CN118910286A - Kit for simultaneously detecting STR gene locus and SNP locus and use method thereof - Google Patents

Abstract

The present invention discloses a kit for simultaneously detecting STR loci and SNP sites and a method for using the kit. The kit includes amplification primers for amplifying the following 132 STR loci and 306 SNP sites, and probes for identifying the following 132 STR loci and 306 SNP sites. The kit of the present invention can simultaneously detect 438 genetic markers (132 STR loci and 306 SNP sites), greatly improving the ability to identify individuals and complex kinship. The method of the present invention based on probe capture detection of STR and SNP typing is not only highly efficient, but also more cost-effective, and the results are reliable, and can be widely used in DNA identification in forensic science.

Description

A kit for simultaneously detecting STR loci and SNP sites and a method for using the kit

Technical Field

The present invention relates to the technical field of gene detection, and in particular to a kit for simultaneously detecting STR loci and SNP sites and a method for using the kit.

Background Art

In the field of forensic evidence, typing tests for sequence polymorphism and length polymorphism of genetic material DNA play an important role in individual identification, paternity testing and even the construction of forensic DNA databases. Short tandem repeats (STRs), also known as microsatellites, refer to continuous repeating DNA units with a length of 2-6 base pairs (bp), which account for at least 3% of the human genome. STRs are highly polymorphic mainly due to the different number of core sequence repetitions. Since they originate from slippage-mismatches between DNA chains that are instantaneously separated during DNA replication, most of them are not affected by selection pressure. At present, STRs have been widely used in the field of forensic medicine and have become the "gold standard" for individual identification and paternity testing.

STR typing test technology, which uses real-time amplification of fluorescently labeled polymerase chain reaction (PCR) primers and capillary electrophoresis (CE) separation based on amplicon size, has become the mainstream method for forensic STR typing due to its high degree of automation, sensitivity, accuracy, and good repeatability. However, the traditional PCR-CE method can only provide variation information based on length, and cannot resolve site mutation information in repetitive sequences and flanking regions. Massively parallel sequencing (MPS), also known as second-generation sequencing, can simultaneously perform genotyping on multiple forensic molecular markers and multiple samples at single base resolution in a single run with high coverage. Therefore, MPS can, in principle, provide more STR polymorphism information, thereby significantly improving the identification efficiency. Another significant advantage of MPS-STR is that MPS can overcome the limitation of primer fragment size, which means that all STRs can be constructed with the smallest feasible primer fragment, thereby improving the amplification efficiency of samples in difficult and complex cases.

Based on the high resolution and compatibility of MPS, other types of molecular markers have gradually been incorporated into the field of forensic genetics, such as single nucleotide polymorphisms (SNPs). Compared with STR markers, SNPs have the advantages of abundant quantity, easy automation, low mutation rate and small PCR amplification fragments. SNPs can also provide individual information about ancestry, phenotype, etc., and play an important role in the new classification and identification projects of forensic evidence practice. It is worth noting that SNP markers are particularly helpful in typing degraded DNA samples and provide investigative clues for certain cases where suspects cannot be identified. With the promotion of the application of MPS in forensic genetics, the composite MPS system integrating STR and SNP genetic markers has been used in forensic practices such as complex kinship identification. However, the number of STR and SNP loci currently available is obviously insufficient to fully characterize the genetic characteristics of the population.

In recent years, the demand for forensic kinship identification has expanded from simple parent-child identification to complex kinship identification, such as the identification of unknown remains in large-scale disasters, the search for missing persons, DNA testing in criminal investigations, and the determination of sibling relationships in inheritance disputes. Although breakthroughs have been made in this field, there is still a gap between existing methods and actual needs due to the reduction in the number of DNA fragments shared by distant relatives, so more genetic markers are needed to improve the ability to identify complex kinship.

Summary of the invention

The purpose of the present invention is to provide a kit for simultaneously detecting STR loci and SNP sites and a method for using the kit in view of the deficiencies in the prior art.

To achieve the above object, the technical solution adopted by the present invention is:

The first aspect is to provide a kit for simultaneously detecting STR loci and SNP sites, the kit comprising amplification primers for amplifying the following 132 STR loci and 306 SNP sites, and probes for identifying the following 132 STR loci and 306 SNP sites;

