A primer composition, kit and application for detecting mitochondrial polymorphic sites based on next-generation sequencing technology

Abstract

The present invention relates to the field of forensic medicine, and particularly to a primer composition, a kit and their applications for detecting mitochondrial polymorphic loci based on next-generation sequencing technology. The primer composition for detecting mitochondrial polymorphic loci provided by the present invention covers 88 polymorphic loci on the human mitochondrial genome, and based on next-generation sequencing technology, a primer composition and a kit capable of detecting 88 mitochondrial polymorphic loci are developed and established. Compared with the previously constructed systems, the primer composition or kit of the present invention can provide more new genetic information. At the same time, the present invention shows higher sensitivity compared with the next-generation sequencing kits for detecting the entire mitochondrial genome. In addition, the mitochondrial polymorphic loci involved in the present invention all have relatively high polymorphic information content and can be applied to aspects such as the study of forensic complex kinship identification and individual recognition.

Description

Technical Field

[0001] The present invention relates to the field of forensic medicine, and particularly to a primer composition, a kit and an application for detecting mitochondrial polymorphic sites based on next-generation sequencing technology. Background Art

[0002] Human mitochondrial DNA (mtDNA) is another genome independent of nuclear chromosomes. Since Margit Nass and Sylvan Nass confirmed the existence of genetic material in chicken oocytes by electron microscopy in the 1960s, domestic and foreign scholars have conducted extensive research on human mtDNA. In 1972, Borst found that mtDNA was a covalently closed circular structure. In 1981, Anderson et al. used sequencing technology to determine the complete mtDNA sequence of humans, which was called the Cambridge reference sequence (CRS), and in 1999, 11 base sequences in the initially published sequence were corrected to form the currently recognized standard reference sequence in forensic science, that is, the revised Cambridge reference sequence (rCRS, GenBank accession number: NC_012920.1).

[0003] Compared with nuclear DNA, mtDNA has different characteristics: ① mtDNA does not conform to Mendelian inheritance laws. It is only passed between mothers and children, showing maternal inheritance, and can record the genetic history of maternal genes; ② Without recombination, it is haploid. During the process of passing on generations, offspring can completely preserve the genetic information of their ancestors; ③ High copy number, making the detection of mtDNA much easier than that of nuclear DNA; ④ It has a closed circular structure and is not easily degraded; ⑤ Due to the lack of protection by histones, mtDNA is easily affected by various factors and undergoes mutations, with a high mismatch rate during replication; ⑥ Mitochondria and mtDNA from sperm enter the cytoplasm of the oocyte after fertilization and will disappear through selective destruction, inactivation or dilution in the early stage of embryo development. Since the 1980s, with the continuous expansion of detection methods and application fields, etc., mtDNA has been widely used in research fields such as systematic evolution, population genetics, anthropology and forensic medicine. For example, in the study of anthropological remains, due to the inherent characteristics of organelles, nucleic acids can be better protected, enabling mtDNA to be more completely preserved. The remains of Tsar Nicholas II were identified through mtDNA sequencing. MtDNA is widely present in eukaryotic cells. Due to its characteristics of maternal inheritance in the human body, high copy number and not being easily degraded, it is often used in forensic medicine for the detection of trace samples, degraded samples and old samples (such as old bloodstains, shed cells, shed hairs, nails, bone or tooth samples, etc.), and can provide effective auxiliary evidence for complex kinship identification and individual recognition (identification of the same person) in forensic practice.

[0004] According to current international guidelines, Sanger sequencing is the preferred method for human forensic mtDNA identification and is highly popular in forensic laboratories worldwide. Some laboratories perform Sanger sequencing on the hypervariable regions I - II fragments of mitochondria, and some have extended it to hypervariable region III fragments and the control region. Massively parallel sequencing (MPS), also known as next-generation sequencing, is a revolutionary change to traditional Sanger sequencing, with characteristics such as high throughput and high accuracy. It can not only verify and deeply study the results of traditional genetic marker typing techniques, but also provide a platform for the detection and application of mtDNA. In order to improve the ability of mtDNA in individual recognition, in the past decade, some studies have extended the analysis to the entire mtDNA genome. However, from the perspective of forensic practice, the key is still to obtain sufficient mtDNA mutation information from limited physical evidence. Summary of the Invention

