A microhaplotype detection system based on NGS platform and its application

Abstract

The present invention belongs to the field of forensic identification, and specifically relates to a micro-haplotype detection system based on the NGS platform and its application. The present invention takes the micro-haplotype of a novel molecular genetic marker as the research object, and systematically selects micro-haplotype molecular genetic markers with high genetic polymorphism in the domestic Han population by performing a meta-analysis of previously reported micro-haplotypes; based on the NGS platform, a synchronous typing detection system is developed for forensic genetics research. The results of the present invention can provide an efficient and practical tool for research such as personal identification, complex kinship identification, mixed sample analysis, and biogeographical origin speculation in forensic genetics.

Description

Technical Field

[0001] The present invention belongs to the field of forensic identification, and specifically relates to a micro-haplotype detection system based on an NGS platform and an application thereof. Background Art

[0002] In forensic genetics research, personal identification and kinship analysis are two main research tasks. In recent years, with the continuous development of detection technology, the research direction of forensic genetics has gradually expanded to mixed sample analysis, biogeographical origin speculation and complex kinship identification. Usually, forensic geneticists use short tandem repeats (STR) to solve the above problems. However, STR has shortcomings in forensic genetics research, such as large amplicon, high mutation rate and interference peaks in PCR, which has certain limitations in practical applications. In response to this, forensic geneticists began to select new molecular genetic markers to solve the above problems. Single nucleotide polymorphism (SNP) and insertion / deletion polymorphism (InDel) as the third generation of molecular genetic markers have the advantages of wide distribution in the genome, low mutation rate and small amplicons. They are widely used by forensic scientists for personal identification, kinship identification, analysis of the number of contributors in mixed samples and ancestry speculation. However, SNPs and InDels are mostly di-allelic variants in a population. Therefore, these two types of genetic markers usually have relatively low genetic polymorphism and cannot be fully used for complex kinship and mixed sample analysis. Therefore, it is urgent to select other types of molecular genetic markers to solve the above problems.

[0003] Microhaplotypes, as a new generation of genetic markers, are composed of multiple SNPs within a DNA region. Compared with existing genetic markers, such as STR, SNP and InDel, microhaplotypes have the advantages of high genetic polymorphism and rich ancestral information in forensic genetics research. To date, forensic scientists have developed multiple microhaplotype detection systems. However, it should be noted that the microhaplotypes included in the currently developed systems usually have lower genetic polymorphism in domestic populations, which provides relatively limited information in terms of personal identification, kinship identification, analysis of the number of contributors to mixed samples, and ethnic origin speculation. Summary of the invention

[0004] To solve the above problems, the present invention provides a micro-haplotype detection system based on the NGS platform, including 33 highly genetically polymorphic micro-haplotype molecular genetic markers and a sex-determining gene (Amelogenin).

[0005] Furthermore, the position information of the highly genetically polymorphic micro-haplotype molecular genetic markers is shown in the table below.

[0006]

[0007] The present invention also provides a primer for amplifying the above-mentioned highly genetically polymorphic micro-haploid molecular genetic marker and Amelogenin, and the nucleotide sequences of the primers are shown in SEQ ID No.1 to SEQ ID No.68.

[0008] The present invention also provides a method for forensic genetics individual identification, kinship analysis, mixed sample analysis and biogeographical origin inference using the above-mentioned micro-haplotype molecular genetic markers and / or primers.

[0009] The present invention has the following beneficial effects:

[0010] 1. Compared with previously reported micro-haplotype loci, the micro-haplotype loci selected by the present invention have higher genetic information content in the domestic Han population, and the average polymorphic information content, personal identification ability, non-paternity exclusion probability and Ae value are greater than 0.8, 0.9, 0.6 and 6, respectively.

[0011] 2. The present invention has high sensitivity and can realize typing detection of 0.125ng DNA sample, which is more suitable for detection and analysis of trace samples.

[0012] 3. The system developed by the present invention can be used for individual identification, biogeographical origin analysis, number estimation of mixed sample contributors, and complex kinship identification research by testing a sample based on the micro-haplotype typing information obtained.