Among them, the 132 STR loci are: D1S1171, D1S1679, D1S1677, D1S1627, D1S1656, D10S2325, D10S1237, D10S1248, D10S1435, D11S4463, D11S1304, D11S2368, TH01, CD4, D12AT A63, D12S391, VWA, D13S325, D13S317, D13S1492, D14S1434, D14S608, D14S1426 ,PENTAE,D15S659,D15S822,D16S3253,D16S539,D16S753,D17S1290,D17S975, D17S976, D17S1294, D17S974, D18S1364, D18S1270, D18S51, D18S535, D18S853, D19S253, D19S433, D2S1360, D2S1338, D2S441, D2S1772, D2S1776, D2S427, D2S1 242. TPOX, D20S161, D20S470, D20S1082, D20S482, D21S1432, D21S11, D21S1270 ,PENTAD,D21S1437,D21S2055,D22S689,D22S534,D22GATA198B05,D22S1045,D 3S1358, D3S1744, D3S4554, D3S3051, D3S4529, D3S4545, D3S1766, D3S2452, D3S 2387, D3S2388, D3S2406, D3S2402, D4S2366, D4S2404, D4S2408, GABRB15, FGA, D 5S818, CSF1PO, D5S2500, D5S2503, D5S1457, D6S474, D6S477, D6S1017, D6S1027 , D7S820, D7S3048, D7S2201, D7S1517, D8S1115, D8S1132, D8S1179, D8S639, LPL , D9S324, D9S925, D9S1118, D9S304, D9S2157, D9S1122, DXS10163, DXS6789, DXS 10161, DXS981, DXS9902, DXS10103, DXS10147, GATA172D05, GATA31E08, HPRTB, DXS6795, DXS6797, DXS6807, DXS6810, DXS7129, DXS7130, DXS9907, DYS533, DYS570, DYS19, DYS390, DYS391, DYS392, DYS425, DYS436, DYS456, DYS458, DYS460;

The 306 SNPs were: rs730123, rs6681639, rs6022, rs7531163, rs859400, rs647347, rs10921915, rs1049607, rs10779650, rs35396665, rs12042900, rs10925256, rs1413212, rs7517833, rs595961, rs12718433, rs75930208, rs7373, rs 1779866, rs2986742, rs7354930, rs681968, rs1281611, rs2991717, rs6671669, rs2799067, rs11191355, rs8946, rs10828545, rs3817419, rs7921673, rs10900297, rs7903315, rs2680327, rs4414169, rs3758626, rs10824095, rs 1059046, rs10887346, rs10881855, rs1977640, rs2566913, rs10502151, rs693147, rs2027767, rs2711823, rs794 5310, rs10501269, rs3829940, rs4939141, rs7932775, rs4944946, rs11604536, rs2196168, rs794533, rs3751000 , rs7929050, rs2764, rs883079, rs1007469, rs478347, rs7962675, rs1909149, rs11171675, rs10747783, rs74861 00, rs11178999, rs10506847, rs10459171, rs2107612, rs959897, rs1058083, rs1865350, rs9515124, rs3751385, r s9319336, rs689338, rs2677595, rs9598152, rs7321823, rs495593, rs12856670, rs319522, rs8003942, rs107937 5. rs5807790, rs7159343, rs2229869, rs946065, rs33943216, rs3759753, rs165932, rs1004667, rs229829, rs879 803, rs4530104, rs2898985, rs700518, rs3848117, rs11558748, rs12442886, rs698500, rs7169902, rs550397, rs 4781878, rs567617218, rs17822931, rs4315319, rs1126933, rs8056865, rs7205345, rs1382387, rs8074840, rs104 6321, rs17855579, rs189899, rs1055086, rs236514, rs4789193, rs8078417, rs2510344, rs7232841, rs8089593, r s1058396, rs3819122, rs4801083, rs17176581, rs6565924, rs9951171, rs1108414, rs2285513, rs7367, rs201457 6. rs2547319, rs7259841, rs2071347, rs1519654, rs12622186, rs9005, rs11888583, rs2716639, rs2920059, rs75 97826, rs164605, rs1540482, rs10497191, rs12613075, rs12464448, rs850889, rs13019672, rs13113, rs2242074, rs4074882, rs2068834, rs2372009, rs10203786, rs13419896, rs12617928, rs2561892, rs6742876, rs4777, rs134 00937, rs1078004, rs2113418, rs2567608, rs734111, rs3215567, rs2868362, rs1000551, rs994487, rs1014803, r s2073383, rs2040411, rs813449, rs2399332, rs1135750, rs876755, rs6439466, rs1586861, rs1131925, rs150205 , rs10470510, rs2270968, rs1017953, rs3747669, rs11128935, rs4493387, rs6802739, rs7374458, rs3774729, rs6 123, rs2765, rs1405154, rs10019345, rs11722834, rs167503, rs1396009, rs218804, rs6857303, rs10024103, rs1 0517844, rs1050145, rs36029731, rs55905558, rs10034991, rs4832887, rs225165, rs270238, rs12640116, rs286 75771, rs1043218, rs344140, rs385194, rs10005781, rs412683, rs1898625, rs7703985, rs2162739, rs10079352, rs39897, rs13182883, rs6191, rs1820795, rs11576001, rs332811, rs295999, rs251934, rs446003, rs641437, rs37 369, rs2672757, rs10072283, rs2406113, rs2928231, rs2371382, rs277984, rs460267, rs4518370, rs3763070, rs 4946582, rs1884255, rs13218440, rs2503107, rs9493627, rs1360947, rs9373491, rs9383976, rs9356399, rs1094 9483, rs1206523, rs1051952, rs719856, rs11242909, rs6933870, rs6934646, rs2305837, rs818269, rs7767965, r s1030428, rs2747737, rs7779997, rs2285914, rs10487320, rs41736, rs2305324, rs321198, rs2272251, rs740136, rs1233555, rs991471, rs917115, rs36170987, rs1050721, rs4723858, rs2053315, rs6973042, rs1860469, rs1061 4. rs12386695, rs1403180, rs1515685, rs298612, rs1383474, rs763869, rs10092983, rs2292975, rs10100663, rs 3735973, rs4237030, rs2912016, rs7814747, rs28435921, rs7831841, rs2308962, rs13279230, rs1063110, rs227 4159, rs7049164, rs2781116, rs1360288, rs2236547, rs7871735, rs10973637, rs4322086, rs6559311, rs8192689.