[0005] The object of the present invention is to provide a primer composition, a kit and an application for detecting mitochondrial polymorphic sites based on next-generation sequencing technology, so as to solve the problems existing in the above-mentioned prior art. The primer composition for detecting mitochondrial polymorphic sites provided by the present invention covers 88 polymorphic sites on the human mitochondrial genome and can provide more new genetic information compared with the previously constructed system.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a primer composition for detecting mitochondrial polymorphic sites based on next-generation sequencing technology. The mitochondrial polymorphic sites include nt73, nt146, nt150, nt152, nt189, nt195, nt199, nt263, nt310, nt489, (CA)n, nt709, nt1541, nt1719, nt1736, nt1811, nt2706, nt3010, nt3348, nt3394, nt3970, nt4216, nt4491, nt4833, nt4883, nt5147, nt5178, nt5417, nt5442, nt5460, nt5465, nt6446, nt6455, nt7028, nt7196, nt7600, nt8020, 9-bp, nt8414, nt8584, nt8684, nt8697, nt8701, nt8793, nt8794, nt8964, nt9090, nt9123, nt9477, nt9540, nt9545, nt9698, nt9824, nt10310, nt10397, nt10398, nt10400, nt10609, nt10873, nt11215, nt11251, nt11719, nt11914, nt12372, nt12705, nt12811, nt13104, nt13708, nt13928, nt14569, nt14668, nt14783, nt15043, nt15301, nt15487, nt15784, nt16126, nt16129, nt16183, nt16223, nt16234, nt16304, nt16311, nt16316, nt16319, nt16327, nt16362 and nt16519;

[0008] The primer composition includes group A primers and group B primers as described in the following table:

[0009]

[0010]

[0011]

[0012]

[0013] The present invention provides the application of the above primer composition in the preparation of a kit for detecting mitochondrial polymorphic loci.

[0014] The present invention provides a kit for detecting mitochondrial polymorphic loci, characterized in that the kit comprises the above primer composition.

[0015] Preferably, the concentrations of the primers in the primer composition are shown in the following table:

[0016]

[0017]

[0018] Preferably, the kit further comprises library construction reagents and sequencing reagents.

[0019] The present invention provides the application of the above primer composition or the above kit in complex kinship identification and / or individual identification.

[0020] The present invention provides the application of the above primer composition or the above kit in detecting degraded forensic samples.

[0021] The present invention provides a method for detecting mitochondrial polymorphic locus typing based on next-generation sequencing technology, comprising:

[0022] After constructing a library for the DNA of the sample to be tested using the above primer composition, sequencing is performed, and the obtained sequencing data is analyzed to obtain mitochondrial polymorphic locus typing.

[0023] Preferably, the sample to be tested includes blood, semen, saliva, bone, tooth, hair, exfoliated cells or human tissue.

[0024] Preferably, the concentration of the DNA of the sample to be tested is 1 ng / μL.

[0025] The present invention discloses the following technical effects:

[0026] The primer composition for detecting mitochondrial polymorphic sites based on next-generation sequencing technology provided by the present invention covers 88 polymorphic sites on the human mitochondrial genome. Based on next-generation sequencing technology, a primer composition and a kit capable of detecting 88 mitochondrial polymorphic sites have been developed and established. Compared with the previously constructed systems (such as the Expressmarker mtDNA-SNP 60 kit of Wuxi Zhongde Meilian Company), the primer composition or kit of the present invention can provide more new genetic information. At the same time, the present invention shows higher sensitivity compared with the next-generation sequencing kits for detecting the entire mitochondrial genome. In addition, the mitochondrial polymorphic sites involved in the present invention all have relatively high polymorphic information content, and can be applied to research directions such as forensic complex kinship identification and individual identification, and can also be applied to the detection of degraded forensic samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0028] Figure 1 Statistical results of the sequencing depth for detecting standard DNA (2800M) at different concentrations by the method provided in Example 2, where Average Alleles Detected represents the average alleles detected (%), and Average Depth of Coverage (DoC) represents the average sequencing depth (DoC). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description of the present invention and the examples are merely illustrative.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] Primers were designed based on 88 mitochondrial polymorphic loci. The specific information of the primers is shown in Table 2 in detail. The said 88 mitochondrial polymorphic loci are derived from mitochondrial polymorphic loci confirmed in existing commercial kits and literature, distributed in each functional region of mitochondria, and have good polymorphism in the Chinese population. Their locus information is shown in Table 1 as follows:

[0036] Table 1 Numbers, chromosomal positions and polymorphic locus information of 88 mitochondrial polymorphic loci

[0037]

[0038]

[0039]

[0040] Table 2 Grouping, numbers, primer sequences and concentrations of amplification primers for 88 mitochondrial polymorphic loci

[0041]

[0042]

[0043]

[0044]

[0045] Note: According to the physical positions of the screened mitochondrial polymorphic loci, multiplex PCR primers were designed using a primer design tool. The design principles include: 1) The annealing temperature is between 58°C and 62°C; 2) Avoid primer dimers and hairpin structures; 3) The GC content is between 20% and 80%; 4) Overlap analysis was performed to reduce the number of primers.

