above 1:19, 1:40, 1:49 and 1:99 samples were tested with the 90 small fragment MH site system constructed according to the above results. The different genotypes of the 90 microsloid loci for 9947A and 2800M standards are shown in table 9. Two of the 90 loci have 10 loci for the same homozygote, 22 loci for the same heterozygote, 2 loci for different homozygotes, 15 loci for 2 heterozygotes (1 allele in total), 9 loci for 1 homozygote 1 different heterozygote, and 32 loci for 1 homozygote 1 heterozygote (1 allele in total). The number of sites at which both mixed samples can be resolved was 44 depending on the genotype (shown bolded in table 7). From the experimental results, the lower components in different mixing ratios can be detected, but some of them cannot be separated from noise, and the following statistics are made. The number of SNP sites which can be resolved according to the SNP site is 60, and the resolvable SNP sites are divided into dilution 20 times (D20), dilution 40 times (D40), dilution 100 times (D100) and dilution 200 times (D200) according to the genotype of the SNP site and the mixing proportion, and the noise level (N) is evaluated by mixing samples which are the same and pure. Calculating the ratio (NL) of the sequencing depth to the total sequencing depth of the lower proportion samples, drawing a box graph for the NL grouping values to show the sequencing efficiency of the system on the mixed samples, and referring to FIG. 8, the result shows that the system can completely distinguish SNP loci with dilution factors less than 100 times from noise interference and most SNP loci with dilution factors of 200 times. That is, the system can complete the detection of smaller contributors with a mixing ratio of 1:49, and can complete the detection of most sites for 1:99.

Table 9: different genotypes of 90 microsloid sites for 9947A and 2800M standards

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Application value of 3.90 micro-haplotype genetic markers in identification of isocybrid/half-cybrid genetic relationship

Based on the frequency data and the simulation data of 90MH loci of the Henan Han population, the identification efficacy of 90MH genetic markers on the relationship between full siblings and half siblings is tested. Using the family 3 software construct, 1000 pairs of isotactic (H1), 1000 pairs of hemi-isotactic (H1) and 1000 pairs of independent individuals (H2) were simulated, and the LR and system performance at different thresholds at the time of detection of 90MH were calculated. The R software is used for drawing a Log10LR distribution density chart and a graph in the relationship, see fig. 9, and the application value of the 90MH genetic markers in genetic relationship identification is evaluated. As a result, it was found that there was little overlap between the LR distribution of isotactic cells and that of unrelated individuals. In the simulation of the whole sibling genetic relationship identification, when LR thresholds are set to 1, 10, 100, 1000 and 10000 respectively, the identification accuracy using the 90MH site system is respectively: 100%,100%,99.60%,99.30% and 98.60%, and the false positive rate was 0.00%. In the simulation of half-sibling genetic relationship identification, when LR thresholds were set to 1, 10, 100, 1000 and 10000, respectively, the identification accuracy using the 90MH site system was: 95.10%,84.80%,62.00%,37.10% and 16.80% false positive rates of 4.20%,1.20%,0.30%,0.00% and 0.00%, respectively, the 90MH system of this study can differentiate half-siblings to some extent.

3. Conclusion of the experiment

The length of the inserted fragment of the constructed NGS sequencing system with 90MH sites is less than 100bp, wherein the Ae values of 80 MH sites are all more than 2, the system can be used in forensic medical practice such as individual identification and complex genetic relationship identification of putrefactive degradation detection materials and mixed detection materials, and the conclusion is also supported by the above examples.

