Kit for detecting micro-haplotype and normal, Y and X chromosome STR gene loci based on next-generation sequencing technology and primer combination thereof

Abstract

The invention discloses a kit for detecting micro-haplotype and STR gene loci of normal, Y and X chromosomes based on a next-generation sequencing technology and a primer combination of the kit. The primer combination provided by the invention consists of 738 types of primers which are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 738. According to the invention, a multi-genetic marker composite detection system containing autosomal STR gene loci, X-STR gene loci, Y-STR gene loci and micro-haplotype gene loci is constructed, and 378 gene loci, namely 55 autosomal STR gene loci, 96 Y-chromosome STR gene loci, 30 X-STR gene loci, 196 micro-haplotype gene loci and 1 gender determination gene locus, can be detected at a time. The method can support forensic medicine applications such as individual identification, paternity test, fine checking of male families, mixed DNA splitting, complex genetic relationship analysis and micro-degradation sample inspection, and provides powerful evidences for practical applications such as case reconnaissance.

Description

Technical Field

[0001] The present invention belongs to the field of forensic identification and kinship identification, and relates to a kit and a primer combination thereof for detecting micro-haplotypes and normal, Y, and X chromosome STR loci based on the second-generation sequencing technology. The kit or primer combination provided by the present invention is integrated with the second-generation sequencing to perform forensic DNA testing and identification and kinship identification. Background Art

[0002] Short tandem repeats (STRs) are the mainstream genetic markers for forensic DNA testing and identification because of their wide distribution on the genome, short sequence length, and good genetic polymorphism. STR genetic markers located on autosomes are widely used in individual identification, parent-child relationship identification, kinship identification, and other testing and identification. The Y chromosome STR genetic markers unique to men have paternal inheritance characteristics and play an important role in paternal family screening, mixed male and female sample testing, and paternal genetic structure research. In addition, X chromosome STR has the characteristics of sex-linked inheritance and is widely used in the identification of complex kinship. With the construction of the STR database, the national standard "Loci and Data Structures Selected for Forensic DNA Databases" (GB / T 41009-2021) was issued in 2021. This standard classifies STR loci into A, B, and C categories, which provides important guidance for the selection of loci when constructing a forensic STR detection system.

[0003] Single nucleotide polymorphism (SNP) has the characteristics of low polymorphism, low mutation rate, and small amplicon fragments that can be designed, which makes it have significant advantages in DNA testing of degraded samples. It is also widely studied in the fields of ethnic origin inference and phenotypic characterization, and plays an important role in forensic DNA testing. Due to the fluorescent color channel limitation of traditional capillary electrophoresis (CE) technology, the number of detectable SNP loci is limited, resulting in the individual identification efficiency of SNP detection kits being inferior to that of STR detection kits. Therefore, SNP is often used as an auxiliary genetic marker of STR.

[0004] In 2013, Professor Kidd proposed a new type of DNA genetic marker, Microhaplotype (MH). Microhaplotype refers to a combination of two or more tightly linked SNPs within a 200bp DNA fragment, which can be used for individual identification, biogeographic ancestry inference, and kinship identification. Since microhaplotype genetic markers contain multiple SNP sites, they have multi-allelic characteristics and are more polymorphic than single SNPs. In addition, microhaplotype genetic markers do not produce STR shadow peaks during the polymerase chain reaction (PCR) process, so they are not interfered by STR shadow peaks in subsequent data analysis, making microhaplotypes an ideal genetic marker for mixed DNA separation in forensic science. Microhaplotypes have the advantages of both STR and SNP genetic markers and have great potential in forensic genetics applications.

[0005] Different forensic genetic markers have different forensic functions, which can be used together or alone in practical applications. By integrating various types of genetic markers with different forensic functions into one detection system, different genetic markers can complement each other while playing their respective functions, significantly improving the forensic application efficiency of the detection system. However, traditional CE technology is based on distinguishing different fluorescence and allele fragment sizes to obtain genotyping, which limits the number and types of genetic markers that can be detected by the multiplex PCR system. In addition, the STR typing results obtained by CE technology currently only have length information, lack sequence information, and have limited detection capabilities for degraded DNA samples. With the development of technology, next-generation sequencing (NGS) technology, also known as massively parallel sequencing, has provided new ideas and options for forensic workers. The advantages of NGS technology, such as high throughput, high speed, integration, and low cost, effectively solve the inherent limitations of traditional CE technology and create unlimited possibilities for the integrated detection of multiple types of forensic genetic markers.

[0006] With the continuous development of NGS technology, many mature detection and analysis technologies have been used in forensic practice. Single genetic marker detection kits for individual identification, parent-child relationship identification, kinship identification, ancestral information inference, phenotype prediction and other practices have been fully promoted and applied. According to the current needs of forensic DNA detection and analysis, integrated detection of multiple genetic markers has become a major trend.

[0007] At present, although forensic second-generation sequencing kits have been commercialized, the genetic markers used in related foreign products are not highly applicable to the Chinese population, and the selected STR loci do not fully cover all Class A and Class B autosomal STR loci and Y chromosome STR loci in the standard "Loci and Data Structure Selected for Forensic DNA Databases" (GB / T 41009-2021). In addition, micro-haplotype genetic markers, which have been widely studied in recent years, have application potential in mixed DNA splitting, individual identification, and kinship analysis, and can complement STRs and SNPs to jointly support forensic DNA detection and analysis. However, there are currently few composite micro-haplotype marker detection systems based on NGS platforms, and the number of micro-haplotype markers in existing detection systems is limited or A e The value is not high and cannot meet the diversified needs of forensic DNA testing. Summary of the invention

[0008] The purpose of the present invention is to provide a kit and a primer combination for detecting micro-haplotypes and normal, Y, and X chromosome STR loci based on the second-generation sequencing technology. The kit or primer combination provided by the present invention is integrated with the second-generation sequencing to perform forensic DNA testing and identification and kinship identification.

[0009] The invention provides a primer combination, which consists of 738 primers; the primers are single-stranded DNA molecules; the nucleotide sequences of the 738 primers are shown in SEQ ID NO: 1 to SEQ ID NO: 738 in sequence.

[0010] The present invention also provides the use of the primer combination in preparing a kit.

[0011] The invention also provides application of the primer combination in forensic DNA testing and identification.

[0012] The present invention also provides the application of the primer combination in paternity testing, kinship testing, individual identification or male pedigree testing.

[0013] The present invention also provides application of the primer combination in gene locus analysis.

[0014] The present invention also provides a kit, comprising the primer combination.

[0015] The present invention also provides a method for preparing the kit, comprising the step of mixing and packaging all the primers in the primer combination.

[0016] The invention also provides application of the kit in forensic DNA testing and identification.

[0017] The present invention also provides the use of the kit in paternity testing, kinship testing, individual identification or male pedigree testing.

[0018] The present invention also provides application of the kit in gene locus analysis.

[0019] The functions of any of the above primer combinations or any of the above kits are as follows (a) or (b) or (c) or (d) or (e) or (f):

[0020] (a) Gene locus analysis;

[0021] (b) Forensic DNA testing and identification;

[0022] (c) individual identification;

[0023] (d) paternity testing;

[0024] (e) Male pedigree identification;

[0025] (f) Identification of kinship.

[0026] The kit may also include other PCR amplification reagents.

[0027] Exemplarily, the PCR amplification reagent may be a DNA polymerase.

[0028] Exemplarily, the PCR amplification reagent may be a premix of a PCR amplification system, such as 2×Master Mix.

[0029] The primer combination is used for locus analysis.

[0030] The kit is used for gene locus analysis.

[0031] The loci are 55 autosomal STR loci, 96 Y-STR loci, 30 X-STR loci, 196 micro-haplotype loci (based on screening of East Asian populations, the Ae values ​​of micro-haplotype markers are all greater than 4, and have efficient mixed DNA splitting capabilities and kinship analysis performance) and 1 sex-determining locus.

[0032] The sex determination locus is: Amelogenin locus.