[0046] This embodiment provides a method for detecting mitochondrial polymorphic loci based on next-generation sequencing technology, including the following steps:

[0047] (1) Extract the DNA of the sample to be tested and dilute the sample concentration to 1 ng / μL;

[0048] (2) Use Library Prep Kit for the first-round multiplex amplification. The PCR amplification system and conditions are shown in Table 3:

[0049] Table 3. The reaction system of the first-round multiplex amplification in library construction

[0050] Component Volume (μL) <![CDATA[ddH2O]]> 8 Enhancer buffer NB (1N) 3.5 Enhancer buffer M 2.5 Primer mixture A or B 5 IGT-EM808 polymerase mixture 10 Sample DNA 1

[0051] Note: Primer mixture A or B is two groups of mixtures of the primers SEQ ID No. 1-110 in Table 2, and the concentration of each primer is shown in Table 2.

[0052] The PCR reaction conditions are: 1) Pre-denaturation at 95°C for 3 min 30 s; 2) Denaturation at 98°C for 20 s; Annealing at 60°C for 8 min; This step is cycled 28 times; 3) Extension at 72°C for 5 min.

[0053] (3) The product of the first-round magnetic bead purification:

[0054] Use Agencourt AMPure XP magnetic beads to purify the first-round PCR product. Before use, the magnetic beads need to be placed at room temperature for 30 min and resuspended to make the magnetic beads uniform.

[0055] (4) The second-round PCR, the reaction system for adapter ligation is shown in Table 4:

[0056] Table 4. The reaction system for adapter ligation in library construction

[0057]

[0058] The PCR reaction conditions are: 1) Pre-denaturation at 95°C for 3 min 30 s; 2) Denaturation at 98°C for 20 s; Annealing at 58°C for 1 min; Extension at 72°C for 30 s; This step is cycled 9 times; 3) Extension at 72°C for 5 min.

[0059] (5) The product of the second-round magnetic bead purification:

[0060] Purify the second-round PCR products using Agencourt AMPure XP magnetic beads. Before use, the magnetic beads need to be placed at room temperature for 30 min and resuspended to make the magnetic beads uniform.

[0061] (6) Purify and quantify the library: Use Qubit and Agilent 2100 Bioanalyzer for library quantification and quality control.

[0062] (7) Sequencing and data analysis: Based on the Illumina platform, sequence the constructed DNA library using MiSeq Reagent Kit V2; for the obtained sequencing data, use Trimmomatic software to remove the sequencing adapters, and then use BWA software to perform sequence alignment to align the sequences with the human reference genome (GRCh37), and use Python tools to obtain mitochondrial polymorphic sites.

[0063] This method can be used as an auxiliary for complex kinship identification and personal identification (identification of the same person).

[0064] Example 2

[0065] This example is the forensic verification work of the method for detecting mitochondrial polymorphic sites based on next-generation sequencing technology provided in Example 1. The specific experiments and results are as follows:

[0066] Calculate the sensitivity, accuracy, repeatability, and forensic population genetics parameters of the method for detecting mitochondrial polymorphic sites based on next-generation sequencing technology provided in Example 1 according to the requirements of the Scientific Working Group for DNA Analysis Methods (SWGDAM, 2016).

[0067] The results show that the method constructed in Example 1 (for 88 mitochondrial polymorphic sites) has high sensitivity. When the DNA input amount is 62.5 pg to 2 ng, complete genotyping of 88 mitochondrial polymorphic sites can be obtained. The sequencing depths at different DNA input amounts are shown in Figure 1 . Verify by Sanger sequencing method, and confirm that the genotypes of all sites in this system are consistent with the next-generation sequencing results. Thus, this method has high accuracy and good repeatability.

[0068] In addition, 500 unrelated Han individuals were detected using the method constructed in Example 1. Complete genotyping of 88 mitochondrial polymorphic loci was obtained for each sample, with an average sequencing depth of 5915×. The lowest average sequencing depth for each locus was 639× (nt11251), and the highest was 8066× (nt9545). Based on the mitochondrial polymorphic locus information of 500 unrelated Han individuals, the method for detecting mitochondrial polymorphic loci based on next-generation sequencing technology provided in Example 1 identified a total of 496 different haplotypes, which were classified into 422 haplotype groups according to the phylogenetic tree. The random match probability was 0.008, the individual identification ability was 0.992, and the haplotype diversity reached 0.994.

[0069] Example 3

[0070] This example is an application of the method for detecting mitochondrial polymorphic loci using next-generation sequencing technology provided in Example 1 in the aspect of identifying individuals with the same maternal lineage.

[0071] Collect evidence samples (samples with unknown individual information) and control samples (samples with known individual information), and extract DNA separately;

[0072] 1. Use the method for detecting mitochondrial polymorphic loci based on next-generation sequencing technology provided in Example 1 to detect the DNA in Step 1. The sequencing results of the 88 mitochondrial polymorphic loci are shown in Table 5 (loci with genotypes inconsistent with rCRS are marked in bold italics).