[0013] 4. The present invention has a high resolution for mixed samples consisting of two contributors. In a 1:49 mixed sample, most of the minor haplotype sites can still be detected in the minor contributor. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 .Evaluation of sequencing performance of the system of the present invention;

[0016] Figure 2.The allele frequency distribution and forensic parameters of the 33 microhaplotype loci in the present invention in the Guizhou Han population;

[0017] Figure 3 .The present invention has the ability to identify different levels of kinship;

[0018] Figure 4 . The sensitivity of the system of the present invention and the analytical performance of mixed samples;

[0019] Figure 5 .The present invention combines artificial intelligence algorithms to analyze the number of contributors to mixed samples;

[0020] Figure 6 .The efficiency of clan origin analysis of 33 microhaplotype loci in the present invention. DETAILED DESCRIPTION

[0021] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] In order to realize the present invention, we adopt the following technical solutions:

[0027] 1. Selection of micro-haplotype molecular genetic markers with high genetic polymorphism in domestic populations

[0028] The present invention mainly conducts a meta-analysis of previously reported micro-haplotype studies and systematically selects micro-haplotype molecular genetic markers using the following principles:

[0029] 1) Each microhaplotype contains at least 3 SNP sites;

[0030] 2) The effective number of alleles (Ae) of the microhaplotype locus in East Asian populations is greater than 5;

[0031] 3) The SNP sites within the microhaplotype are about 100 bp apart;

[0032] 4) The microhaplotypes on the same chromosome are more than 1 Mb apart.

[0033] Based on the above principles, 33 micro-haplotype molecular genetic markers were preliminarily selected. The basic information of the selected 33 micro-haplotype molecular genetic markers is shown in Table 1.

[0034] Table 1 Basic information of 33 microhaplotype molecular genetic markers

[0035]

[0036] 2. Development of a synchronous typing detection system based on the NGS platform

[0037] In order to realize the synchronous typing detection of the selected micro-haplotype molecular genetic markers, the present invention is based on the NGS platform and adopts multiple PCR technology to develop a composite amplification system for the detection of micro-haplotype molecular genetic markers. First, based on the site information of the micro-haplotype in Table 1, the present invention obtains the sequence information of 200bp upstream and downstream of each micro-haplotype genetic marker by querying the ensembl database. Next, the primers of the micro-haplotype site are designed using the Oligo tool. In addition, in the system development, the present invention also adds a sex determination site (Amelogenin) for the sex determination of the sample to be tested. The primer sequences and amplicon sizes of 33 micro-haplotype molecular genetic markers and an Amelogenin site are shown in Table 2.

[0038] Table 2 Primer sequences and amplicon sizes for 33 microhaplotype molecular genetic markers and one Amelogenin locus

[0039]

[0040] Based on the obtained primer sequences, the present invention continuously optimizes the primer mixing ratio of each site, and ultimately achieves the synchronous typing detection of the selected 33 micro-haplotype molecular genetic markers and the Amelogenin gene.

[0041] The detection process of 33 micro-haplotype molecular genetic markers and Amelogenin loci in the present invention is as follows:

[0042] 1) First, library construction was performed, including two-step PCR reactions. The first round of PCR system included 9μL ddH2O, 3.5μL Enhancer buffer NB (1N), 2.5μL Enhancer buffer M, 5μL Primer mixture, 10μL IGT-EM808 polymerase mixture and 1ng DNA sample. The thermal cycle conditions were pre-denaturation at 95℃ for 3min30s; followed by 98℃20s, 55℃1min, 60℃1min and 65℃2min, for a total of 22 cycles; reaction at 72℃ for 5min, and finally storage at 4℃. After completing the first round of PCR, the amplified product was purified using a magnetic bead DNA purification kit. Next, the second round of PCR was performed to add adapter sequences and sequencing primers. The reagents included in the second round of PCR were 13.5μL purified PCR product, 2.5μL Enhancer buffer M, 2μL UDI Index (5μM), 10μL IGT-EM808 polymerase mixture and 2μL ddH2O. The PCR thermal cycle conditions included 95°C pre-denaturation for 3 min 30 s, followed by 98°C for 20 s, 58°C for 1 min, and 72°C for 30 s, for a total of 9 cycles, followed by reaction at 72°C for 5 min, and finally storage at 4°C. Similarly, the second round of PCR reaction products were purified using a magnetic bead DNA purification kit.

[0043] 2) After completing the library construction, take 1 μL of the library and use the Qubit dsDNA HS Assay kit to Quantitative analysis was performed on a 3.0 Fluorometer device.

[0044] 3) The quantified DNA library was added to the DNBSEQ-T7 sequencing platform for sequencing.