[0033] The 55 autosomal STR loci are as follows: D1S1656, D1GATA113, D2S1338, D2S1360, D2S1776, D2S441, TPOX, D3S1358, D3S1744, D3S3045, D3S3053, D3S4529, D4S2364, D4S2366, D4S2408, FGA, CSF1PO, D5S818, D6S1017, D6S1043, D6S 474, D6S477, D7S1517, D7S3048, D7S820, D8S1115, D8S1132, D8S1179, D9S1122, D10S1248, D10S1435, D10 S2325, D11S2368, D11S4463, TH01, D12S391, vWA, D13S317, D13S325, D14S1434, D14S608, D15S659, Penta E. D16S539, D17S974, D18S51, D18S535, D19S253, D19S433, D20S1082, D20S482, D21S11, PentaD, D22GATA198B05, D22S1045.

[0034] The 96 Y-STR loci are as follows: DYS19, DYS385a / b, DYF387S1a / b, DYS388, DYS389-I / II, DYS390, DYS391, DYS392, DYS393, DYF399S1a / b / c, DYF404S1a / b, DYS426, DYS435, DYS437, DYS438, DYS439, DYS443, DYS444 , DYS447, DYS448, DYS449, DYS450, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459a / b, DYS460, DY S461, DYS476, DYS481, DYS502, DYS504, DYS508, DYS510, DYS511, DYS512, DYS513, DYS518, DYS520, D YS522, DYS525, DYS527a / b, DYS530, DYS531, DYS533, DYS538, DYS541, DYS549, DYS552, DYS556, DYS 557, DYS561, DYS565, DYS568, DYS570, DYS571, DYS572, DYS573, DYS576, DYS578, DYS585, DYS587, DY S590, DYS593, DYS596, DYS612, DYS613, DYS616, DYS622, DYS626, DYS627, DYS630, DYS635, DYS638, D YS640, DYS641, DYS643, DYS644, DYS645, DYS710, DYS712, DYS720, DYS724, Y-GATA-A10, Y-GATA-H4.

[0035] The 30 X-STR loci are as follows: DXS101, DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10159, DXS10162, DXS10164, DXS6789, DXS6795, DXS6800, DXS6804, DXS6807, DXS6809, DXS6810, DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS9895, DXS9902, DXS9907, GATA165B12, GATA31E08, GATA172D05, and HPRTB.

[0036] The 196 microhaplotype loci are as follows: mh01WL-005, mh01WL-006, mh01WL-008, mh01WL-010, mh01WL-011, mh01WL-012, mh01WL-013, mh01WL-019, mh01WL-033, mh01WL-037, mh01WL-038, mh01WL-041, mh01WL-042, mh01WL-043, mh01WL-044, mh01WL-045, mh01WL-047, mh02WL-002, mh02WL-003, mh02WL-005, mh02WL-009, mh02WL-011, mh02WL-014, mh02WL-015, mh02WL-017, mh02WL-020, mh02WL-028, mh02WL-029, mh02WL-031, mh02WL-032, mh02WL-035, mh02WL-036, mh02WL-038, mh02WL-040, mh02WL-041, mh02WL-044, mh02WL-049, mh02WL-050, mh03WL-003, mh03WL-006, mh03WL-009, mh03WL-010, mh03WL-011, mh03WL-012, mh03WL-013, mh03WL-015, mh03WL-016, mh03WL-017, mh03WL-021, mh03WL-022, mh03WL-029, mh03WL-035, mh03WL-038, mh03WL-039, mh04WL-004, mh04WL-005, mh04WL-007, mh04WL-009, mh04WL-012, mh04WL-022, mh04WL-023, mh04WL-026, mh04WL-027, mh04WL-028, mh04WL-030, mh04WL-031, mh04WL-033, mh04WL-035, mh04WL-036, mh04WL-037, mh05WL-001, mh05WL-002, mh05WL-003, mh05WL-004, mh05WL-006, mh05WL-008, mh05WL-010, mh05WL-013, mh05WL-019, mh05WL-024, mh05WL-025, mh05WL-026, mh05WL-030, mh05WL-031, mh05WL-033, mh06WL-004, mh06WL-005, mh06WL-007, mh06WL-012mh06WL-027, mh06WL-029, mh06WL-032, mh06WL-034, mh07WL-004, mh07WL-005, mh07WL-006, mh07WL-007, mh07WL-009, mh07WL-012, mh07WL-014, mh07WL-015, mh07WL-016, mh07WL-020, mh07WL-022, mh07WL-023, mh07WL-034, mh07WL-036, mh08WL-003, mh08WL-005, mh08WL-008, mh08WL-009, mh08WL-0 11、mh08WL-012、mh08WL-018、mh08WL-023、mh08WL-024、mh08WL-026、mh09WL-003、mh09WL-004、mh09WL-005、mh09WL-006、mh09WL-008、mh09WL-013、mh09WL-015、mh10WL-005、mh10WL-007、mh10WL-012、mh10WL-013、mh10WL-015、mh10WL-021、mh10WL-022、mh11WL-002、mh11WL-004、mh11WL-005、mh11W L-007、mh11WL-008、mh11WL-009、mh11WL-010、mh11WL-011、mh11WL-013、mh11WL-020、mh12WL-002、mh12WL-005、mh12WL-006、mh12WL-009、mh12WL-01 0、mh12WL-016、mh12WL-022、mh12WL-023、mh12WL-025、mh13WL-001、mh13WL-003、mh13WL-005、mh13WL-010、mh13WL-013、mh13WL-014、mh13WL-016、mh13WL-016 14WL-004, mh14WL-005, mh14WL-008, mh14WL-009, mh14WL-013, mh14WL-016, mh14WL-017, mh15WL-001, mh15WL-003, mh15WL-004, mh15WL-015, mh15WL-017, mh15WL-018, mh15WL-019, mh16WL-003, mh16WL-004, mh16WL-005, mh16WL-008, mh16WL-013, mh16WL-018, mh17WL-004, mh17WL-005, mh17WL-010,mh17WL-011, mh17WL-015, mh17WL-016, mh18WL-002, mh18WL-013, mh19WL-001, mh20WL-003, mh20WL-007, mh20WL-008, mh20WL-009, mh20WL-012, mh21WL-002, mh21WL-004, mh21WL-007, mh22WL-004, mh22WL-006. ,

[0037] The SNPs in each microhaplotype locus are shown in the rightmost column of Table 4 .

[0038] The nucleotide sequences of each locus in the hg38 (GRCh38.p14) human reference genome are shown in Figure 1 to Figure 369 .

[0039] The SNPs in each microhaplotype locus are Figures 174 to 369 Nucleotides are shown in red font.

