CN116377084A - High-efficiency autosomal micro-haplotype genetic marker system, and detection primer and kit thereof - Google Patents

Abstract

The invention discloses a high-efficiency autosomal micro-haplotype genetic marker system, and a detection primer and a kit thereof. The genetic marker system comprises 50 micro-haplotype loci, and in addition, the invention also provides a primer for detecting the genetic marker system, and the primer sequence is shown in SEQ ID NO. 1-104. The invention combines a multiplex PCR targeting capture sequencing technology and a homemade typing scheme (Python and R) to construct a high-efficiency 50plex MH panel based on MPS for forensic DNA analysis. And the potential of the panel in forensic applications including sensitivity, accuracy, polymorphism, forensic parameters, single source degradation samples, mixed degradation samples and species specificity is comprehensively explored.

Description

High-efficiency autosomal micro-haplotype genetic marker system, and detection primer and kit thereof

Technical Field

The invention belongs to the technical field of forensic identification, and particularly relates to a high-efficiency autosomal micro-haplotype genetic marker system, and a detection primer and a kit thereof.

Background

Microsoid (MHs) is a novel genetic marker proposed by Kidd laboratories in 2013 to supplement DNA genotyping tools currently used in forensic genetics. They are characterized by the presence of two or more closely related SNPs within 300,bp and by the presence of three or more alleles (haplotypes). Thus, they provide more information than single SNPs and exhibit lower recombination rates over such short distances (assuming on average 1% recombination per megabase and no recombination hotspots within the site). Microshenotypes do not preferentially amplify certain alleles within a locus because all alleles at a locus are the same size. MHs has no stutter pseudopeaks, lower mutation rate, and fewer alleles compared to STRs. A large number of MHs can approach the recognition capability of a group of STRs and provide valuable information in individual recognition, mixed plaque interpretation, ancestral prediction, paternity identification, and medical diagnostic applications. They are therefore becoming increasingly popular in the forensic DNA field and are used in various related studies.

Currently, massively Parallel Sequencing (MPS) is the dominant method of detection MHs. Sanger sequencing is a "gold standard" for DNA sequencing. However, when two or more loci are heterozygous, it is impossible to determine the cis-trans relationship, i.e., haplotype phase, between individual SNP alleles in genomic DNA. Our previous studies showed that Capillary Electrophoresis (CE) platform, although capable of typing MHs, can only separate MHs consisting of two SNPs, with low detection throughput at a time. However, MPS can make up for the deficiencies of Sanger sequencing and CE platforms. It can identify each MH allele at a specific site by cloning and sequencing each amplicon of each DNA strand present in the sample, whether it is single-sourced or mixed in origin. In addition, MPS platform has higher sequencing throughput, and hundreds of thousands of variations can be detected simultaneously. Thus, it makes it possible for forensic analysis of MH defined by multiple SNPs, while the combination of different SNP alleles within a single short site can provide a greater probability of individual identification. Therefore, the MPS technique can clone and sequence the male parent haplotype on the male parent chromosome and the female parent chromosome, and greatly enhances the characterization capability of the forensic MH.

The international reported panel has successfully developed a new optimized MH set. Thus, more and more identities, ancestors, and effective MHs of mixed plaques have recently been published and offered to the global forensic community. Analysis of these markers and population genetic data will serve as the basis for MH, DNA analysis in case work in the future. When an individual of interest (POI) cannot be excluded as a possible component of forensic biological evidence, population-specific allele frequencies are used to estimate the statistical weight of the evidence. Similar to conventional STRs, the use of MH sequencing in individual case work requires the development of large and appropriate Allele Frequency (AF) datasets. Although Kidd's laboratory has collected AF data of the original MH in the global population and uploaded it to ALFRED (allele frequency database). ALFRED, however, does not include MH, AF of the chinese southwest han population (chang city), which would hamper relevant forensic application studies. Although the MicroHapDB established by stadag et al contains the basic parameters of 412 MHs in 26 populations, these parameters are based on statistical inferences and are not accurate enough to be practical in forensics. Furthermore, microHapDB does not provide data for "the original pool of sites for MH", which makes it difficult to meet different research requirements.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a high-efficiency autosomal micro-haplotype genetic marker system, a detection primer and a kit thereof, and a screen of the invention178A in the thousand people genome data were selected _e Candidate micro haplotypes above 4 are reached. Specific primers are designed for the 178 candidate sites, and a set of 128 micro-haplotype site multiplex detection system with high recognition capability is compounded. And, after correction, the 50MHs which are finally reserved have good repeatability, extremely high sensitivity and stability.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a high-efficiency autosomal micro-haplotype genetic marker system comprising at least one of the following tables, the specific information being as in table 1 (in ascending order of chromosome and physical location):