The second aspect is to provide a method for detecting STR and SNP typing based on probe capture using the above-mentioned kit, comprising the following steps:

S1, extract sample genomic DNA to obtain sample DNA;

S2, screening of STR and SNP genetic markers;

S3, design probes for the target loci screened in S2;

S4, preparing a small fragment library A using the sample DNA, and detecting the library concentration and fragment size of the small fragment library A;

S5, hybridize the biotin-labeled probe designed by S3 with the small fragment library A obtained by S4 to capture the target DNA fragment;

S6, the hybridization reaction products are enriched by streptavidin-coated magnetic beads and eluted to obtain purified target DNA products;

S7, PCR amplification of the purified target DNA product to construct sequencing library B;

S8, high-throughput sequencing to obtain sequence information;

S9, sequencing data analysis, obtaining the genotypes of target STR and SNP genetic markers;

S10, accuracy assessment of STR typing by next-generation sequencing;

S11, application of complex kinship identification.

Furthermore, S4 specifically includes the following steps:

S41, firstly fragment the sample DNA, perform end repair and A-tailing, and then screen with magnetic beads to obtain blunt-end DNA fragments;

S42, connecting the oligonucleotide adapter to the end of the fragment to obtain a DNA fragment with an adapter;

S43, PCR amplification and purification to obtain the small fragment library A, and detection of the library concentration and fragment size of the small fragment library A.

Furthermore, S8 specifically includes the following steps:

S81, quality filtering of raw sequencing data to remove low-quality short reads;

S82, align high-quality reads to the human genome sequence, sort the genome coordinates, and mark repetitive sequences;

S83, perform quality control on the BAM file obtained in S82;

S84, genotyping of all target STR loci and SNP sites.

Furthermore, S10 specifically includes the following steps:

S101, using CE to detect at least one STR locus to obtain its typing information;

S102, compare the consistency of next-generation sequencing and CE results.

The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

The kit of the present invention can simultaneously detect 438 genetic markers (132 STR loci and 306 SNP sites), greatly improving the ability to identify individuals and complex kinship relationships.

The method of the present invention based on probe capture detection of STR and SNP typing is not only highly efficient but also more cost-effective and has reliable results, and can be widely used in DNA identification in forensic science.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

The DNA in the peripheral blood of 50 Han people in Liyang was used as samples to construct the STR and SNP typing system based on probe capture detection and test its identification ability. The specific process is as follows:

S1: DNA was extracted from the peripheral blood of 50 Liyang Han people to obtain sample DNA

The specific operation was as follows: peripheral blood was collected and stored in EDTA anticoagulant tubes, sample DNA was extracted from the blood samples using the commercially available QIAamp DNA Bloodmini Kit (Qiagen, Hilden, Germany), and the DNA concentration was determined using Qubit ^™ dsDNA HS Assay Kit (ThermoFisher Scientific, Carlsbad, USA).

S2: Screening STR loci

The specific operation is as follows: First, 51 core STR loci were selected from three commercially available STR kits, namely SiFaSTR ^TM 23-plex (Beijing ReadGene Technology Co., Ltd.), AGCU NC 21+1 (Wuxi Sino-German Meilian Biotechnology Co., Ltd.) and Goldeneye ^TM DNAID 22NC (Beijing Basepoint Cognitive Technology Co., Ltd.). Other STR loci of forensic interest were retrieved from the public STR databases STRBase (https://strbase-archive.nist.gov) and WebSTR (https://webstr.ucsd.edu) and published literature on STR analysis of the Han population;