[0040] In the primer combination, according to the primer sequence (i.e., from SEQ ID NO: 1 to SEQ ID NO: 738), the molar ratios of each primer are as follows: 0.02: 0.02: 0.02: 0.02: 0.05: 0.05: 0.04: 0.04: 0.04: 0.04: 0.05: 0.05: 0.02: 0.02: 0.02: 0.02: 0.02: 0.06: 0.06: 0.04: 0.04: 0.03: 0.03: 0.02: 0.02: 0.03: 0.03: 0.04: 0.04: 0.04: 0.03: 0.03: 0.02: 0.02: 0.03: 0.03: 0.04: 0.04: 0.04: 0.03: 0.03: 0.02: 0.02: 0.03: 0.03: 0.04: 0.04: 0.02: 0.02: 0.06: 0.06 : 0.06: 0.06: 0.03: 0.03: 0.07: 0.07: 0.03: 0.03: 0.06: 0.06: 0.04: 0.04: 0.09: 0.09: 0.05: 0.05: 0.02: 0.02: 0.05: 0.05: 0.03: 0.03: 0.03: 0.03: 0.03: 0.02: 0.02: 0.02: 0.02: 0.03: 0.03: 0.03: 0.02: 0.07: 0.07: 0.04: 0.04: 0.04: 0.04: 0.04: 0.02: 0.04: 0.04: 0.03:0.03:0.02:0.02:0.04:0.04:0.04:0.04:0.08:0.08:0.02:0.02:0.02:0.02:0.02:0.05:0.05:0.02:0.17:0.17:0.23:0.23:0.04:0.04:0.05:0.05:0.12:0.12:0.05:0.05:0.02:0.02:0.05:0.05:0.28:0.28:0.05:0.05:0.03:0.03:0.03:0.03:0.03:0.07:0.07:0.02:0.02:0 .03:0.03:0.31:0.31:0.04:0.04:0.04:0.04:0.37:0.37:0.02:0.02:0.34:0.34:0.20:0.20:0.15:0.15:0.13:0.13:0.14:0.14:0.02:0.02:0.03:0.03:0.05:0.05:0.05:0.04:0.04:0.02:0.02:0.04:0.04:0.03:0.03:0.03:0.03:0.06:0.06:0.06:0.04:0.04:0.16:0.16:0.02：0.02：0.06：0.06：0.02：0.02：0.02：0.02：0.14：0.14：0.05：0.05：0.03：0.03：0.19：0.19：0.03：0.03：0.03：0.03：0.06：0.06：0.05：0.05：0.04：0.04：0.17：0.17：0.05：0.05：0.18：0.18：0.15：0.15：0.13：0.13：0.04：0.04：0.34：0.34：0.34：0.34：0.16：0.16：0.12：0.12：0.06：0.06：0.10：0.10：0.05：0.05：0.02：0.02：0.07：0.07：0.04：0.04：0.04：0.04：0.06：0.06：0.20：0.20：0.04：0.04：0.08：0.08：0.10：0.10：0.04：0.04：0.04：0.04：0.30：0.30：0.17：0.17：0.08：0.08：0.03：0.03：0.18：0.18：0.03：0.03：0.04：0.04：0.04：0.04：0.03：0.03：0.15：0.15：0.03：0.03：0.10：0.10：0.1：0.1：0.07：0.07：0.07：0.07：0.07：0.07：0.05：0.05：0.11：0.11：0.1：0.1：0.09：0.09：0.09：0.09：0.06：0.06：0.11：0.11：0.05：0.05：0.12：0.12：0.08：0.08：0.08：0.08：0.12：0.12：0.07：0.07：0.03：0.03：0.07：0.07：0.05：0.05：0.04：0.04：0.21：0.21：0.04：0.04：0.04：0.04：0.04：0.04：0.09：0.09：0.07：0.07：0.04：0.04：0.07：0.07：0.07：0.07：0.04：0.04：0.06：0.06：0.07：0.07：0.08：0.08：0.02：0.02：0.02：0.02：0.01：0.01：0.06：0.06：0.02：0.02：0.03：0.03：0.02：0.02：0.04：0.04：0.01：0.01：0.02：0.02：0.01：0.01：0.02：0.02：0.03：0.03：0.03：0.03：0.03：0.03：0.02：0.02：0.02：0.02：0.02：0.02：0.02：0.02：0.03：0.03：0.03：0.03：0.02：0.02：0.03：0.03：0.03：0.03：0.02：0.02：0.01：0.01：0.01：0.01：0.05：0.05：0.09：0.09：0.01：0.01：0.02：0.02：0.03：0.03：0.06：0.06：0.02：0.02：0.05：0.05：0.03：0.03：0.02：0.02：0.02：0.02：0.06：0.06：0.06：0.06：0.02：0.02：0.04：0.04：0.04：0.04：0.02：0.02：0.02：0.02：0.05：0.05：0.02：0.02：0.02：0.02：0.04：0.04：0.05：0.05：0.04：0.04：0.03：0.03：0.02：0.02：0.02：0.02：0.04：0.04：0.03：0.03：0.01：0.01：0.03：0.03：0.02：0.02：0.04：0.04：0.02：0.02：0.02：0.02：0.03：0.03：0.03：0.03：0.02：0.02：0.05：0.05：0.03：0.03：0.03：0.03：0.02：0.02：0.05：0.05：0.03：0.03：0.01：0.01：0.02：0.02：0.04：0.04：0.04：0.04：0.05：0.05：0.04：0.04：0.01：0.01：0.05：0.05：0.03：0.03：0.03：0.03：0.02：0.02：0.02：0.02：0.07：0.07：0.02：0.02：0.05：0.05：0.04：0.04：0.05：0.05：0.04：0.04：0.05：0.05：0.01：0.01：0.04：0.04：0.03：0.03：0.07：0.07：0.06：0.06：0.03：0.03：0.03：0.03：0.03：0.03：0.01：0.01：0.02：0.02：0.02：0.02：0.05：0.05：0.02：0.02：0.05：0.05：0.01：0.01：0.05：0.05：0.05：0.05：0.05：0.05：0.02：0.02：0.02：0.02：0.04：0.04：0.04：0.04：0.05：0.05：0.02：0.02：0.01：0.01：0.04：0.04：0.05：0.05：0.05：0.05：0.03：0.03：0.04：0.04：0.04：0.04：0.03：0.03：0.06：0.06：0.02：0.02：0.02：0.02：0.02：0.02：0.03：0.03：0.02：0.02：0.02：0.02：0.03：0.03：0.03：0.03：0.09：0.09：0.05：0.05：0.02：0.02：0.02：0.02：0.03：0.03：0.04：0.04：0.03：0.03：0.09：0.09：0.06：0.06：0.03：0.03：0.09：0.09：0.01：0.01：0.04：0.04：0.02：0.02：0.04：0.04：0.07：0.07：0.05：0.05：0.03：0.03：0.03：0.03：0.02：0.02：0.05：0.05：0.03：0.03：0.05：0.05：0.01：0.01：0.07：0.07：0.04：0.04：0.02：0.02：0.02：0.02：0.04：0.04：0.03：0.03：0.02：0.02：0.03：0.03：0.02：0.02：0.02：0.02：0.05：0.05：0.04：0.04：0.02：0.02：0.03：0.03：0.05：0.05：0.01：0.01：0.04：0.04：0.03：0.03：0.02：0.02：0.02：0.02：0.01：0.01：0.05：0.05：0.03：0.03：0.03：0.03：0.05：0.05：0.03：0.03：0.02：0.02：0.02：0.02：0.02：0.02：0.03：0.03：0.01：0.01：0.02：0.02：0.02：0.02：0.06：0.06：0.02：0.02：0.07：0.07：0.02：0.02。.