TABLE 150 micro haplotype information

a represents a group previously reported

A primer for amplifying the high-efficiency autosomal micro-haplotype genetic marker system, which consists of 104 primers; the primer is a single-stranded DNA molecule; the primer sequences are shown in SEQ ID NO. 1-104.

The use of the above primers in forensic identification.

A forensic identification kit comprising the above primer.

Further, the kit can be used for individual identification or genetic relationship identification.

Further, buffers, DNA polymerase and dntps are included.

The application method of the kit comprises the following steps: extracting genome DNA of a sample to be detected as a template, then carrying out multiplex PCR amplification, carrying out a linker sequence PCR reaction on the obtained amplified product to obtain an amplified library, and carrying out quantitative and second-generation sequencing detection analysis on the amplified library to obtain a typing result of the micro-haplotype locus. Wherein, the concentration of the genome DNA is larger than 18 ng/. Mu.L, and the concentration of each primer is 5-10 mu.M.

Further, the multiplex PCR reaction system is as follows: enhancer buffer NB (1N) 3.5. Mu.L, enhancer buffer M2.5.5. Mu.L, primer pool 5. Mu.L, sample 1ng-5 ng/reaction tube, quantitative result of DNA concentration Qubit (Thermo Fisher), IGT-EM808 polymelase 10. Mu.L, and finally ddH ₂ O is added to 30 mu L;

the multiplex PCR reaction procedure was: 210s at 95 ℃;98 ℃ for 20s; at 60℃for 4min,22 cycles, and finally at 72℃for 5min.

Further, the linker sequence PCR reaction system is: PCR product mixture 13.5.5. Mu.L, enhancer buffer M2.5.5. Mu.L, CDI Primer 2. Mu. L, IGT-EM808 polymerase 10. Mu.L, and finally ddH ₂ O is added to 30 mu L;

the linker sequence PCR reaction procedure was: 210s at 95 ℃;98 ℃ for 20s;58 ℃ for 1min; 30s at 72 ℃; for 22 cycles, at last 72℃for 5min.

The invention also protects application of the primer in preparation of a forensic identification kit.

The invention also protects application of the primer in preparation of a genetic relationship identification kit.

The invention also protects application of the primer in genetic relationship identification.

The invention also provides a preparation method of the kit, which comprises the step of mixing and packaging all the primers in the primers.

The invention has the beneficial effects that:

the 50 micro-haplotype genetic markers provided by the invention combine the advantages of STR and SNP, and have the advantages of no amplification deviation, no stutter peak, short fragment, short amplicon, low mutation rate, low recombination rate and high polymorphism. The provided primers can specifically amplify the 50 micro haplotype sites in the same system, the primer pairs can not interfere with each other, the requirement of multiple PCR can be met, the stability and the accuracy of forensic identification analysis are further improved, the operation steps of forensic identification are simplified, and the primers are particularly effective in detecting degradation mixed samples and have high application value.