While the present invention has been particularly described with reference to exemplary embodiments, it is not intended to be construed as limiting the scope of the invention. And it should be noted that modifications, equivalents, or improvements thereto may be made by those skilled in the art without departing from the scope or spirit of the present invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (4)

1. A set of 90 micro-haplotype site combinations for forensic detection, wherein the 90 micro-haplotype sites are: MH01WHD-001, MH01WHD-002, MH01WHD-003, MH01WHD-004, MH01WHD-005, MH01WHD-006, MH01WHD-007, MH01WHD-008, MH02WHD-001, MH02WHD-002, MH02WHD-003, MH02WHD-005, MH02WHD-006, MH03WHD-001, MH03WHD-002, MH03WHD-003, MH03WHD-004, MH03WHD-005, MH03WHD-006, MH03WHD-007, MH03WHD-008, MH MH04WHD-001, MH04WHD-002, MH04WHD-003, MH04WHD-004, MH04WHD-005, MH05WHD-001, MH05WHD-002, MH05WHD-003, MH06WHD-001, MH06WHD-002, MH07WHD-001, MH07WHD-003, MH07WHD-004, MH08WHD-001, MH08WHD-002, MH08WHD-003, MH08WHD-004, MH08WHD-005, MH09WHD-001, MH09WHD-002 MH09WHD-003, MH09WHD-004, MH10WHD-001, MH10WHD-002, MH10WHD-003, MH10WHD-004, MH10WHD-005, MH10WHD-006, MH10WHD-007, MH11WHD-001, MH11WHD-002, MH11WHD-003, MH11WHD-004, MH11WHD-005, MH11WHD-006, MH11WHD-007, MH11WHD-008, MH12WHD-001, MH12WHD-002, MH12WHD-003, MH12WHD-004, MH13WHD-001, MH13WHD-002, MH14WHD-001, MH14WHD-002, MH14WHD-003, MH14WHD-004, MH15WHD-001, MH16WHD-002, MH16WHD-003, MH16WHD-004, MH16WHD-005, MH16WHD-006, MH17WHD-001, MH17WHD-002, MH17WHD-003, MH18WHD-001, MH18WHD-002, MH18WHD-003, MH19WHD-001, MH19WHD-002, MH19WHD-003, MH20WHD-001, MH20WHD-002, MH21WHD-001, MH21WHD-002.