[0041] In the primer combination, according to the primer sequence (i.e., from SEQ ID NO: 1 to SEQ ID NO: 738), the working concentrations of each primer are as follows (all units are μM): 0.02, 0.02, 0.02, 0.02, 0.05, 0.05, 0.04, 0.04, 0.04, 0.04, 0.05, 0.05, 0.02, 0.02, 0.02, 0.02, 0.02, 0.06, 0.06, 0.04, 0.04, 0.03, 0.03, 0.02, 0.02, 0.03, 0.03, 0.04, 0.04, 0.04, 0.03, 0.03, 0.02, 0.02, 0.03, 0.03, 0.04, 0.04, 0.04, 0.03, 0.03, 0.02, 0.02, 0.03, 0.03, 0.04, 0.04, 0.02, 0.02, 0.06, 0.06, 0.06, 0.03, 0.03, 0.07, 0.07, 0.03, 0.03, 0.06, 0.04, 0.04, 0.09, 0.09, 0.05, 0.05, 0.02, 0.02, 0.05, 0.05, 0.03, 0.03, 0.03, 0.03, 0.02, 0.02, 0.02, 0.03, 0.03, 0.03, 0.02, 0.02, 0.03, 0.03, 0.03, 0.03, 0.02, 0.07, 0.07, 0.04, 0.04, 0.04, 0.04, 0.02, 0.02, 0 .04, 0.04, 0.03, 0.03, 0.02, 0.02, 0.04, 0.04, 0.04, 0.04, 0.08, 0.08, 0.02, 0.02, 0.02, 0.02, 0.05, 0.05, 0.02, 0.17, 0.17, 0.23, 0.23, 0.04, 0.04, 0.05, 0.05, 0.12, 0.12, 0.05, 0.05, 0.02, 0.02, 0.05, 0.05, 0.28, 0.28, 0.05, 0.05, 0.03, 0.03, 0.03, 0.03, 0.03, 0.07, 0.07, 0 .02, 0.02, 0.03, 0.03, 0.31, 0.31, 0.04, 0.04, 0.04, 0.04, 0.37, 0.37, 0.02, 0.02, 0.34, 0.34, 0.20, 0.20, 0.15, 0.15, 0.13, 0.13, 0.14, 0.14, 0.02, 0.02, 0.03, 0.03, 0.05, 0.05, 0.05, 0.04, 0.04, 0.02, 0.02, 0.04, 0.04, 0.03, 0.03, 0.03, 0.03, 0.06, 0.06, 0.06, 0.04, 0.04, 0.16、0.16、0.02、0.02、0.06、0.06、0.02、0.02、0.02、0.02、0.14、0.14、0.05、0.05、0.03、0.03、0.19、0.19、0.03、0.03、0.03、0.03、0.06、0.06、0.05、0.05、0.04、0.04、0.17、0.17、0.05、0.05、0.18、0.18、0.15、0.15、0.13、0.13、0.04、0.04、0.34、0.34、0.34、0.34、0.16、0.16、0.12、0.12、0.06、0.06、0.10、0.10、0.05、0.05、0.02、0.02、0.07、0.07、0.04、0.04、0.04、0.04、0.06、0.06、0.20、0.20、0.04、0.04、0.08、0.08、0.10、0.10、0.04、0.04、0.04、0.04、0.30、0.30、0.17、0.17、0.08、0.08、0.03、0.03、0.18、0.18、0.03、0.03、0.04、0.04、0.04、0.04、0.03、0.03、0.15、0.15、0.03、0.03、0.10、0.10、0.1、0.1、0.07、0.07、0.07、0.07、0.07、0.07、0.05、0.05、0.11、0.11、0.1、0.1、0.09、0.09、0.09、0.09、0.06、0.06、0.11、0.11、0.05、0.05、0.12、0.12、0.08、0.08、0.08、0.08、0.12、0.12、0.07、0.07、0.03、0.03、0.07、0.07、0.05、0.05、0.04、0.04、0.21、0.21、0.04、0.04、0.04、0.04、0.04、0.04、0.09、0.09、0.07、0.07、0.04、0.04、0.07、0.07、0.07、0.07、0.04、0.04、0.06、0.06、0.07、0.07、0.08、0.08、0.02、0.02、0.02、0.02、0.01、0.01、0.06、0.06、0.02、0.02、0.03、0.03、0.02、0.02、0.04、0.04、0.01、0.01、0.02、0.02、0.01、0.01、0.02、0.02、0.03、0.03、0.03、0.03、0.03、0.03、0.02、0.02、0.02、0.02、0.02、0.02、0.02、0.02、0.03、0.03、0.03、0.03、0.02、0.02、0.03、0.03、0.03、0.03、0.02、0.02、0.01、0.01、0.01、0.01、0.05、0.05、0.09、0.09、0.01、0.01、0.02、0.02、0.03、0.03、0.06、0.06、0.02、0.02、0.05、0.05、0.03、0.03、0.02、0.02、0.02、0.02、0.06、0.06、0.06、0.06、0.02、0.02、0.04、0.04、0.04、0.04、0.02、0.02、0.02、0.02、0.05、0.05、0.02、0.02、0.02、0.02、0.04、0.04、0.05、0.05、0.04、0.04、0.03、0.03、0.02、0.02、0.02、0.02、0.04、0.04、0.03、0.03、0.01、0.01、0.03、0.03、0.02、0.02、0.04、0.04、0.02、0.02、0.02、0.02、0.03、0.03、0.03、0.03、0.02、0.02、0.05、0.05、0.03、0.03、0.03、0.03、0.02、0.02、0.05、0.05、0.03、0.03、0.01、0.01、0.02、0.02、0.04、0.04、0.04、0.04、0.05、0.05、0.04、0.04、0.01、0.01、0.05、0.05、0.03、0.03、0.03、0.03、0.02、0.02、0.02、0.02、0.07、0.07、0.02、0.02、0.05、0.05、0.04、0.04、0.05、0.05、0.04、0.04、0.05、0.05、0.01、0.01、0.04、0.04、0.03、0.03、0.07、0.07、0.06、0.06、0.03、0.03、0.03、0.03、0.03、0.03、0.01、0.01、0.02、0.02、0.02、0.02、0.05、0.05、0.02、0.02、0.05、0.05、0.01、0.01、0.05、0.05、0.05、0.05、0.05、0.05、0.02、0.02、0.02、0.02、0.04、0.04、0.04、0.04、0.05、0.05、0.02、0.02、0.01、0.01、0.04、0.04、0.05、0.05, 0.05, 0.05, 0.03, 0.03, 0.04, 0.04, 0.04, 0.03, 0.03, 0.06, 0.06, 0.02, 0.02, 0.02, 0.02, 0.02, 0.02, 0.03, 0.03, 0.02, 0.02, 0.02, 0.03, 0.03, 0.03, 0.03, 0.03, 0.09, 0.09, 0.05, 0.05, 0.02, 0.02, 0.02, 0.03, 0.03, 0.04, 0.04, 0.03, 0.03, 0.09, 0.09, 0.06, 0.06, 0.03, 0.03, 0.09, 0.09, 0.01, 0.01, 0.04, 0.04, 0.02, 0.02, 0.04, 0.07, 0.07, 0.05, 0.05, 0.03, 0.03, 0.03, 0.02, 0.02, 0.05, 0.05, 0.03, 0.03, 0.05, 0.05, 0.03, 0.03, 0.05, 0.05, 0.0 1, 0.01, 0.07, 0.07, 0.04, 0.04, 0.02, 0.02, 0.02, 0.02, 0.04, 0.04, 0.03, 0.03, 0.02, 0.02, 0.03, 0.03, 0.02, 0.02, 0.02, 0.05, 0.05, 0.04, 0.04, 0.02, 0.02, 0.03, 0.03, 0.05, 0.01, 0.01, 0.04, 0.04, 0.03, 0.03, 0 .02, 0.02, 0.02, 0.02, 0.01, 0.01, 0.05, 0.05, 0.03, 0.03, 0.03, 0.03, 0.05, 0.05, 0.03, 0.03, 0.02, 0.02, 0.02, 0.02, 0.02, 0.03, 0.03, 0.01, 0.01, 0.02, 0.02, 0.02, 0.02, 0.06, 0.06, 0.02, 0.02, 0.07, 0.07, 0.02, 0.02. .

[0042] Any of the above applications specifically includes the following steps:

[0043] (1) taking the test DNA and performing multiplex PCR amplification using the primer combination;

[0044] (2) After completing step (1), DNA purification is performed and then a library is prepared;

[0045] (3) taking the library prepared in step (2) and sequencing it;

[0046] (4) Perform data analysis on the sequencing results to obtain information on each locus.

[0047] The information of a locus refers to the typing information of the locus.

[0048] For STR loci, the information of the locus refers to the typing information (length typing and / or core repeat structure) of the locus.

[0049] For microhaploid loci, the locus information refers to the nucleotides of each SNP.

[0050] Forensic DNA testing and identification is performed based on the information of the genetic loci.

[0051] Individual identification is performed based on the information of the genetic loci.

[0052] Paternity testing is performed based on information from the genetic loci.

[0053] Male pedigree identification was performed based on the information of the gene loci.

[0054] The relationship is identified based on the information of the gene loci.

[0055] Specifically, the sequencing may be next generation sequencing.

[0056] Specifically, the sequencing may be high-throughput sequencing.