[0045] Finally, the obtained offline data was analyzed. The raw file fastq sequence obtained by sequencing was preprocessed using Trimmomatic software to remove the adapter sequence and low-quality sequence. For the clean sequence obtained, the present invention uses BWA software to obtain SNP and InDel variation information by comparing it with the GRCh38 reference genome. Based on the clean sequence obtained by sequencing each sample, the present invention uses perl software to obtain the typing information of each microhaplotype molecular genetic marker, and the analysis parameters are the sequencing depth of the site>20 and the sequencing depth of the minor allele / total depth>15%.

[0046] Example

[0047] The present invention takes microhaplotype molecular genetic markers as the research object, and through meta-analysis of previously reported microhaplotype sites, systematically selects molecular genetic markers that are highly genetically polymorphic in domestic populations; docking with the NGS platform, through multiplex PCR combined with rolling circle sequencing technology, the synchronous typing detection of the selected microhaplotype molecular genetic markers is achieved.

[0048] The reagents involved in the present invention mainly include 1N Enhancer buffer NB, Enhancer buffer M, Primer mixture of micro-haplotype loci, IGT-EM808 polymerase mixture, 5μM UDI Index, purification magnetic beads and Qubit dsDNA HS Assay reagent; the instruments and equipment involved include VeritiPro PCR amplifier, magnetic stand, 3.0Fluorometer and DNBSEQ-T7 sequencer.

[0049] For a sample to be tested, the specific detection steps are as follows:

[0050] 1) Construction of sequencing library. Take an individual DNA sample (1ng) and configure the first round of PCR system as follows:

[0051]

[0052]

[0053] PCR conditions were as follows:

[0054]

[0055] After completing PCR, the amplified product was purified using a magnetic bead purification kit.

[0056] Next, the purified amplified product was used for the second round of PCR. The ratios of each system were as follows:

[0057]

[0058] The reaction conditions for the second round of PCR are as follows:

[0059]

[0060] Similarly, the second-round PCR products were purified using a magnetic bead DNA purification kit.

[0061] 2) Quantification and sequencing of sequencing library. After completing the library construction, take 1 μL of the constructed library and use the QubitdsDNA HS Assay kit to Quantification was performed on a 3.0 Fluorometer device. Based on the obtained DNA quantification results, all samples were mixed together and then placed on the DNBSEQ-T7 sequencing platform to complete the sequencing reaction.

[0062] 3) Microhaplotype typing. The obtained offline data were analyzed. The raw fastq sequence obtained by sequencing was preprocessed using Trimmomatic software to remove adapter sequences and low-quality sequences. For the clean sequence obtained, BWA software was used to obtain SNP and InDel variation information by comparing it with the GRCh38 reference genome. Based on the clean sequence obtained by sequencing each sample, the present invention obtains the typing information of each microhaplotype molecular genetic marker based on perl software (analysis parameters: sequencing depth>20 and sequencing depth of minor alleles / total depth>15%).

[0063] Figure 1 What is shown is the sequencing result of 201 Guizhou Han individuals using the present invention. As can be seen from the figure, the average sequencing depth of the 33 micro-haplotype sites selected by the present invention is above 937. In addition, these sites show a higher average allele balance, and the average allele balance of most sites is above 0.7. In summary, it shows that the system developed by the present invention has a higher sequencing performance.

[0064] Figure 2 Displayed are the allele frequencies and forensic parameter distributions of 33 selected microhaplotype molecular genetic markers in the Guizhou Han population (201 cases). Figure 2 The results of A showed that the number of alleles of the selected 33 micro-haplotype molecular genetic markers in the Guizhou Han population ranged from 6 to 27. In addition, these 33 micro-haplotype molecular genetic markers showed high polymorphic information content, individual identification ability and non-paternal exclusion probability in the Guizhou Han population ( Figure 2B). The cumulative individual identification ability and cumulative non-paternity exclusion probability of 33 micro-haplotype molecular genetic markers in the Guizhou Han population were 1-5.62E-43 and 1-1.60E-15, respectively. These results show that the system developed by the present invention can be well used for individual identification and paternity testing of Guizhou Han individuals.

[0065] Based on the obtained allele frequency information in the Guizhou Han population, the present invention uses Familias software to simulate individuals with different kinship relationships and evaluates the insight efficiency of the system developed by the present invention on these kinship relationships ( Figure 3 ). The results show that for father-son relationships and full sibling relationships, the system developed by the present invention can well distinguish between father-son relationships, full sibling relationships and unrelated individuals. For second-level kinship relationships such as half-siblings, grandparents and grandchildren, and uncles and nephews, there is a certain overlap between the likelihood rates obtained and unrelated individuals. Despite this, most individuals can be identified as correct relatives or unrelated individuals. For third-level kinship relationships, there is a high overlap between the likelihood rate distribution diagram obtained for first-degree cousins ​​and unrelated individuals. In summary, it is shown that the system developed by the present invention has a high identification efficiency for first-degree and second-degree blood relationships, and can also distinguish between third-degree blood relationships and unrelated individuals to a certain extent.