The system provided by the invention contains 50 autosomal micro haplotypes in total, after Bonferroni correction, all 50MHs are reserved for example operation analysis, and the selected 50 sites have good repeatability, extremely high sensitivity and stability. Meanwhile, 50 sites of 137 Chinese southwest Han nationality irrelevant individuals, 6 non-degradation mixed samples, 8 single-source degradation samples and 10 mixed degradation samples are detected, and compared with common autosomes STRs, SNPs or published MH panels, the average A of 50plex MH panels is that _e The value is 4.192, the genetic polymorphism is higher, and the genetic polymorphism has higher value in forensic applications such as individual identification, degradation detection, mixed spot interpretation, relative analysis and the like. The invention further shows that the system identified by the invention has high efficiency, strong detection capability of degradation and mixed samples, and good application potential in forensic practical examination cases.

Drawings

FIG. 1 shows depth of coverage (DoCs) and Allele Coverage Ratios (ACRs) for 50 MH; wherein, (a) the left axis blue bar indicates overall DoC in ascending order, the blue dotted line indicates average DoC, the right axis orange solid line indicates overall ACR, and the orange dotted line indicates average ACR; (B) no correlation between DoC and ACR;

FIG. 2 shows allele frequencies of 50MHs for 137 individuals unrelated to Han nationality in southwest China;

FIG. 3 shows three examples of analysis methods of MH-27 (MH 3-38814227/233/234/273) in a random sample (number 152); the whole figure shows our protocol, sanger sequencing and IGV obtained genotypes from top to bottom, where the black box represents the target SNP and the screenshot shows only the physical location and length of the target MH;

FIG. 4 is a graph showing degradation degree of single and mixed DNA;

FIG. 5 is an STR pattern of degraded single DNA and degraded mixed DNA.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

EXAMPLE 1 micro-haplotype screening

1. Selection of MH

Site screening work was performed on 1000G data (PHASE 3) using MH screening software (R language and Python script) in combination with PHASE software. Information on hg19 human genome: http:// hgdownload. Soe. Ucsc. Edu/goldenPath/hg19/bigZips. Thousands of genome population genotyping data: https:// ftp trace: ncbi:. Nih. Gov/1000 genome/ftp/release/20130502/all. Chr 1-22. Phase3_shape 2_mvncall_integrated_v5a.201301282. Genetypes.

Only genotype data of 105 southern han nationality people (CHS) in the thousand people genome are retained. Extraction of 80 bp-containing two or more SNPs in CHS and efficient allele factors (A _e ) MH with a value > 3 and estimating the theoretical value of population haplotype frequency. A is that _e The calculation formula is as follows: a is that _e ＝1/∑p _i ² The method comprises the steps of carrying out a first treatment on the surface of the Wherein p is _i The frequency of the haplotype found in all sample data.

On this basis, candidates MHs are screened according to the following criteria: 1) All MHs SNPs must show Minimal Allele Frequencies (MAFs) in the dbSNP database>0；2)A _e The value is equal to or greater than 4 because A _e High MH can enhance individual identification, mixed plaque interpretation and affinity analysis; 3) Taking each autosomal unit and taking A in all overlapping sequences of each group _e Maximum MH; 4) MHs having a significant repetitive motif in the base sequence is removed; 5) Selecting an initial MHs set with physical position more than or equal to 10Mb, and avoiding bitsLinkage Disequilibrium (LD) between points; 6) Only MH was retained which was able to design primers and successfully multiplex amplified. Finally reserve theory A _e 178 candidate micro-haplotypes more than or equal to 4 are compounded into a set of 128 micro-haplotype locus multiple detection system with high recognition capability based on the candidate micro-haplotypes.

After optimization, the final system contains 50 autosomal micro haplotypes (specific information is shown in Table 1), A _e Ranging from 1.503 to 7.547, average 4.192. Of 50MH, 10A _e Less than 3.0,8A _e More than or equal to 3.0, 24A _e Not less than 4.0,3A _e Not less than 5.0,3A _e Not less than 6.0,2A _e ≥7.0。

Example 2 design of detection primers

1. Primer design

After 178 candidate micro-haplotypes are obtained, primers are designed for each locus based on hg19 for the physical position information of the micro-haplotypes. Based on thermodynamic stability, a plurality of specific primers are designed on both sides of the region of interest (ROI); the amplicon is 120-200bp, and the coverage rate is 100%. And then primer dimerization and nonspecific amplification are evaluated by using primer evaluation software, designed and synthesized primers are tested, and primers with poor detection effect are evaluated and replaced. And by searching experimental conditions, most primer amplification conditions are as consistent as possible, and enzyme with good multiplex amplification efficiency is screened for amplification so as to meet experimental requirements.