[0057] Specifically, the reaction system for the multiplex PCR amplification is (20 μL): composed of 10 μL 2×Master Mix, 1 μ test DNA, the primer combination and nuclease-free water. In the reaction system, the concentration of each primer is shown in the fifth column of Table 2.

[0058] Specifically, the reaction procedure of the multiplex PCR amplification is: 95°C for 5 min; 95°C for 30 s, 59°C for 1 min 30 s, 72°C for 2 min, 21 cycles; 72°C for 5 min.

[0059] Specifically, the DNA library was prepared using TruSeq DNA PCR-Free HT Library Prep Kit according to the instructions.

[0060] Specifically, the sequencing is carried out using Reagent Kit v3-600 cycles Sequencing reagents in MiSeqFGx TM Forensic genomic analysis is performed systematically.

[0061] Specifically, the data analysis method includes the following steps:

[0062] After sequencing, MiSeq FGxTM The forensic genome analysis system automatically generates fastq files, and the fastq files are analyzed using STRaitRazor3.0 software;

[0063] Before running the STRaitRazor 3.0 software, enter the configuration files of each locus in the excel table. The configuration files of the loci are as follows: ① Locus name; ② Chromosome information; ③ 12 bases after the forward primer; ④ The reverse complementary sequence of the first 12 bases of the reverse primer; ⑤ The repeating unit of the core sequence of the locus; ⑥ The number of bases in a repeating unit; ⑦ The number of bases in the flanking sequence, and each part is separated by the Tab key. Enter the 378 loci in this format. After the gene configuration files of the 378 loci are entered, run the STRaitRazor 3.0 software to generate the allsequences.txt file. The file gives the following five parts of information: ① Locus name; ② Genotyping; ③ Number of amplicon bases; ④ Amplicon sequence information; ⑤ Sequencing depth.

[0064] The core repeat structure of the sequences of 181 STR loci was constructed according to the STR sequence guide for Y-STR loci published by the International Society for Forensic Genetics (ISFG) (https: / / strider.online), the NIST STR database (https: / / strbase.nist.gov / Loci / FactSheet) and the national standard "Nomenclature of sequence polymorphism STR" (GB / T 44393-2024).

[0065] The information of SNPs in 196 microhaplotype loci was obtained based on the sequencing results.

[0066] Deoxyribonucleic acid (DNA) genetic markers are important molecular markers in forensic practice. Short tandem repeat sequence genetic markers can be used for individual identification, paternity testing and other practical operations. Single nucleotide polymorphisms are widely used in ethnic origin inference and phenotypic characterization. In recent years, micro-haplotype (marker) research has become increasingly widespread in the field of forensic medicine, and has good mixed DNA splitting efficiency. However, in traditional capillary electrophoresis detection technology, multiple genetic markers need to be detected separately, which has limitations such as limited number of detected genetic markers, large sample consumption, complicated experimental procedures and poor correlation of genetic information, and it is difficult to achieve micro-haplotype testing. Compared with CE technology, second-generation sequencing technology has the characteristics of high throughput and integration, supports the simultaneous detection of multiple genetic markers, and is more suitable for the integrated detection of multiple types of forensic genetic markers. At present, there are limited studies and reports on NGS integrated detection technology at home and abroad. Although there are integrated detection kits dedicated to forensic medicine, there are problems such as small number of genetic markers or single type, and low applicability to the Chinese population.

[0067] The present invention is based on the second-generation sequencing platform MiSeq FGx forensic genome analysis system, and constructs a multi-genetic marker composite detection system including autosomal STR loci, X-STR loci, Y-STR loci and micro-haplotype loci. 378 loci can be detected at a time, including 55 autosomal STR loci, 96 Y chromosome STR loci, 30 X-STR loci, 196 micro-haplotype loci and 1 gender determination locus Amelogenin. It contains all 29 A and B autosomal STR loci and all 35 A and B Y-STR loci. The present invention is based on NGS technology to achieve integrated detection of four genetic markers, and utilizes the advantages of high throughput, high speed, integration and low cost of NGS to achieve one-time detection of a biological sample to obtain four genetic marker information, effectively improve detection efficiency, and save limited on-site biological samples. At the same time, the integrated detection of four genetic markers is conducive to multi-dimensional mining and exploration of genetic information, realizing information association between different genetic markers, thereby realizing the association of biological evidence. The present invention can support forensic applications such as individual identification, paternity testing, detailed screening of male family lines, mixed DNA splitting, complex kinship analysis, and testing of trace degraded samples, providing powerful evidence for practical applications such as case investigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 It is the target sequence of a sex-determining locus (Amelogenin locus) in the reference genome.

[0069] Figures 2 to 56 Target sequences of 55 autosomal STR loci in the reference genome.

[0070] Figure 57 to Figure 143 Target sequences of 96 Y-STR loci in the reference genome.

[0071] Figures 144 to 173 are the target sequences of 30 X-STR loci in the reference genome.

[0072] Figures 174 to 369 Target sequences of 196 microhaplotype loci in the reference genome.

[0073] Figure 1 to Figure 369 In the figure, the underlined region is the region corresponding to the primer.

[0074] Figures 174 to 369 In the figure, the nucleotides in red font are the nucleotides corresponding to the SNP. DETAILED DESCRIPTION

[0075] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Among the STR loci, two-copy loci are counted as two STR loci, and three-copy loci are counted as three STR loci. The reference genome refers to the GRCh38 human genome (see the UCSC Genome Browser database), the website is as follows: http: / / hgdownload.soe.ucsc.edu / goldenPath / hg38 / bigZips / hg38.2bit.

[0076] Some degenerate bases are involved in the primers. The meanings of the degenerate bases are shown in Table 1.

[0077] Table 1

[0078] Degenerate bases meaning R A or G Y C or T M A or C K G or T S G or C W A or T H A or T or C B G or T or C V G or A or C D G or A or T N A or T or C or G

[0079] Example 1: Screening and establishment of primer combinations and composite systems

[0080] 1. Locus Screening

[0081] Based on the results of the previous analysis, combined with multiple existing databases, a large amount of sequence information based on East Asian populations was obtained, and 226 STR loci, 202 microhaplotype loci and 1 sex determination locus (Amelogenin locus) were preliminarily screened. The Reagent Kit V3 sequencing kit is a double-end 300bp sequencing mode (forward detection 300bp, reverse detection 300bp), which can detect up to 600bp DNA fragments. Since base errors are prone to occur in the splicing process of forward and reverse sequences, forward and reverse sequence splicing is generally not performed, so only 300bp DNA fragments can be detected. The sequence information of 226 STR loci was checked. In the reference genome, the core repeat region sequence lengths of loci DYS526b, DYS547, DYS711, and DYS713 were all greater than 280bp. The flanking sequences of the above loci have a small adjustable range when designing primers, which is not conducive to designing primers. Therefore, loci DYS526a / b, DYS547, DYS711, and DYS713 were deleted. After locus screening, 221 STR loci, 202 micro-haplotype loci, and 1 sex determination locus remained.

[0082] 2. Design and selection of primers for next-generation sequencing

[0083] The amplification efficiency and specificity of primers in the composite amplification system determine the composite amplification effect, so primer design and screening are key. When designing primers, select the same parameters for screening and keep the annealing temperature as consistent as possible to ensure the specificity and balance of composite amplification.

[0084] Based on the screened 221 STR loci, 202 microhaplotype loci and 1 sex determination locus, the reference genome was used to obtain the sequence information of each locus and primers were designed. The primer design region was within 200 bp of the upstream and downstream flanking sequences of the target fragment. The amplicon length was controlled within 300 bp, and at least 2 pairs of primers were designed for each locus.

[0085] The principles of primer design are as follows: ① Primer length: 24±7bp. If the primer sequence is too short, mismatches are likely to occur. If the primer sequence is too long, the extension temperature will be too high, which is not conducive to the Taq DNA polymerase reaction, thereby affecting the amplification efficiency; ② GC content: between 40%-60%. When the sequence primer design of the locus is difficult, it can be appropriately adjusted to 35%-65%; ③ Annealing temperature: between 58℃-62℃, and the temperature range can be appropriately relaxed; ④ Sequence structure: no palindromic structure, avoid hairpin structure; ⑤ 3' end base requirements: Since the base pairing at the 3' end of the primer affects the extension of the enzyme, in order to ensure the specificity and amplification efficiency of the primer, the 3' terminal base of the primer should be strictly complementary to the template DNA sequence.