In summary, a total of 132 STR loci were screened: D1S1171, D1S1679, D1S1677, D1S1627, D1S1656, D10S2325, D10S1237, D10S1248, D10S1435, D11S4463, D11S1304, D11S2368, TH01, CD4, D 12ATA63, D12S391, VWA, D13S325, D13S317, D13S1492, D14S1434, D14S608, D14S 1426, PENTAE, D15S659, D15S822, D16S3253, D16S539, D16S753, D17S1290, D17S9 75. D17S976, D17S1294, D17S974, D18S1364, D18S1270, D18S51, D18S535, D18S8 53. D19S253, D19S433, D2S1360, D2S1338, D2S441, D2S1772, D2S1776, D2S427, D 2S1242, TPOX, D20S161, D20S470, D20S1082, D20S482, D21S1432, D21S11, D21S1 270. PENTAD, D21S1437, D21S2055, D22S689, D22S534, D22GATA198B05, D22S1045 , D3S1358, D3S1744, D3S4554, D3S3051, D3S4529, D3S4545, D3S1766, D3S2452, D 3S2387, D3S2388, D3S2406, D3S2402, D4S2366, D4S2404, D4S2408, GABRB15, FGA , D5S818, CSF1PO, D5S2500, D5S2503, D5S1457, D6S474, D6S477, D6S1017, D6S10 27. D7S820, D7S3048, D7S2201, D7S1517, D8S1115, D8S1132, D8S1179, D8S639, LP L, D9S324, D9S925, D9S1118, D9S304, D9S2157, D9S1122, DXS10163, DXS6789, DX S10161, DXS981, DXS9902, DXS10103, DXS10147, GATA172D05, GATA31E08, HPRTB , DXS6795, DXS6797, DXS6807, DXS6810, DXS7129, DXS7130, DXS9907, DYS533, DYS570, DYS19, DYS390, DYS391, DYS392, DYS425, DYS436, DYS456, DYS458, DYS460.

S3: Screening SNP sites

The specific operations were as follows: sample data of the Chinese Han population were downloaded and screened from the 1000 Genomes Project (https://www.internationalgenome.org), and related individuals (all second- and third-degree relatives and children of first-degree relatives) were removed;

The Hardy-Weinberg equilibrium (HWE) was calculated using vcftools--hardy, with the overall heterozygosity of all samples as the background. The loci with P>0.05 in the Han population were retained, including SNPs, insertions and deletions (INDELs), and structural variations (SVs);

Perform genome-wide screening. Exclude sites located within 100 kb upstream or downstream of coding/non-coding genes and sites located in the REPEATS region; the heterozygosity of candidate sites in the Han population is required to be greater than 0.3; use "vcftools--weir-fst-pop" to calculate the population differentiation index (F _ST ) of each site, and retain sites with F _ST less than 0.01;

Only SNP sites were retained for the above screened sites, and the distance between adjacent SNP sites was no less than 10 kb;

The linkage disequilibrium (LD) calculations of the above loci were performed using the “LD” function in the R package “genetics” for final screening;