The analysis experiment shows that at tm=60 ℃,22 cycles have the most sites, the typing is accurate and the sequencing depth is uniform. Optimized PCR amplification reaction procedure: 3min 30s at 95 ℃;98 ℃ for 20s at 60 ℃ for 4min,22 cycles; 72 ℃ for 5min; preserving at 4 ℃.

Primers were designed and tested for 178 candidate microshenotype sites, and MPS-based protocols allowed primers to be designed and 128 MHs to be tested in multiple in a single assay. 6 rounds of optimization are performed, and MHs with more nonspecific amplification products, large amplification and sequencing deviation among different samples and low sequencing coverage rate are eliminated. 50MHs were reserved to ensure optimal system performance of the panel and distributed over 21 autosomes (no target MH on chr22 after 6 rounds of optimization). 1-5 MH (average 2.38) were observed on each autosome, each MH contained 3-15 SNPs (total 251, average 4.83), the length of the markers was 11-81bp (average 65.58 bp), and the amplicon was 123-198bp (average 156.02 bp). The final multiplex amplification system retained an effective primer set of 50 microshutters, comprising 104 primers (where mh8-93440810/858/861/885 has 3 pairs of different primers) for detection of 50 microshutters. The lengths of the 50 micro haplotypes and the target sequence information are shown in Table 1 (each SNP is marked in turn by bold underlines in the table; marked SNPs are single nucleotides, if two SNPs are adjacent, the same underlines are shown in the table), the detailed information of 104 primers is shown in Table 2, the forward primer number of the position of the detection number sequence 1 in Table 2 is SEQ ID NO.1, and the reverse primer is SEQ ID NO.2, and thus the sequence is numbered downwards to SEQ ID NO.104.

TABLE 2 primer information for 50 micro haplotypes and concentration in multiplex PCR reaction System

a represents a group previously reported

Example 3 detection of micro-haplotype sites based on second generation sequencing technology

1. DNA sample preparation

Standard sample: 2800M Control DNA (Promega, madison, wis., USA).

Random independent samples: peripheral blood, blood sample was collected from a female volunteer, number 152.

Extracting genome DNA of a random irrelevant sample, and diluting the genome DNA with TE buffer solution until the DNA concentration is 1 ng/. Mu.L, thus obtaining the template solution. Taking a standard sample, and diluting the standard sample with TE buffer solution until the DNA concentration is 1 ng/. Mu.L, thus obtaining the template solution. Together, the two template solutions.

2. Library preparation and second generation sequencing

1. Template solution was taken and subjected to a first round of multiplex PCR (2 hours) using

The microsloid site set (Beijing Dongsheng Innovative Biotechnology Co., ltd.) was operated according to its instructions with amplicons between 120-200bp, and a first round of magnetic bead purification (35 min) was performed. The second round included the linker sequence PCR reaction. Amplified libraries were obtained by introducing the next generation sequencing adapter sequences of Illumina company to both sides of the amplified product (25 minutes). After the second round of bead purification (35 min), qubit was used ^TM The dsDNA HS Assay kit is used for carrying out strict concentration measurement on a sample, then a Qsep400 full-automatic nucleic acid protein analysis system is used for carrying out quality inspection, and then the sample is subjected to IlluminaAmplicon targeted capture using PE1128 double-ended sequencing mode on NovaSeq 6000 system.