[0066] Figure 4 The results show the sensitivity of the present invention and the mixed sample analysis performance. Figure 4 As can be seen in A, even when the amount of template DNA drops to 125 pg, all sites in the present invention can be detected, indicating that the system developed by the present invention has a high sensitivity and is suitable for the detection and analysis of old and trace samples. Figure 4 B shows the allele detection of two mixed samples composed of different mixing ratios by the system developed by the present invention. The results show that for mixed samples of 1:1 to 1:9 (or 9:1), all sites can be detected in the smaller contributors. Although there is allele loss at some sites in the mixed samples of 1:19 or 19:1, most of the sites can still be detected in the smaller contributors. For mixed samples of 1:49 or 49:1, it can also be found that about 1 / 3 of the sites can be detected in the smaller contributors. In summary, the system developed by the present invention has a high identification ability for two-person mixed samples composed of different mixing ratios. Even in mixed samples of 1:49 or 49:1, more than 1 / 3 of the sites can still be detected in the minor contributors.

[0067] For the analysis of the number of contributors in mixed samples, the present invention uses the R software package to simulate 2000 mixed samples consisting of 1-5 contributors based on the frequency information of 33 micro-haplotype sites observed in the Guizhou Han population. Next, based on the sum, mean, median of the number of alleles observed at all sites and the number of alleles observed at each site, the present invention respectively evaluated the performance of naive Bayes, random forest, decision tree, XGBoost, CART and linear discriminant analysis algorithms in analyzing the number of contributors in mixed samples. First, the samples were randomly divided into training sets and test sets in a ratio of 3:1, and the training set was used for model construction and model parameter optimization. Finally, the test set was used to evaluate the actual performance of the model. The results are as follows. Figure 5 As shown. In the test set, it can be seen that most of the mixed samples can be identified as the correct number of contributors. Among them, for the random forest algorithm, we found that only 13 samples had the number of contributors predicted incorrectly, indicating that the random forest algorithm has the best performance in parsing the number of contributors of mixed samples. In summary, the system developed by the present invention combines machine learning algorithms, especially the random forest algorithm, to well implement the parsing of the number of contributors of mixed samples.

[0068] In addition, the present invention also repeatedly tested different types of samples from the same individual and found that these samples could obtain the same genetic typing, indicating that the system developed by the present invention has good repeatability.

[0069] The 33 micro-haplotype sites selected in the present invention were analyzed for species specificity by searching the 200 bp upstream and downstream sequences and uploading the obtained sequences to the BLAST tool in NCBI. The results obtained showed that the sites selected by the present invention were only detected in the human genome, indicating that the system developed by the present invention has a high degree of species specificity and will not be interfered by samples of other species at the crime scene.

[0070] Figure 6 The results of population genetics analysis of different intercontinental populations using the 33 microhaplotype loci of the present invention are shown. Figure 6 The results of the multidimensional scaling analysis of A showed that the groups from the five major continents, such as Europe, East Asia, South Asia, Africa and America, especially the groups other than America, the remaining four continental groups formed four group clusters respectively, and these group clusters were consistent with their continental origins. Figure 6 B shows the results of the phylogenetic tree of these intercontinental groups. Similarly, the five intercontinental groups formed five branches, and the intercontinental origins of these branches and these groups were also completely consistent. Figure 6C shows the results of the genetic structure analysis of these intercontinental populations. From the figure, it can be seen that different intercontinental populations show different distributions of genetic components, such as Africa is mainly green genetic components, East Asian populations are mainly blue genetic components, European populations are mainly orange genetic components, and South Asian populations are mainly red genetic components. The above results show that the 33 micro-haplotype molecular genetic markers selected by the present invention can better realize the ethnic origin analysis of populations in Europe, East Asia, South Asia, Africa and the Americas.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer for amplifying highly genetically polymorphic microhaploid molecular genetic markers and sex-determining genes, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID No.1 to SEQ ID No.68; The basic information of the highly genetically polymorphic micro-haplotype molecular genetic markers is shown in the following table: 。 2. A method for forensic genetics personal identification or kinship identification, characterized in that: The primers as claimed in claim 1 were used for identification.

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