[0086] Primer screening was performed, and based on the results, primers that amplified more than two DNA fragments from the autosomal STR loci, single-copy Y-STR loci, X-STR loci, and microhaploid loci were deleted; primers that amplified more than two DNA fragments from the two-copy Y-STR loci were deleted; and primers that amplified more than three DNA fragments from the three-copy Y-STR loci were deleted.

[0087] 3. Construction and optimization of the composite amplification system

[0088] 1. Obtain optimized primers for 65 loci through construction and optimization of the composite amplification system

[0089] Complete the construction of the composite detection system of autosomal STR loci (A and B), Y-chromosome STR loci (A and B) and sex-determining loci (Amelogenin loci) in the "Loci and Data Structure Selected for Forensic DNA Database" (GB / T 41009-2021), evaluate the amplification efficiency of the target fragments of the system, and conduct targeted primer interference investigation to optimize the detection system of 65 core preferred loci. 65 core preferred loci: 29 autosomal STR loci (A and B), 35 Y-STR loci (A and B) and sex-determining loci (Amelogenin locus). Primers for the 65 core preferred loci will be combined to verify the performance of the primer combination. Primers were optimized based on sequencing results. The principles of primer optimization are as follows: ① If there is primer interference only between two loci, the primers of loci with high sequencing depth are retained first, and the primers of loci with low sequencing depth are replaced; ② If there is interference between the primers of a single locus and multiple loci, the primers of the locus are directly replaced. After replacing the primers, the primer concentration of each locus is adjusted according to the sequencing depth, and the performance of the primer combination is verified again. Primer combinations for detecting 65 loci were obtained.

[0090] 2. Construction and optimization of the composite detection system of 65 loci and Y-STR loci

[0091] The primer combination obtained in step 1 was combined with the primers used to detect 82 Y-STR loci to verify the performance of the primer combination. Primer optimization was performed based on the sequencing results. The primer optimization principles are as follows: ① If there is interference between the newly added Y-STR loci and one of the 65 core preferred loci, but the sequencing depth of the core preferred loci is not significantly reduced, the primers of the Y-STR loci are replaced; ② If there is interference between the newly added Y-STR loci and one of the 65 core preferred loci, resulting in a significant reduction in the sequencing depth of the core preferred loci or no detection, the Y-STR loci are deleted; ③ If there is interference between the newly added Y-STR loci and multiple loci in the 65 core preferred loci, the Y-STR loci are deleted; ④ If there is primer interference between two newly added Y-STR loci, the Y-STR loci with high sequencing depth are retained first; ⑤ If there is primer interference between a single newly added Y-STR locus and multiple Y-STRs, the Y-STR loci are deleted. After completing locus deletion and primer replacement, adjust the concentration of each primer according to the sequencing depth and verify the performance of the primer combination again. Primer combinations for detecting 126 loci were obtained (61 Y-STR loci were added relative to the 65 core preferred loci in step 1).

[0092] 3. Construction and optimization of a composite detection system for 126 loci and autosomal STR loci

[0093] The primer combination obtained in step 2 was combined with the primers used to detect 43 autosomal STR loci to verify the performance of the primer combination. Primer optimization was performed based on the sequencing results. The primer optimization principles were as follows: ① If there was interference between the newly added autosomal STR loci and one of the 65 core preferred loci, but the sequencing depth of the core preferred loci was not significantly reduced, the primers of the autosomal STR loci were replaced; ② If there was interference between the newly added autosomal STR loci and one of the 65 core preferred loci, resulting in a significant reduction in the sequencing depth of the core preferred loci or no detection, the autosomal STR loci were deleted; ③ If there was interference between the newly added autosomal STR loci and multiple loci in the 65 core preferred loci, the autosomal STR loci were deleted; ④ If there was primer interference between two newly added autosomal STR loci, the autosomal STR loci with high sequencing depth were preferentially retained; ⑤ If there was primer interference between a single newly added autosomal STR locus and multiple non-core preferred STR loci, the autosomal STR loci were deleted. After completing locus deletion and primer replacement, the concentration of each primer was adjusted according to the sequencing depth, and the performance of the primer combination was verified again. Primer combinations for detecting 152 loci were obtained (26 autosomal STR loci were added relative to the 126 loci in step 2).

[0094] 4. Construction and optimization of the composite detection system of microhaplotype loci and STR loci

[0095] The primers used to detect 202 micro-haplotype loci were divided into 5 groups (groups 1 to 3 were used to detect 40 loci each, and groups 4 and 5 were used to detect 41 loci each). The primer combinations obtained in step 3 were combined with the primers used to detect each group to verify the performance of the primer combination. Primers were optimized based on the sequencing results. The primer optimization principles were as follows: ① If there was interference between the micro-haplotype loci and the STR loci, the micro-haplotype loci were deleted; ② If there was primer interference between two micro-haplotype loci, the micro-haplotype loci with a high Ae value were retained; ③ If there was interference between a single micro-haplotype locus and multiple micro-haplotype loci, the micro-haplotype loci were deleted. After the locus deletion was completed, the remaining micro-haplotype loci were compounded with the STR loci, the concentration of each primer was adjusted according to the sequencing depth, and the performance of the primer combination was verified again. Primer combinations for detecting 348 loci were obtained (196 microhaplotype loci were added compared to the 152 loci in step 4).

[0096] 5. Construction and optimization of the composite detection system of 348 loci and X-STR loci

[0097] The primer combination obtained in step 4 was combined with the primers used to detect 36 X-STR loci to verify the performance of the primer combination. Primer optimization was performed based on the sequencing results. The primer optimization principles are as follows: ① If there is interference between the X-STR loci and the 65 core preferred loci, the X-STR loci are deleted; ② If there is interference between the X-STR loci and the non-core preferred autosomal STR loci and / or Y-STR loci, the X-STR loci are deleted; ③ If there is primer interference between the X-STR loci, the X-STR loci with high sequencing depth are retained; ④ If there is interference between the X-STR loci and the micro-haplotype loci, the micro-haplotype loci are deleted. After the locus deletion is completed, the concentration of each primer is adjusted according to the sequencing depth, and the performance of the primer combination is verified again. A primer combination for detecting 378 loci was obtained (30 X-STR loci were added relative to the 348 loci in step 4).

[0098] The composite system finally constructed in this example is used to detect the following loci: 55 autosomal STR loci, 96 Y-STR loci, 30 X-STR loci, 196 micro-haplotype loci and 1 sex determination locus (Amelogenin locus), a total of 378 loci. These loci include all A and B autosomal STR loci (29 loci) and all A and B Y-STR loci (35 loci) recorded in "Loci and Data Structure Selected for Forensic DNA Database" (GB / T 41009-2021). Among the STR loci, the DYS385a / b locus, DYF387S1a / b locus, DYS389-I / DYS389-II locus, DYF404S1a / b locus, DYS459a / b locus, DYS527a / b locus and DYS724 locus are all two-copy loci, counted as 2 loci respectively; the DYF399S1a / b / c locus is a three-copy locus, counted as 3 loci; the other loci are all single-copy loci, counted as 1 locus respectively.

[0099] Example 2: Locus information and primer information

[0100] The primer combination obtained in Example 1 consists of 738 primers (369 primer pairs) for detecting 378 loci, and the information of each primer in the primer combination is shown in Table 2. In Table 2, from left to right, the first column is the locus name, the second column is the primer name, the third column is the primer number in the sequence table, the fourth column is the primer sequence, and the fifth column is the working concentration of the primer.

[0101] The target sequences of the primer pairs in Table 2 in the reference genome are shown in Figure 1 to Figure 369 .