Wherein, illuminaThe flow of NovaSeq 6000 platform PE1128 double-end capture sequencing was as follows: the constructed library and PhiX were denatured and diluted with reference to Protocol a of NovaSeq 6000System Denature and Dilute Libraries Guide (DoCument #1000000106351 v03), the sample table was compiled with reference to Illumina Experiment Manager Software Guide (DoCument #112831335v 08) using IEM software, and on-machine sequencing was performed with reference to NovaSeq 6000Sequencing System Guide (DoCument #1000000019358v14 Material#20023471). The sequencing Reagent selected for this experiment was NovaSeq 6000s4 Reagent kit v1.5, PE1128 double-ended sequencing.

2. Site typing test results

The invention captures 50MH of 137 Chinese southwest Han nationality irrelevant individuals altogether, and sequences the MH to obtain complete MH alleles, and the result of allelic typing of each locus is shown in Table 3. In Table 3, exemplified is a microsloid type 1 (mh 1-12898591/610/647/654/667) which consists of 5 SNPs in sequence (see Table 1). Wherein, the genotype corresponding to allele 1 of 2800M is "GCCAC", the genotype corresponding to allele 2 is "GCCAC" (homozygote), and the 5 SNPs on one chromosome are G, C, C, A, C in sequence, and the 5 SNPs on the other homologous chromosome are G, C, C, A, C in sequence. In addition, the genotype corresponding to allele 1 of random sample 152 was "GCTGG", the genotype corresponding to allele 2 was "GCCAC" (heterozygote), and it was indicated that the 5 SNPs on one chromosome were G, C, T, G, G in sequence, and the 5 SNPs on the other homologous chromosome were G, C, C, A, C in sequence. Therefore, based on the detection results of table 3, the standard 2800M and the random irrelevant sample 152 all obtain complete site typing, which indicates that the invention can meet the requirements of forensic physical evidence inspection.

TABLE 3 typing test results for 50 micro-haplotypes

a represents a group previously reported

3. HWE results at sites MH 1-50

After Bonferroni correction, no significant deviation in HWE (p=0.05/50=0.001) was observed for the 50MHs (table 4). Wherein, when calculating HWE, the effective allele is 1/Σpiwith the reciprocal of homozygosity ² Wherein p is _i The p-value of Hardy-Weinberg equilibrium (HWE) was calculated for the frequency of allele i occurrence and summing all alleles at MH, then using Arlequin v3.5 software.

These samples were genotyped with 1.825-25.992ng of input DNA using all 50MHs DoC and ACR to evaluate panel sequencing performance. The overall DoC was 1259.20-23200.80 ×, average 7928.39 ± 4990.952 × (fig. 1A). Overall ACR0.74-0.95, average value of 0.90+ -0.045, and 96% of MHs (48/50) allele balance ratio of 80% (FIG. 1A). There is no correlation between DoCs and ACR (linear correlation coefficient R ² = 0.0771, fig. 1B). The results showed that our 50plex MH panel had very good sequencing performance.

TABLE 4 HWE results of 50MH for 137 Chinese southwest Han nationality-independent individuals sequenced

Wherein, p=0.05/50=0.001; a represents what has been previously reported.

Example 4 calculation of population genetics parameters

1. Haplotype (i.e., allele) frequencies of the above 50MHs were calculated, as shown in fig. 2 and table 5 (where a represents a previous report).

As shown in fig. 2 and table 5, each MH had 2-23 alleles (average 7), with 3 MH having 2-3 alleles, 4 MH having 4 alleles, 15 MH having 5 alleles, 12 MH having 6 alleles, 3 MH having 7 alleles, 13 MH having 8 and above alleles, and all 350 alleles had a frequency ranging from 0.004 to 0.803.

2. Obtaining the above 50 forensic statistical parameters MHs, including homozygosity (Hom), heterozygosity (Het), A _e Value, match Probability (MP), cumulative Match Probability (CMP), recognition probability (DP), cumulative recognition probability (CDP), exclusion Probability (PE), cumulative exclusion probability (CPE), polymorphic Information Content (PIC), and typical father index (TPI).