2. A primer pair combination for detecting 90 micro-haplotype markers, which is characterized in that the 90 micro-haplotype markers are the 90 micro-haplotype markers in claim 1, and a primer pair for amplifying MH01WHD-001 consists of primers shown in SEQ ID NO.1 and SEQ ID NO.2 in a sequence table; the primer pair for amplifying MH01WHD-002 consists of primers shown as SEQ ID NO.3 and SEQ ID NO.4 in a sequence table; the primer pair of MH01WHD-003 consists of primers shown as SEQ ID NO.5 and SEQ ID NO.6 in a sequence table; the primer pair of MH01WHD-004 consists of primers shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence table; the primer pair of MH01WHD-005 consists of primers shown as SEQ ID NO.9 and SEQ ID NO.10 in a sequence table; the primer pair of MH01WHD-006 consists of primers shown as SEQ ID NO.11 and SEQ ID NO.12 in a sequence table; the primer pair of MH01WHD-007 consists of primers shown as SEQ ID NO.13 and SEQ ID NO.14 in a sequence table; the primer pair of MH01WHD-008 consists of primers shown as SEQ ID NO.15 and SEQ ID NO.16 in a sequence table; the primer pair of MH02WHD-001 consists of primers shown as SEQ ID NO.17 and SEQ ID NO.18 in a sequence table; the primer pair of MH02WHD-002 consists of primers shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence table; the primer pair of MH02WHD-003 consists of primers shown as SEQ ID NO.21 and SEQ ID NO.22 in a sequence table; the primer pair of MH02WHD-004 consists of primers shown as SEQ ID NO.23 and SEQ ID NO.24 in the sequence table; the primer pair of MH02WHD-005 consists of primers shown as SEQ ID NO.25 and SEQ ID NO.26 in the sequence table; the primer pair of MH02WHD-006 consists of primers shown as SEQ ID NO.27 and SEQ ID NO.28 in a sequence table; the primer pair of MH03WHD-001 consists of primers shown as SEQ ID NO.29 and SEQ ID NO.30 in a sequence table; the primer pair of MH03WHD-002 consists of primers shown as SEQ ID NO.31 and SEQ ID NO.32 in the sequence table; the primer pair of MH03WHD-003 consists of primers shown as SEQ ID NO.33 and SEQ ID NO.34 in a sequence table; the primer pair of MH03WHD-004 consists of primers shown as SEQ ID NO.35 and SEQ ID NO.36 in the sequence table; the primer pair of MH03WHD-005 consists of primers shown as SEQ ID NO.37 and SEQ ID NO.38 in a sequence table; the primer pair of MH03WHD-006 consists of primers shown as SEQ ID NO.39 and SEQ ID NO.40 in a sequence table; the primer pair of MH03WHD-007 consists of primers shown as SEQ ID NO.41 and SEQ ID NO.42 in a sequence table; the primer pair of MH03WHD-008 consists of primers shown as SEQ ID NO.43 and SEQ ID NO.44 in a sequence table; the primer pair of MH04WHD-001 consists of primers shown as SEQ ID NO.45 and SEQ ID NO.46 in a sequence table; the primer pair of MH04WHD-002 consists of primers shown as SEQ ID NO.47 and SEQ ID NO.48 in the sequence table; the primer pair of MH04WHD-003 consists of primers shown as SEQ ID NO.49 and SEQ ID NO.50 in a sequence table; the primer pair of MH04WHD-004 consists of primers shown as SEQ ID NO.51 and SEQ ID NO.52 in the sequence table; the primer pair of MH04WHD-005 consists of primers shown as SEQ ID NO.53 and SEQ ID NO.54 in the sequence table; the primer pair of MH05WHD-001 consists of primers shown as SEQ ID NO.55 and SEQ ID NO.56 in a sequence table; the primer pair of MH05WHD-002 consists of primers shown as SEQ ID NO.57 and SEQ ID NO.58 in the sequence table; the primer pair of MH05WHD-003 consists of primers shown as SEQ ID NO.59 and SEQ ID NO.60 in a sequence table; the primer pair of MH06WHD-001 consists of primers shown as SEQ ID NO.61 and SEQ ID NO.62 in the sequence table; the primer pair of MH06WHD-002 consists of primers shown as SEQ ID NO.63 and SEQ ID NO.64 in the sequence table; the primer pair of MH07WHD-001 consists of primers shown as SEQ ID NO.65 and SEQ ID NO.66 in a sequence table; the primer pair of MH07WHD-002 consists of primers shown as SEQ ID NO.67 and SEQ ID NO.68 in the sequence table; the primer pair of MH07WHD-003 consists of primers shown as SEQ ID NO.69 and SEQ ID NO.70 in a sequence table; the primer pair of MH07WHD-004 consists of primers shown as SEQ ID NO.71 and SEQ ID NO.72 in the sequence table; the primer pair of MH08WHD-001 consists of primers shown as SEQ ID NO.73 and SEQ ID NO.74 in a sequence table; the primer pair of MH08WHD-002 consists of primers shown as SEQ ID NO.75 and SEQ ID NO.76 in the sequence table; the primer pair of MH08WHD-003 consists of primers shown as SEQ ID NO.77 and SEQ ID NO.78 in a sequence table; the primer pair of MH08WHD-004 consists of primers shown as SEQ ID NO.79 and SEQ ID NO.80 in the sequence table; the primer pair of MH08WHD-005 consists of primers shown as SEQ ID NO.81 and SEQ ID NO.82 in the sequence table; the primer pair of MH09WHD-001 consists of primers shown as SEQ ID NO.83 and SEQ ID NO.84 in the sequence table; the primer pair of MH09WHD-002 consists of primers shown as SEQ ID NO.85 and SEQ ID NO.86 in the sequence table; the primer pair of MH09WHD-003 consists of primers shown as SEQ ID NO.87 and SEQ ID NO.88 in the sequence table; the primer pair of MH09WHD-004 consists of primers