[0073] Table 5 Sequencing results of 88 mitochondrial polymorphic loci in Example 3

[0074]

[0075]

[0076] 2. mtDNA is maternally inherited, and different individuals from the same maternal lineage should have the same mtDNA sequence. The test results recorded in Table 5 indicate that the mtDNA sequence characteristics of the evidence sample and the control sample are inconsistent. Based on the existing data and the mtDNA sequencing results, it is excluded that the evidence sample and the control sample are from the same maternal lineage.

[0077] Example 4

[0078] This example is a comparison of the analysis performance of the method for detecting mitochondrial polymorphic loci based on next-generation sequencing technology provided in Example 1 and the Expressmarker mtDNA-SNP 60 kit of the capillary electrophoresis platform for aged and degraded samples.

[0079] The Expressmarker mtDNA-SNP 60 kit based on the capillary electrophoresis platform uses 3 reaction tubes and can simultaneously amplify 60 mitochondrial polymorphic loci. Due to its relatively high required DNA input and limited number of polymorphic loci, when applied to forensic practice, it is often impossible to obtain sufficient effective information.

[0080] In this example, 2 cases of old bone samples (stored at room temperature for 9 years) were collected and detected using the method for detecting mitochondrial polymorphic loci provided in Example 1 based on next-generation sequencing technology (for 88 mitochondrial polymorphic loci) and the Expressmarker mtDNA-SNP 60 kit respectively, and the detection efficacies of the two were compared. The results showed that using the method provided in Example 1, complete genotypes of 88 mitochondrial polymorphic loci of the 2 samples were obtained (Table 6), while for these 2 samples detected by the Expressmarker mtDNA-SNP 60 kit, genotype results of only 24 and 11 loci were obtained respectively. The results confirmed that the method provided in Example 1 had significantly better detection effect on old degraded samples than the Expressmarker mtDNA-SNP 60 kit on the capillary electrophoresis platform.

[0081] Table 6 Sequencing results of 88 mitochondrial polymorphic loci in Example 4

[0082]

[0083]

[0084] As can be seen from the above examples, the primer composition, kit and method provided by the present invention provide a new detection means for forensic actual work such as the identification of complex kinship, individual identification and degraded forensic samples in the forensic research in China.

[0085] The above-described examples are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer composition for detecting mitochondrial polymorphic sites based on next-generation sequencing technology, characterized in that, The mitochondrial polymorphic loci include nt73, nt146, nt150, nt152, nt189, nt195, nt199, nt263, nt310, nt489, (CA)n, nt709, nt1541, nt1719, nt1736, nt1811, nt2706, nt3010, nt3348, nt3394, nt3970, nt4216, nt4491, nt4833, nt4883, nt5147, nt5178, nt5417, nt5442, nt5460, nt5465, nt6446, nt6455, nt7028, nt7196, nt7600, nt8020, 9-bp, nt8414, nt8584, nt8684, nt8697, nt8701, nt8793, nt8794, nt8964, nt9090, nt9123, nt9477, nt9540, nt9545, nt9698, nt9824, nt10310, nt10397, nt10398, nt10400, nt10609, nt10873, nt11215, nt11251, nt11719, nt11914, nt12372, nt12705, nt12811, nt13104, nt13708, nt13928, nt14569, nt14668, nt14783, nt15043, nt15301, nt15487, nt15784, nt16126, nt16129, nt16183, nt16223, nt16234, nt16304, nt16311, nt16316, nt16319, nt16327, nt16362 and nt16519; The primer composition includes Group A primers and Group B primers as described in the following table:

2. Use of the primer composition according to claim 1 in the preparation of a kit for detecting mitochondrial polymorphic loci.

3. A kit for detecting mitochondrial polymorphic sites, characterized in that, The kit includes the primer composition according to claim 1.

4. The kit according to claim 3, characterized in that, The concentrations of the primers in the primer composition are as shown in the following table:

5. The kit according to claim 4, wherein The kit further includes library construction reagents and sequencing reagents.

6. Use of the primer composition according to claim 1 or the kit according to any one of claims 3-5 in complex kinship identification and / or individual identification.

7. Use of the primer composition according to claim 1 or the kit according to any one of claims 3-5 in the detection of degraded forensic samples.

8. A method for detecting mitochondrial polymorphic locus typing based on next-generation sequencing technology, characterized in that, Comprising: After constructing a library for the DNA of the sample to be tested using the primer composition according to claim 1, sequencing is performed, and the obtained sequencing data is analyzed to obtain mitochondrial polymorphic locus typing.

9. The method according to claim 8, wherein The sample to be tested includes blood, semen, saliva, bone, teeth, hair, exfoliated cells or human tissue.

10. The method according to claim 8, characterized in that, The concentration of the DNA of the sample to be tested is 1 ng / μL.

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