Based on allele frequencies (Table 5), forensic parameters (Table 6) show, hom, het and A _e 0.133 to 0.665 (average 0.266), 0.335 to 0.867 (average 0.734), and 1.1283 to 7.547 (average 4.192), respectively. Of the 50MHs,<3.0A _e There are 10, 8, 24, 3, 6.0, and 2 for > 3.0, 4.0, 5.0.

Furthermore, we have observed that MP, CMP, DP, CDP, PE, CPE, PIC and TPI are 0.032-0.484 (average 0.127), 0.999180791, 0.516-0.968 (average 0.873), 1-3.109 ×10, respectively ^-49 0.086-0.747 (average 0.481), 1-8.727 ×10 ^-16 0.308-0.855 (average 0.692) and 0.770-4.029 (average 2.018) (Table 6). The results show that the 50plex MH panel screened by the invention has exceeded the commonly used 23 STRs or 52 SNPs and the efficacy of other reported MH panels, which shows that our panel may have good prospects in future individual identification, mixed plaque interpretation, relatedness detection and non-invasive prenatal paternity test (NIPPT).

TABLE 5 allele frequencies of 50MHs for 137 Chinese southwest Han nationality-independent individuals sequenced

TABLE 6 forensic parameters of 50MHs of 137 individuals unrelated to Han nationality in southwest China

Wherein a represents a group previously reported.

Example 5 verification of sensitivity, accuracy and specificity

1. Sensitivity and accuracy detection

10,5,1,0.5,0.25 and 0.125ng of standard 2800M are entered into MPS platform and are found in IlluminaNovaSeq 6000 system. 18 samples (1 sample x 6 gradients x 3 replicates) were placed on the same NovaSeq 6000 chip. For three replicates of 2800M inputs (10, 5,1,0.5,0.25 and 0.125 ng), we detected complete typing of all 50MHs at 0.25 ng. In the third repetition of 0.125ng, loss of MH-37 was observed only once, reading 20X, below the analysis threshold of 25X.

An unrelated sample (accession number 152) and sites MH-4 and MH-27 were randomly selected for Sanger sequencing. The bam original file obtained from MPS was input into Integrative Genomics Viewer (IGV), the target MH was genotyped, and finally the obtained MH genotype was compared with the MH genotype obtained by IGV and Sanger sequencing simultaneously. No inconsistent haplotypes between Sanger sequencing, IGV or our analysis protocols were observed in the MH sites analyzed and in unrelated individuals. FIG. 3 shows genotypes corresponding to three analysis methods for random MH in random samples, and shows 100% identity.

2. Specific detection

The specificity was assessed by testing common animal DNA, specifically: multiple PCR targeted capture sequencing was performed on animal DNA samples of cats, cattle, chickens, ducks, fish, pigs, rabbits and sheep using the same method as human DNA, with an input DNA amount of 3.753-6.1286ng, and the results are shown in Table 7.

As shown in Table 7, 1. Mu.L of DNA input for all 8 animal DNA samples did not obtain a complete genotype, and for animal DNA, only 2-8MHs were detected per DNA, with only 25 of MHs containing 1-4 alleles. The current data indicate that our panel acquires incomplete genotyping for different animal samples and the signal is very low, so the species specificity of the 50plex assay is sufficient to meet the needs of conventional personal work.

TABLE 7 summary of MHs genotypes and DoCs for each animal sample based on the MPS platform

Example 6 detection of simulated Single Source degradation and Mixed degradation samples