shown as SEQ ID NO.89 and SEQ ID NO.90 in the sequence table; the primer pair of MH10WHD-001 consists of primers shown as SEQ ID NO.91 and SEQ ID NO.92 in a sequence table; the primer pair of MH10WHD-002 consists of primers shown as SEQ ID NO.93 and SEQ ID NO.94 in the sequence table; the primer pair of MH10WHD-003 consists of primers shown as SEQ ID NO.95 and SEQ ID NO.96 in a sequence table; the primer pair of MH10WHD-004 consists of primers shown as SEQ ID NO.97 and SEQ ID NO.98 in the sequence table; the primer pair of MH10WHD-005 consists of primers shown as SEQ ID NO.99 and SEQ ID NO.100 in the sequence table; the primer pair of MH10WHD-006 consists of primers shown as SEQ ID NO.101 and SEQ ID NO.102 in a sequence table; the primer pair of MH10WHD-007 consists of primers shown as SEQ ID NO.103 and SEQ ID NO.104 in a sequence table; the primer pair of MH11WHD-001 consists of primers shown as SEQ ID NO.105 and SEQ ID NO.106 in a sequence table; the primer pair of MH11WHD-002 consists of primers shown as SEQ ID NO.107 and SEQ ID NO.108 in the sequence table; the primer pair of MH11WHD-003 consists of primers shown as SEQ ID NO.109 and SEQ ID NO.110 in a sequence table; the primer pair of MH11WHD-004 consists of primers shown as SEQ ID NO.111 and SEQ ID NO.112 in the sequence table; the primer pair of MH11WHD-005 consists of primers shown as SEQ ID NO.113 and SEQ ID NO.114 in a sequence table; the primer pair of MH11WHD-006 consists of primers shown as SEQ ID NO.115 and SEQ ID NO.116 in a sequence table; the primer pair of MH11WHD-007 consists of primers shown as SEQ ID NO.117 and SEQ ID NO.118 in the sequence table; the primer pair of MH11WHD-008 consists of primers shown as SEQ ID NO.119 and SEQ ID NO.120 in a sequence table; the primer pair of MH12WHD-001 consists of primers shown as SEQ ID NO.121 and SEQ ID NO.122 in a sequence table; the primer pair of MH12WHD-002 consists of primers shown as SEQ ID NO.123 and SEQ ID NO.124 in the sequence table; the primer pair of MH12WHD-003 consists of primers shown as SEQ ID NO.125 and SEQ ID NO.126 in a sequence table; the primer pair of MH12WHD-004 consists of primers shown as SEQ ID NO.127 and SEQ ID NO.128 in the sequence table; the primer pair of MH13WHD-001 consists of primers shown as SEQ ID NO.129 and SEQ ID NO.130 in the sequence table; the primer pair of MH13WHD-002 consists of primers shown as SEQ ID NO.131 and SEQ ID NO.132 in the sequence table; the primer pair of MH14WHD-001 consists of primers shown as SEQ ID NO.133 and SEQ ID NO.134 in a sequence table; the primer pair of MH14WHD-002 consists of primers shown as SEQ ID NO.135 and SEQ ID NO.136 in the sequence table; the primer pair of MH14WHD-003 consists of primers shown as SEQ ID NO.137 and SEQ ID NO.138 in a sequence table; the primer pair of MH14WHD-004 consists of primers shown as SEQ ID NO.139 and SEQ ID NO.140 in the sequence table; the primer pair of MH15WHD-001 consists of primers shown as SEQ ID NO.141 and SEQ ID NO.142 in the sequence table; the primer pair of MH16WHD-001 consists of primers shown as SEQ ID NO.143 and SEQ ID NO.144 in the sequence table; the primer pair of MH16WHD-002 consists of primers shown as SEQ ID NO.145 and SEQ ID NO.146 in the sequence table; the primer pair of MH16WHD-003 consists of primers shown as SEQ ID NO.147 and SEQ ID NO.148 in a sequence table; the primer pair of MH16WHD-004 consists of primers shown as SEQ ID NO.149 and SEQ ID NO.150 in the sequence table; the primer pair of MH16WHD-005 consists of primers shown as SEQ ID NO.151 and SEQ ID NO.152 in the sequence table; the primer pair of MH16WHD-006 consists of primers shown as SEQ ID NO.153 and SEQ ID NO.154 in a sequence table; the primer pair of MH17WHD-001 consists of primers shown as SEQ ID NO.155 and SEQ ID NO.156 in a sequence table; the primer pair of MH17WHD-002 consists of primers shown as SEQ ID NO.157 and SEQ ID NO.158 in the sequence table; the primer pair of MH17WHD-003 consists of primers shown as SEQ ID NO.159 and SEQ ID NO.160 in a sequence table; the primer pair of MH18WHD-001 consists of primers shown as SEQ ID NO.161 and SEQ ID NO.162 in a sequence table; the primer pair of MH18WHD-002 consists of primers shown as SEQ ID NO.163 and SEQ ID NO.164 in the sequence table; the primer pair of MH18WHD-003 consists of primers shown as SEQ ID NO.165 and SEQ ID NO.166 in a sequence table; the primer pair of MH19WHD-001 consists of primers shown as SEQ ID NO.167 and SEQ ID NO.168 in a sequence table; the primer pair of MH19WHD-002 consists of primers shown as SEQ ID NO.169 and SEQ ID NO.170 in the sequence table; the primer pair of MH19WHD-003 consists of primers shown as SEQ ID NO.171 and SEQ ID NO.172 in a sequence table; the primer pair of MH20WHD-001 consists of primers shown as SEQ ID NO.173 and SEQ ID NO.174 in a sequence table; the primer pair of MH20WHD-002 consists of primers shown as SEQ ID NO.175 and SEQ ID NO.176 in the sequence table; the primer pair of MH21WHD-001 consists of primers shown as SEQ ID NO.177 and SEQ ID NO.178 in the sequence table; the primer pair of MH21WHD-002 consists of the primers shown in SEQ ID No.179 and SEQ ID No.180 in the sequence list.