[0102] Based on the reference genome, the length of the amplified products of 378 loci ranged from 113 to 299 bp. Among them, TPOX, D2S1776, D3S4529, D4S2366, D4S2408, D6S474, D10S1435, vWA, D17S974, Penta The amplicon length of the D, D22S1045, DXS6795, DXS7132, DXS10162, GATA165B12, HPRTB, DYS388, DYS392, DYS460, DYS538, DYS570, DYS641, and DYS643 loci was ≤150 bp; the amplicon length of the D1S1656, D1GATA113, D4S2364, D6S1017, D6S1043, D7S3048, D8S1115, D8S1132, D8S1179, D9S1122, vWA, D12S391, D13S317, D13S325, D14S1434, Penta E. D16S539, D18S51, D18S535, D19S253, D20S482, D22GATA198B05, DXS6789, DXS6800, DXS6804, DXS7133, DXS7423, DXS7424, DXS9895, DXS9902 , DXS9907, DXS10079, GATA31E08, GATA172D05, DYS19, DYS389-I, DYF399S1a / b / c, DYF404S1a / b, DYS426, DYS438, DYS443, DYS458, DYS459a / b, D The amplicon lengths of the YS476, DYS481, DYS508, DYS525, DYS530, DYS531, DYS556, DYS561, DYS565, DYS573, DYS576, DYS578, DYS590, DYS596, DYS612, DYS613, DYS622, DYS644, DYS645, DYS710, Y-GATA-A10, Y-GATA-H4, mh04WL-012, mh05WL-003, mh05WL-025, mh09WL-004, and mh18WL-002 loci were >150 bp and ≤200 bp. Loci with smaller amplicon lengths are beneficial for testing difficult, degraded, and old samples.

[0103] Table 2

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] Note: Primers with odd numbers in their names are forward primers, and primers with even numbers in their names are reverse primers.

[0120] Example 3: Application of primer combination

[0121] Test sample: 2800M. 2800M is a positive control DNA (single-source male human genomic DNA) for Human STR Analysis, Promega, product catalog number DD7101.

[0122] 1. Multiplex PCR amplification using primer combinations and DNA purification

[0123] 1. Take the test sample and dilute it with nuclease-free water to make the DNA concentration 1 ng / μL, which is the template solution.

[0124] 2. Take the template solution prepared in step 1 and perform multiple PCR amplification using a primer combination (consisting of 738 primers, the sequences are shown in Table 2) to obtain PCR amplification products.

[0125] The amplification system (20 μL) consists of: 10 μL 2×Master Mix, 1 μL template solution prepared in step 1, 2 μL primer combination solution, and 7 μL nuclease-free water. The primer combination solution is used to provide all primers in the primer combination. In this reaction system, the concentration of each primer is shown in the rightmost column of Table 2.

[0126] 2×Master Mix: contains 40mM Tris-HCl, 3.2mM MgCl2, 100mM KCl, 0.04g / 100ml NaN3, 6.4% (volume percentage) glycerol, 0.4% (volume percentage) Tween 20, 1.6mg / mL bovine serum albumin,

[0127] 400 mM dNTP, 0.2 U / μL Taq DNA polymerase (Roche product, catalog number 11147633103), and the balance is water.

[0128] Amplification program: 95°C for 5 min; 95°C for 30 s, 59°C for 1 min and 30 s, 72°C for 2 min, 21 cycles; 72°C for 5 min; store at 4°C.

[0129] 3. Take the PCR amplification product obtained in step 2 and use PCR purification kit and operate according to the instructions to obtain the purified product of PCR amplification product. PCR purification kit (MinElute PCR Purification Kit): QIAGEN, product catalog number is 28006.

[0130] 4. Take the purified product obtained in step 3 and use Qubit TM dsDNA quantification kit (follow the instructions) and DNA was quantified using a 3.0 fluorescence quantifier to obtain the DNA concentration of the purified product.

[0131] Qubit TM dsDNA quantification kit: Thermo Fisher Scientific, product catalog number is Q32854.

[0132] 3.0 Fluorometer: Thermo Fisher Scientific.

[0133] 2. Library Preparation

[0134] Take the purified product obtained in step 1, 3, and use the TruSeq DNA PCR-Free HT Library Prep Kit according to the instructions (the library preparation process includes: end repair, purification of end repair products, A-tail ligation, Adapter ligation, and purification of ligation products), then use the KAPA Library Quantification Kit to quantify the library, and then perform library homogenization to obtain a library solution.

[0135] TruSeq DNA PCR-Free HT Library Prep Kit: Illumina, product catalog number is FC-121-3003. KAPA Library Quantification Kit: KAPA, product catalog number is KK4824.

[0136] 3. Sample test

[0137] Take the library solution prepared in step 2 and use Reagent Kit v3-600 cycles Sequencing reagents in MiSeq FGx TM The sequencing was performed using the Forensic Genome Analysis System (a product of Illumina).

[0138] Reagent Kit v3-600 cycles sequencing reagents: Illumina, product catalog number is MS-102-3003.

[0139] After sequencing, MiSeq FGx TM The forensic genome analysis system automatically generates fastq files, and the fastq files are analyzed using the STRaitRazor3.0 software. Before running the STRaitRazor 3.0 software, enter the configuration files of each locus in the excel table. The locus configuration files are as follows: ① locus name; ② chromosome information; ③ 12 bases after the forward primer; ④ the reverse complementary sequence of the first 12 bases of the reverse primer; ⑤ the repeating unit of the core sequence of the locus; ⑥ the number of bases in a repeating unit; ⑦ the number of bases in the flanking sequence, and each part is separated by the Tab key. Enter the 378 loci in this format. After the 378 loci gene configuration files are entered, run the STRaitRazor 3.0 software to generate the allsequences.txt file. The file gives the following five parts of information: ① locus name; ② genotyping; ③ the number of amplicon bases; ④ amplicon sequence information; ⑤ sequencing depth.

[0140] The core repeat structure of the sequences of 181 STR loci was constructed according to the STR sequence guide for Y-STR loci published by the International Society for Forensic Genetics (ISFG) (https: / / strider.online), the NIST STR database (https: / / strbase.nist.gov / Loci / FactSheet) and the national standard "Nomenclature of sequence polymorphism STR" (GB / T 44393-2024).

[0141] Result of the sex-determining locus (Amelogenin locus): XY.

[0142] The results of 181 STR loci are shown in Table 3. In Table 3, from left to right, the first column is the locus name, the second column is the length typing based on the core repeat structure, and the third column is the core repeat structure. For loci located on autosomes: if two rows of results are displayed in the table (e.g., D1S1656), it represents heterozygous type, and there are two sequencing results at the same time (i.e., the two chromosomes of a pair of homologous chromosomes each have one sequencing result); if one row of results is displayed in the table (e.g., D1GATA113), it represents homozygous type, and there is only one sequencing result (i.e., the two chromosomes of a pair of homologous chromosomes have the same sequencing result). For loci located on the Y chromosome: if two copies of the locus are displayed in the table as two rows of results (e.g., DYS385a / b), it means that two sequencing results were obtained (i.e., there are two amplicons); if three copies of the locus are displayed in the table as three rows of results (e.g., DYF399S1a / b / c), it means that three sequencing results were obtained (i.e., there are three amplicons).

[0143] Table 3 Length and repeat structure of 181 STR alleles in 2800M positive control

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] Note: N represents any nucleotide, and its subscript number represents the number of nucleotides (N and the number after it represent a base sequence, and the number after it represents the number of bases in the sequence), for example, N48 represents 48 arbitrary nucleotides; the nucleotides represented by regular characters are included in the length typing, and the nucleotides represented by italic characters are not included in the length typing; "—" means no result is obtained;

[0150] Note: Two sequencing results were obtained for the D5S818 locus. The core repeat sequence was the same, but the downstream flanking sequence of one allele was present [rs25768A>G] while the other was not, thus distinguishing the two amplicons.