1. Analog single source degradation sample

The two DNA were diluted to a concentration of 5 ng/. Mu.L and treated with DNase I (Thermo Fisher Scientific, california, USA), respectively. mu.L of intact DNA (5 ng/. Mu.L) was combined with 3.75. Mu.L of 10 XMgCl ₂ Buffer (Thermo Fisher Scientific, california, USA) was mixed. Adding 0.6. Mu.L of 0.3U/. Mu.L DNase I to the mixture, incubating at 37℃and removing 10. Mu.L of degraded DNA from the incubated mixture at predetermined time intervals (2.5, 5, 10 and 15 minutes, respectively) and placing it in separate 0.2mlIn sterile amplification tubes (Axygen Scientific, union City, calif.). EDTA (1.6. Mu.L, 30 mM) was added immediately to each tube and incubated at 65℃for 10min to stop DNA degradation. The extent of degradation was then assessed using the AGCU EX22 kit (Applied ScienTech, jiangsu, china) on an ABI 31280Genetic Analyzer (Applied Biosystems, foster City, calif., USA) and using the High Sensitivity DNA kit on an Agilent 2100Bioanalyzer (Agilent Technologies, santa Clara, calif., USA) according to manufacturer's instructions. For MPS, 1 μl was used for each DNase I treated sample.

2. Simulated mixed degradation sample

One of the above single-source degradation samples was set as the secondary DNA and fixed at 0.5ng, and the other was set as the primary DNA. The primary DNA having different degradation times was added to the corresponding secondary DNA to form a mixture at a ratio of 1:10. The subsequent degradation degree evaluation and detection process is the same as the process of the single-source degradation sample. For MPS, 1 μL was used per 1:10 mixed degradation.

3. Degradation outcome detection

(1) The length of the DNA fragment is between 120 and 320bp after different DNase I treatment time (2.5, 5, 10 and 15 minutes). The extent of degradation of the single and mixed samples was then measured using an Agilent 2100bioanalyzer (Agilent Technologies, santa Clara, CA, USA), and individual DNAs C51, C131 and a 1:10 mixture thereof were randomly taken and treated with DNase I at 37 ℃ for 2.5, 5, 10 and 15min, respectively. Then 1 μl was collected separately and the corresponding electropherograms (from left to right) were obtained using a High Sensitivity DNA kit (agilent technologies, california, usa) and the results are shown in fig. 4.

In FIG. 4 (A), for C51, the degraded fragment was dispersed at 2.5min, concentrated at 200bp at 5min, and then concentrated at about 150 bp. (B) For C131, the degraded fragments were dispersed at 2.5min, concentrated at 150bp at 5min, and then concentrated in shorter fragments. (C) Under the conditions of 1:10-C51+C131, the degraded fragments were dispersed at 2.5min, concentrated at 150bp at 5min, and then concentrated in shorter fragments. From this, it can be seen from the detection results of fig. 4 that the degraded single sample and the mixed sample were treated with DNase I, and an ideal simulated degradation state was obtained.

(2) Random single DNA C51, C131 and a 1:10 mixture thereof were treated with DNase I at 37℃for 2.5, 5, 10 and 15min, respectively. 1. Mu.L each was taken and the corresponding electropherograms (FIG. 5, left to right) were obtained using the AGCUEX 22 kit (Applied ScienTech, jiangsu, china). In FIG. 5, (A) the peak height of C51 decreases significantly at 2.5min, from 320bp at 5min, and then gradually; (B) The peak height of C131 decreases from 280bp at 2.5min, from 120bp at 5min, and then gradually. (C) For 1:10-C51+C131, peak heights decreased from 280bp at 2.5min, from 150bp at 5-10min, and from 120bp at 15min. However, the STR kit cannot obtain a complete profile at different degradation times, regardless of whether a single sample or a mixed sample is used for degradation.

Long-STR genotyping failed when random single-source DNA was treated with DNase I at 37℃for 2.5, 5, 10 and 15 minutes (FIGS. 5A and B). In contrast, 50-MH panel based on MPS successfully obtained the complete alleles of all single-source degraded DNA (Table 8). Long STR genotyping failed when the simulated two-person mixture was treated with DNase I at 37 ℃ for 2.5, 5, 10 or 15 minutes (fig. 5C). However, 50-MH panel based on MPS successfully achieved complete typing of major, minor DNA in 6 degradation mixtures of 1:10-2.5 and 1:10-5. In the other 4 degradation mixtures of 1:10-10 and 1:10-15, only 1-4 unique alleles (effective alleles) were deleted. The overall detection rate was 93% -100% (table 8). From this, the results show that 50plex MHs are more efficient than CE-STRs in sequencing and genotyping single-source degraded DNA and mixed degraded DNA, and are expected to be applied to noninvasive prenatal paternity test in the future.