In summary, a total of 306 SNP loci were screened: rs730123, rs6681639, rs6022, rs7531163, rs859400, rs647347, rs10921915, rs1049607, rs10779650, rs35396665, rs12042900, rs10925256, rs1413212, rs7517833, rs595961, rs12718433, rs75930208, rs 7373, rs1779866, rs2986742, rs7354930, rs681968, rs1281611, rs2991717, rs6671669, rs2799067, rs11191355, rs8946, rs10828545, rs3817419, rs7921673, rs10900297, rs7903315, rs2680327, rs4414169, rs3758626, rs10824 095, rs1059046, rs10887346, rs10881855, rs1977640, rs2566913, rs10502151, rs693147, rs2027767, rs2711823 , rs7945310, rs10501269, rs3829940, rs4939141, rs7932775, rs4944946, rs11604536, rs2196168, rs794533, rs37 51000, rs7929050, rs2764, rs883079, rs1007469, rs478347, rs7962675, rs1909149, rs11171675, rs10747783, rs 7486100, rs11178999, rs10506847, rs10459171, rs2107612, rs959897, rs1058083, rs1865350, rs9515124, rs3751 385, rs9319336, rs689338, rs2677595, rs9598152, rs7321823, rs495593, rs12856670, rs319522, rs8003942, rs1 079375, rs5807790, rs7159343, rs2229869, rs946065, rs33943216, rs3759753, rs165932, rs1004667, rs229829, r s879803, rs4530104, rs2898985, rs700518, rs3848117, rs11558748, rs12442886, rs698500, rs7169902, rs55039 7. rs4781878, rs567617218, rs17822931, rs4315319, rs1126933, rs8056865, rs7205345, rs1382387, rs8074840, r s1046321, rs17855579, rs189899, rs1055086, rs236514, rs4789193, rs8078417, rs2510344, rs7232841, rs80895 93. rs1058396, rs3819122, rs4801083, rs17176581, rs6565924, rs9951171, rs1108414, rs2285513, rs7367, rs201 4576, rs2547319, rs7259841, rs2071347, rs1519654, rs12622186, rs9005, rs11888583, rs2716639, rs2920059, r s7597826, rs164605, rs1540482, rs10497191, rs12613075, rs12464448, rs850889, rs13019672, rs13113, rs22420 74. rs4074882, rs2068834, rs2372009, rs10203786, rs13419896, rs12617928, rs2561892, rs6742876, rs4777, rs 13400937, rs1078004, rs2113418, rs2567608, rs734111, rs3215567, rs2868362, rs1000551, rs994487, rs101480 3. rs2073383, rs2040411, rs813449, rs2399332, rs1135750, rs876755, rs6439466, rs1586861, rs1131925, rs150 205. rs10470510, rs2270968, rs1017953, rs3747669, rs11128935, rs4493387, rs6802739, rs7374458, rs3774729, rs6123, rs2765, rs1405154, rs10019345, rs11722834, rs167503, rs1396009, rs218804, rs6857303, rs10024103, rs10517844, rs1050145, rs36029731, rs55905558, rs10034991, rs4832887, rs225165, rs270238, rs12640116, rs2 8675771, rs1043218, rs344140, rs385194, rs10005781, rs412683, rs1898625, rs7703985, rs2162739, rs1007935 2. rs39897, rs13182883, rs6191, rs1820795, rs11576001, rs332811, rs295999, rs251934, rs446003, rs641437, rs 37369, rs2672757, rs10072283, rs2406113, rs2928231, rs2371382, rs277984, rs460267, rs4518370, rs3763070, rs4946582, rs1884255, rs13218440, rs2503107, rs9493627, rs1360947, rs9373491, rs9383976, rs9356399, rs109 49483, rs1206523, rs1051952, rs719856, rs11242909, rs6933870, rs6934646, rs2305837, rs818269, rs7767965, rs1030428, rs2747737, rs7779997, rs2285914, rs10487320, rs41736, rs2305324, rs321198, rs2272251, rs740136 , rs1233555, rs991471, rs917115, rs36170987, rs1050721, rs4723858, rs2053315, rs6973042, rs1860469, rs106 14. rs12386695, rs1403180, rs1515685, rs298612, rs1383474, rs763869, rs10092983, rs2292975, rs10100663, rs 3735973, rs4237030, rs2912016, rs7814747, rs28435921, rs7831841, rs2308962, rs13279230, rs1063110, rs227 4159, rs7049164, rs2781116, rs1360288, rs2236547, rs7871735, rs10973637, rs4322086, rs6559311, rs8192689.

S4: Design probes for target loci

The start/stop position of the probe was adjusted to control the melting temperature, thereby optimizing the probe length. Finally, 310 STR probes and 306 SNP probes were designed, with an average length of 120 bp. The above probes can be obtained by existing design software, which is common knowledge in the art and will not be described in detail in this application.

S5: The sample DNA was fragmented into fragments with an average size of 350 bp using a Covaris ultrasonic device (Covaris, Woburn, MA, USA); the small fragment library A was prepared using the KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA, USA) according to the manufacturer's instructions, with an initial DNA amount of 500 ng;

Specifically, the DNA fragments after ultrasonic disruption are purified by magnetic beads, and then the ends are repaired and A-tailed to obtain blunt-end DNA fragments; oligonucleotide adapters are connected, and PCR amplification (the amplification primers used can be obtained from existing design software, which is common knowledge in the field and will not be described in detail in this application) and purification are performed to obtain a small fragment library A; qPCR quantification is performed using the Universal KAPA library quantification kit;

S6: Mix the biotin-labeled probe with the small fragment library A and perform hybridization capture according to the Twist Fast Hybridization Target Enrichment Protocol;

The specific operations are:

Mix the small fragment library A in equal amounts with 500 ng of each library, and prepare the reaction system according to Table 1:

Table 1

Reaction system Dosage (μL) Mixed DNA (1.5 μg) 1 TwistProbePanel 4 UniversalBlockers 8 BlockerSolution 5

The reaction system was mixed thoroughly and dried at 60° C. using a SpeedVac (Genevac, UK) system.

Add 20 μL Fast Hybridization Mix and 30 μL Hybridization Enhancer to the dried hybridization reaction library and set up the PCR reaction program according to Table 2:

Table 2

temperature time 95℃ 5min 60℃ 4h

After the incubation, the hybridization reaction solution was added to 200 μL of purified streptavidin magnetic bead suspension and shaken at room temperature for 30 min;

Take out the PCR tube, remove the supernatant, add 200 μL of Fast Wash Buffer 1 preheated to 70°C, place in a 70°C constant temperature bath and incubate for 5 min for rinsing, repeat twice;

Take out the PCR tube, remove the supernatant, add 200 μL of Fast Wash Buffer 2 preheated to 48°C, place in a 48°C constant temperature bath and incubate for 5 min for rinsing. After rinsing, add 22.5 μL of ddH ₂ O for elution;

Add 2.5 μL Amplification Primers and 25 μL EquinoxLibrary Amp Mix to the above magnetic bead suspension and set up the PCR reaction program according to Table 3:

Table 3

After the PCR reaction was completed, 90 μL of DNA Purification Beads was taken to purify the product, and then 30 μL of ddH ₂ O was added for elution to construct sequencing library B.