The method for obtaining the 90 micro-haplotype sites is characterized by comprising the following steps: the method comprises the following steps:

step one: based on polymorphic SNP loci recorded in a thousand-person genome database, programming a Perl program, and screening loci with polymorphic SNP in a flanking region of 100bp of the SNP loci; next, these sites were further selected according to the following criteria:

a. the selected SNP locus is required to be positioned in a pseudogene, a gene intron or a non-coding region, so that the influence of potential pathogenic causes of individuals is avoided;

b. constructing MH haplotypes in each region by adopting PHASE v2.1 software, wherein SNP loci in a single MH region have different allele frequency distribution, and the linkage disequilibrium between two SNPs is checked that r2 is more than 0.6;

c. the screened MH locus accords with the Hardy-Winberg equilibrium rule in the east Asia population, and the MH locus on the same chromosome does not deviate from linkage disequilibrium test;

d. the screened MH locus has higher polymorphism in east Asia population and is not contained in ALFRED and MicroHapDB databases;

step two: evaluating genetic polymorphism and forensic application value of 26 individuals 2504 in 5 different areas of Africa, south Asia, east Asia, europe and America of candidate sites, calculating parameters by adopting STRAF software, including He, PIC, CMP and CPE, and selecting the site with the highest forensic parameters in the east Asia group;

step three: optimizing a system site: the system site amplification compatibility is good, and the GC content is between 45% and 55%; and the system site number is controlled within 100 by considering the test efficiency and economy.

4. The use of 90 microscale haplotype markers for forensic detection according to claim 1 in spoilage detection, unbalanced mixture detection, complex genetic identification, and family border inference.