TABLE 8 50-MH typing results of degraded Single and Mixed DNA

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.

Claims (9)

1. A high-efficiency autosomal micro-haplotype genetic marker system is characterized in that the genetic marker system comprises mh1-53568101/116/144/162/171, mh1-115990063/085/092/136, mh1-143149431/462/477/488, mh1-218265683/713/718, mh1-228465346/366/370/403, mh2-44347304/305/365/384, mh2-81081505/507/509/511/513/551, mh2-138693814/815/821/826/880/883/890/891, mh2-191143118/127/132/173, mh2-221689038/091/092, mh3-59679857/882/894/906/908/910/917, mh4-23972751/767/768/827, mh4-36376702/708/712/764/771/772/775/777, mh4-57792067/078/128, mh4-68303172/190/201/203/205/207, mh4-155137770/820/823/832/835/838, mh5-19151590/592/594/596/662, mh5-124540406/413/416/430/483, mh5-159013018/027/028/029/045, mh6-42614552/554/556/560/562/564, mh6-52812768/817/820/842, mh6-152930711/754/755/767/769/771, mh7-75440052/101/107, mh8-93440810/858/861/885, mh9-565917/950/986, mh9-103969852/888/923, mh10-47631395/405/406/419/457/462, mh10-103933361/362/363/404/415/424/425/432, mh11-36130910/939/957/972, mh12-52863017/038/044/091, mh12-64140424/439/446/449/482/491, mh13-54060827/881/892, mh13-81151788/799/862/863, mh13-101767111/127/134/184, mh14-20373007/019/034/086, mh14-41782002/007/054/067/072, mh14-52334164/203/205/242, mh14-80876061/108/111, mh15-61692639/665/687/718, mh16-13097710/734/737/740/743/746/749/752/755/758/773, mh16-31847392/393/427/451, mh16-83973898/918/956/965, mh17-31320658/695/712/713, mh17-41401201/202/203/204/225/229/231/232/242/244/247/251/261/265/269, mh, mh18-108028/029/044/045/059/060/077/080/089/091/094/108, mh18-50534370/395/412/448, mh18-62548474/486/493/513/519/532, mh19-33764649/678/692/718/723/726, mh20-16024597/641/644 and mh21-9829987/30023/30024/30060/30066.

2. A primer for amplifying the high-efficiency autosomal micro-haplotype genetic marker system according to claim 1, wherein the primer sequence is shown in SEQ ID No. 1-104.

3. Use of a primer as claimed in claim 2 in forensic identification.

4. A kit for forensic identification comprising the primer of claim 2.

5. The kit of claim 4, wherein the kit is useful for performing individual identification or genetic relationship identification.

6. The kit of claim 4, further comprising a buffer, a DNA polymerase, and dntps.

7. The method of using the kit of claim 4, wherein the genomic DNA of the sample to be tested is extracted as a template, multiplex PCR amplification is performed by using the kit, the obtained amplified product is subjected to a linker sequence PCR reaction to obtain an amplified library, and quantitative and second-generation sequencing detection analysis is performed on the amplified library to obtain the typing result of the micro-haplotype locus.

8. The use according to claim 7, characterized in that

Multiplex PCR amplification was performed with multiplex amplicon kits.

9. The method of claim 7, wherein the linker sequence PCR reaction system is: PCR product mixture 13.5. Mu.L, enhancer buffer M2.5.5. Mu.L, CDI Primer 2. Mu. L, IGT-EM808 polymerase 10. Mu.L, and finally ddH ₂ O is added to 30 mu L;

the linker sequence PCR reaction procedure was: 210s at 95 ℃;98 ℃ for 20s;58 ℃ for 1min; 30s at 72 ℃; for 22 cycles, at last 72℃for 5min.

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