Quantification was performed using qPCR and quality control was performed using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA).

S7: High-throughput sequencing

The sequencing library B in S6 was sequenced using Illumina NovaSeq 6000 to obtain sequencing data in FASTQ format with a sequence length of 150 bp;

S8: Sequencing data analysis to obtain genotypes of target STR loci and SNP sites

The specific operation is as follows: fastp (0.23.4) was used to perform quality control on the sequencing data obtained by S7, and bases with quality lower than Q20 and reads with length less than 50 bp were removed.

Speedseq align (0.1.2) was used to align the filtered sequences to the human reference genome (human_g1k_v37_decoy) to generate sorted BAM files with repeat sequence markers. Qualimap bamqc (2.3) was used to perform quality control on each BAM file. Bedtools (2.28.0) was used to calculate the sequencing depth and coverage of each target locus.

SNP detection was performed according to the GATK standard process; specifically, first, GATK HaplotypeCaller was run in GENOTYPE_GIVEN_ALLELES mode, the target SNP site was a known allele, the target genomic region including the 40 bp flanking regions on both sides was the target region, all target SNPs were genotyped, and the genotype, sequencing depth, mutation frequency and other information of each locus were obtained;

STR detection; specifically, first remove duplicate reads generated by PCR amplification or sequencing errors; use samtools and seqtk (https://github.com/lh3/seqtk) to extract reads overlapping the target genomic region from the original FASTQ file; use STRait Razor v2.5 to call default parameters to analyze the above read sequences to obtain a length-based STR profile; if the heterozygous balance ratio (Hb) of autosomal STR (A-STR) and X chromosome STR (X-STR) is > 0.25, the STR is considered to be heterozygous; otherwise, the STR is considered to be homozygous. The Y chromosome STR (Y-STR) genotype follows the minor allele frequency/major allele frequency ≥ 0.35 to ensure its paternal inheritance homozygosity. STR loci with a sequencing depth lower than 30× were excluded from subsequent analysis;

In summary, after quality filtering, a total of 80 A-STRs, 17 X-STRs, 10 Y-STRs, and 292 A-SNPs were used for the construction of the subsequent composite system.

S9: Accuracy assessment of STR typing by next-generation sequencing

The specific operation was as follows: 50 DNA samples were subjected to conventional STR typing analysis according to the instructions using the commercially available SiFaSTR ^™ 23-plex kit, AGCUNC 21+1 kit, and Goldeneye ^™ DNA ID 22NC kit, with 1 ng of DNA template added to each reaction; PCR products were amplified in a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific, Carlsbad, USA); capillary electrophoresis was performed on an ABI 3500XL capillary sequencer (Thermo Fisher Scientific); and the target STR loci were analyzed using GeneMapper ID-X1.5 (Thermo Fisher Scientific), with a threshold of 50 RFU.

The consistency of STR genotypes generated by MPS and traditional PCR-CE methods was evaluated. Among them, the typing results of 35 out of 41 STR loci were completely consistent, and only 6 STRs (D6S477, D6S474, D3S1744, D18S853, D15S659, and D14S608) had locus loss or allele loss in at least one sample due to insufficient sequencing depth or too high Hb threshold.

In summary, the high-throughput sequencing method based on probe capture not only has high throughput but also high typing accuracy.

S10: DNA sensitivity analysis

Specifically, DNA from a male was extracted and diluted to generate six gradients of 200 ng, 100 ng, 50 ng, 10 ng, 5 ng and 1 ng for library construction and high-throughput sequencing. Each DNA sample was sequenced five times on the same instrument.

When the DNA content was as low as 10 ng, 85.2% of the STR loci could be accurately detected; when the DNA content was as low as 5 ng, 95.61% of the SNP sites could be accurately detected.

S11: Hybrid DNA Analysis

Specifically, five different ratios of DNA mixtures were prepared from genomic DNA from two male individuals at a ratio of 1:1, 2:1, 4:1, 9:1, and 19:1, and two single-source DNA samples for control were used for library preparation and high-throughput sequencing. The DNA input amount was 500 ng.

Compared with single-source samples, non-overlapping A-STR and A-SNP alleles from minor contributors were detected at greater than 87% and 82% in 1:1 and 4:1 mixtures, respectively.

S12: Species-specific analysis

Specifically, non-human DNA was extracted from the peripheral blood of two rabbits, two chickens, two ducks, two pigs, one mouse and one cow for PCR amplification and high-throughput sequencing. The DNA input for each reaction was 500 ng.