[0151] The results of 196 micro-haplotype loci are shown in Table 4. In Table 4, from left to right, the first column is the locus name, the third column is the several linkage disequilibrium SNPs in the micro-haplotype locus, and the second column is the linkage genotype based on the several linkage disequilibrium SNPs in the third column. For micro-haplotype loci: If two rows of results are displayed in the table (for example, mh01WL-005), it represents a heterozygous type (that is, each chromosome of a pair of homologous chromosomes has a sequencing result), and the alleles carried by the 5 SNPs (rs11119867 / rs11119868 / rs4951574 / rs11583051 / rs7550051) in the locus on one chromosome are CCCGA, and the alleles carried by the 5 SNPs (rs11119867 / rs11119868 / rs4951574 / rs11583051 / rs7550051) on the other chromosome are CCCGA, respectively. The alleles carried by the 5 SNPs in the locus are CCCGG in sequence; if a row of results is displayed in the table (for example, mh01WL-011), it represents the homozygous type (that is, the two chromosomes of a pair of homologous chromosomes are the same sequencing results), and the alleles carried by the 4 SNPs (rs16846350 / rs74139746 / rs12093903 / rs3820638) in the locus on the two chromosomes are TGAC in sequence.

[0152] Table 4 Typing results of 196 microhaplotype loci in 2800M positive control

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] Example 4: Consistency of primer combination and CE platform test results

[0162] Test samples: 2800M or 9948. 2800M is the positive control DNA (Single-source male human genomic DNA) for Human STR Analysis, Promega, product catalog number DD7101. 9948 (Control DNA 9948) is the male positive control DNA, Qiagen, product catalog number 386041. Take the test sample and dilute it with nuclease-free water to make the DNA concentration 1 ng / μL, which is the template solution.

[0163] Take the template solution and perform detection according to the method of Example 3.

[0164] Take the template solution and use DNATyper TM 25 test kit (Ministry of Public Security Identification Center), DNATyper TM X19 test kit (Ministry of Public Security Identification Center) and Microreader TM 40Y Direct ID Kit (product of Yuewei Gene Co., Ltd.) was used and capillary electrophoresis was performed according to the instructions to obtain the typing of each locus in the kit.

[0165] The results of the autosomal STR loci are shown in Table 5. In Table 5, NGS represents the results of the test according to the method of Example 3, and CE represents the results of the test using DNATyper TM The results showed that the DNATyper TM For the 15 autosomal loci that overlapped among the 25 kits, the results of NGS and CE were consistent.

[0166] The results of the X chromosome STR locus are shown in Table 6. In Table 6, NGS represents the results of the test according to the method of Example 3, and CE represents the results of the test using DNATyper TM The results of the X19 kit test showed that TMOf the 16 X-STR loci overlapped by the X19 kit, the NGS and CE results for 15 loci were consistent, and only the NGS and CE results for the DXS6804 locus were inconsistent. The NGS typing results for 2800M and 9948 were one repeat unit shorter than the CE typing results, which was probably caused by the inconsistent STR sequence naming rules between NGS and CE.

[0167] The results of the Y chromosome STR loci are shown in Table 7. In Table 7, NGS represents the results of the detection according to the method of Example 3, and CE represents the results of the detection using a Microreader. TM The results of the 40Y Direct ID kit test showed that the TM For the 37 Y-STR loci overlapped by the 40Y Direct ID kit, the results of NGS and CE were consistent.

[0168] Table 5. Verification of consistency between NGS and CE platforms for autosomal STR loci

[0169]

[0170] Table 6 X chromosome STR locus NGS and CE platform consistency verification

[0171]

[0172] Table 7 Verification of consistency between NGS and CE platforms for Y chromosome STR loci

[0173]

[0174]

[0175] Example 5: Consistency of primer combinations and NGS platform test results

[0176] Test sample: 2800M.

[0177] Take the test sample and dilute it with nuclease-free water to make the DNA concentration 1 ng / μL, which is the template solution.

[0178] Take the template solution and perform detection according to the method of Example 3.

[0179] The template solution was taken and the library preparation was completed using the STRSeqTyper122 second-generation sequencing STR typing reagent (Identification Center of the Ministry of Public Security) according to the instructions of the kit. Sequencing was completed on the MiSeq FGx platform, and data analysis was performed using the ForensicTyper forensic genetic marker second-generation sequencing data analysis software to obtain the length typing and sequence information of the 122 STR loci.

[0180] The results show that the detection results of the method in Example 3 are consistent with the results of the 106 loci overlapped by the STRSeqTyper122 second-generation sequencing STR typing reagent, and the results are shown in Table 3. The 106 loci are as follows: Amelogenin, CSF1PO, D10S1248, D10S1435, D10S2325, D11S4463, D12S391, D13S317, D13S325, D14S1434, D14S608, D15S659, D16S539, D17S974, D18S51, D18S535, D19S253, D19S433, D1GATA113, D1S1656, D20S1082, and D20S482. , D21S11, D22gata198B05, D22S1045, D2S1338, D2S1360, D2S1776, D2S441, D3S1358, D3S1744, D3S3045, D3S3053, D3S 4529, D4S2364, D4S2366, D5S818, D6S1043, D6S474, D6S477, D7S1517, D7S3048, D7S820, D8S1132, D8S1179, D9S1122, DXS10103, DXS6789, DXS6800, DXS6804, DXS6809, DXS7132, DXS7423, DXS7424, DXS9895, DXS9902, DYF399S1a / b / c, DY F404S1a / b, DYS19, DYS385a / b, DYS388, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS 439, DYS448, DYS456, DYS458, DYS460, DYS481, DYS504, DYS508, DYS520, DYS531, DYS533, DYS549, DYS552, DYS557, DY S570, DYS576, DYS593, DYS596, DYS622, DYS635, DYS641, DYS643, DYS645, FGA, GATA165B12, GATA31E08, HPRTB, Penta D, Penta E, TH01, TPOX, vWA, Y-GATA-H4.

[0181] Example 6: Repeatability of primer combination detection results

[0182] Test sample (set up three parallel samples): 2800M.

[0183] Three parallel samples were taken respectively and tested according to the method of Example 3.

[0184] The results are consistent and the same as those in Table 3.

[0185] Example 7: Sensitivity of primer combination detection

[0186] Test sample: 2800M.

[0187] Take the test sample and dilute it with nuclease-free water to make the DNA concentration 1 ng / μL, 0.5 ng / μL, 0.25 ng / μL, 0.125 ng / μL, 0.0625 ng / μL or 0.03125 ng / μL, which are the respective template solutions.

[0188] Take each template solution and perform detection according to the method of Example 3.

[0189] Correct results were obtained for each template solution (ie, the same results as those in Tables 3 and 4).

[0190] The results showed that the sensitivity of the primer combination detection was at least 0.03125 ng / μL.

[0191] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A primer combination, consisting of 738 primers; the primers are single-stranded DNA molecules; the nucleotide sequences of the 738 primers are shown in SEQ ID NO: 1 to SEQ ID NO:

738.

2. Use of the primer combination according to claim 1 in preparing a kit; The functions of the kit are as follows (a) or (b) or (c) or (d) or (e) or (f): (a) Gene locus analysis; (b) Forensic DNA testing and identification; (c) individual identification; (d) paternity testing; (e) Male pedigree identification; (f) Identification of kinship.

3. Use of the primer combination described in claim 1 in forensic DNA testing and identification.

4. Use of the primer combination according to claim 1 in paternity testing, kinship testing, individual identification or male pedigree testing.

5. Use of the primer combination according to claim 1 in gene locus analysis.

6. A kit comprising the primer combination according to claim 1.

7. The method for preparing the kit according to claim 6, comprising the step of mixing and packaging all the primers in the primer combination.

8. Use of the kit according to claim 6 in forensic DNA testing and identification.

9. Use of the kit according to claim 6 in paternity testing, kinship testing, individual identification, or male pedigree testing.

10. Use of the kit according to claim 6 in gene locus analysis.

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