The genotyping results showed that neither STR loci nor SNP sites were detected in non-human DNA samples.

S13: Identification of complex kinship relationships

Specifically, first, a machine simulation is performed based on the allele frequency of each locus to generate 10,000 unrelated individuals. Then, two individuals are randomly selected from the 10,000 simulated individuals as the father and mother, and their two children are generated according to the genotypes of the parents to form a pair of full siblings. Similarly, a pair of half-siblings is generated by two randomly selected fathers and one mother. In order to simulate a pair of cousins, a pair of grandparents is randomly selected from 10,000 simulated individuals, and their two children are paired with two randomly selected unrelated individuals, and the two children generated are regarded as cousins. The above steps are repeated 10,000 times to generate 10,000 pairs of first-degree relatives (represented by full siblings), 10,000 pairs of second-degree relatives (represented by half siblings), 10,000 pairs of third-degree relatives (represented by cousins) and 10,000 pairs of unrelated individuals. The system's identification efficiency for complex kinship relationships is evaluated by calculating the likelihood ratio (LR). The results are shown in Table 4:

Table 4

The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. A kit for simultaneously detecting STR loci and SNP sites, characterized in that the kit comprises amplification primers for amplifying the following 132 STR loci and 306 SNP sites, and probes for identifying the following 132 STR loci and 306 SNP sites; Among them, the 132 STR loci are: D1S1171, D1S1679, D1S1677, D1S1627, D1S1656, D10S2325, D10S1237, D10S1248, D10S1435, D11S4463, D11S1304, D11S2368, TH01, CD4, D12AT A63, D12S391, VWA, D13S325, D13S317, D13S1492, D14S1434, D14S608, D14S1426 ,PENTAE,D15S659,D15S822,D16S3253,D16S539,D16S753,D17S1290,D17S975, D17S976, D17S1294, D17S974, D18S1364, D18S1270, D18S51, D18S535, D18S853, D19S253, D19S433, D2S1360, D2S1338, D2S441, D2S1772, D2S1776, D2S427, D2S1 242. TPOX, D20S161, D20S470, D20S1082, D20S482, D21S1432, D21S11, D21S1270 ,PENTAD,D21S1437,D21S2055,D22S689,D22S534,D22GATA198B05,D22S1045,D 3S1358, D3S1744, D3S4554, D3S3051, D3S4529, D3S4545, D3S1766, D3S2452, D3S 2387, D3S2388, D3S2406, D3S2402, D4S2366, D4S2404, D4S2408, GABRB15, FGA, D 5S818, CSF1PO, D5S2500, D5S2503, D5S1457, D6S474, D6S477, D6S1017, D6S1027 , D7S820, D7S3048, D7S2201, D7S1517, D8S1115, D8S1132, D8S1179, D8S639, LPL , D9S324, D9S925, D9S1118, D9S304, D9S2157, D9S1122, DXS10163, DXS6789, DXS 10161, DXS981, DXS9902, DXS10103, DXS10147, GATA172D05, GATA31E08, HPRTB, DXS6795, DXS6797, DXS6807, DXS6810, DXS7129, DXS7130, DXS9907, DYS533, DYS570, DYS19, DYS390, DYS391, DYS392, DYS425, DYS436, DYS456, DYS458, DYS460; The 306 SNPs were: rs730123, rs6681639, rs6022, rs7531163, rs859400, rs647347, rs10921915, rs1049607, rs10779650, rs35396665, rs12042900, rs10925256, rs1413212, rs7517833, rs595961, rs12718433, rs75930208, rs7373, rs 1779866, rs2986742, rs7354930, rs681968, rs1281611, rs2991717, rs6671669, rs2799067, rs11191355, rs8946, rs10828545, rs3817419, rs7921673, rs10900297, rs7903315, rs2680327, rs4414169, rs3758626, rs10824095, rs 1059046, rs10887346, rs10881855, rs1977640, rs2566913, rs10502151, rs693147, rs2027767, rs2711823, rs794 5310, rs10501269, rs3829940, rs4939141, rs7932775, rs4944946, rs11604536, rs2196168, rs794533, rs3751000 , rs7929050, rs2764, rs883079, rs1007469, rs478347, rs7962675, rs1909149, rs11171675, rs10747783, rs74861 00, rs11178999, rs10506847, rs10459171, rs2107612, rs959897, rs1058083, rs1865350, rs9515124, rs3751385, r s9319336, rs689338, rs2677595, rs9598152, rs7321823, rs495593, rs12856670, rs319522, rs8003942, rs107937 5. rs5807790, rs7159343, rs2229869, rs946065, rs33943216, rs3759753, rs165932, rs1004667, rs229829, rs879 803, rs4530104, rs2898985, rs700518, rs3848117, rs11558748, rs12442886, rs698500, rs7169902, rs550397, rs 4781878, rs567617218, rs17822931, rs4315319, rs1126933, rs8056865, rs7205345, rs1382387, rs8074840, rs104 6321, rs17855579, rs189899, rs1055086, rs236514, rs4789193, rs8078417, rs2510344, rs7232841, rs8089593, r s1058396, rs3819122, rs4801083, rs17176581, rs6565924, rs9951171, rs1108414, rs2285513, rs7367, rs201457 6. rs2547319, rs7259841, rs2071347, rs1519654, rs12622186, rs9005, rs11888583, rs2716639, rs2920059, rs75 97826, rs164605, rs1540482, rs10497191, rs12613075, rs12464448, rs850889, rs13019672, rs13113, rs2242074, rs4074882, rs2068834, rs2372009, rs10203786, rs13419896, rs12617928, rs2561892, rs6742876, rs4777, rs134 00937, rs1078004, rs2113418, rs2567608, rs734111, rs3215567, rs2868362, rs1000551, rs994487, rs1014803, r s2073383, rs2040411, rs813449, rs2399332, rs1135750, rs876755, rs6439466, rs1586861, rs1131925, rs150205 , rs10470510, rs2270968, rs1017953, rs3747669, rs11128935, rs4493387, rs6802739, rs7374458, rs3774729, rs6 123, rs2765, rs1405154, rs10019345, rs11722834, rs167503, rs1396009, rs218804, rs6857303, rs10024103, rs1 0517844, rs1050145, rs36029731, rs55905558, rs10034991, rs4832887, rs225165, rs270238, rs12640116, rs286 75771, rs1043218, rs344140, rs385194, rs10005781, rs412683, rs1898625, rs7703985, rs2162739, rs10079352, rs39897, rs13182883, rs6191, rs1820795, rs11576001, rs332811, rs295999, rs251934, rs446003, rs641437, rs37 369, rs2672757, rs10072283, rs2406113, rs2928231, rs2371382, rs277984, rs460267, rs4518370, rs3763070, rs 4946582, rs1884255, rs13218440, rs2503107, rs9493627, rs1360947, rs9373491, rs9383976, rs9356399, rs1094 9483, rs1206523, rs1051952, rs719856, rs11242909, rs6933870, rs6934646, rs2305837, rs818269, rs7767965, r s1030428, rs2747737, rs7779997, rs2285914, rs10487320, rs41736, rs2305324, rs321198, rs2272251, rs740136, rs1233555, rs991471, rs917115, rs36170987, rs1050721, rs4723858, rs2053315, rs6973042, rs1860469, rs1061 4. rs12386695, rs1403180, rs1515685, rs298612, rs1383474, rs763869, rs10092983, rs2292975, rs10100663, rs 3735973, rs4237030, rs2912016, rs7814747, rs28435921, rs7831841, rs2308962, rs13279230, rs1063110, rs227 4159, rs7049164, rs2781116, rs1360288, rs2236547, rs7871735, rs10973637, rs4322086, rs6559311, rs8192689. 2. A method for detecting STR and SNP typing based on probe capture using the kit according to claim 1, characterized in that it comprises the following steps: S1, extract sample genomic DNA to obtain sample DNA; S2, screening of STR and SNP genetic markers; S3, design probes for the target loci screened in S2; S4, preparing a small fragment library A using the sample DNA, and detecting the library concentration and fragment size of the small fragment library A; S5, hybridize the biotin-labeled probe designed by S3 with the small fragment library A obtained by S4 to capture the target DNA fragment; S6, the hybridization reaction products are enriched by streptavidin-coated magnetic beads and eluted to obtain purified target DNA products; S7, PCR amplification of the purified target DNA product to construct sequencing library B; S8, high-throughput sequencing to obtain sequence information; S9, sequencing data analysis, obtaining the genotypes of target STR and SNP genetic markers; S10, accuracy assessment of STR typing by next-generation sequencing; S11, application of complex kinship identification. 3. The method according to claim 2, characterized in that S4 specifically comprises the following steps: S41, firstly fragment the sample DNA, perform end repair and A-tailing, and then screen with magnetic beads to obtain blunt-end DNA fragments; S42, connecting the oligonucleotide adapter to the end of the fragment to obtain a DNA fragment with an adapter; S43, PCR amplification and purification to obtain the small fragment library A, and detection of the library concentration and fragment size of the small fragment library A. 4. The method according to claim 2, characterized in that S8 specifically comprises the following steps: S81, quality filtering of raw sequencing data to remove low-quality short reads; S82, align high-quality reads to the human genome sequence, sort the genome coordinates, and mark repetitive sequences; S83, perform quality control on the BAM file obtained in S82; S84, genotyping of all target STR loci and SNP sites. 5. The method according to claim 2, characterized in that S10 specifically comprises the following steps: S101, using CE to detect at least one STR locus to obtain its typing information; S102, compare the consistency of next-generation sequencing and CE results.