Kit for detecting 103 gene loci based on next-generation sequencing technology and special primer combination thereof

Abstract

The invention discloses a kit for detecting 103 gene loci based on a next-generation sequencing technology and a special primer combination thereof. The invention provides a primer combination which is composed of 188 primers. The primer is a single-stranded DNA molecule; the nucleotide sequences of the 188 primers are sequentially shown as a sequence 1 to a sequence 188 in a sequence table. The invention also provides application of the primer combination in preparation of a kit. The invention also provides a kit which comprises the primer combination. When the primer combination provided by the invention is used for detecting STR typing of 103 gene loci, the typing result is accurate. The method has great application and popularization values for forensic identification and genetic relationship identification.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic medicine and relates to a kit for detecting 103 gene loci based on the second generation sequencing technology and a special primer combination thereof, wherein the 103 gene loci consist of 102 Y-STR gene loci and one sex determination gene locus. Background Art

[0002] Short tandem repeat (STR) is a DNA fragment composed of 2-6 oligonucleotides as repeating units in the human genome. It has length polymorphism and is an ideal DNA marker for forensic individual identification and paternity testing. Human Y chromosome short tandem repeat (Y-chromosome short tandem repeat, Y-STR) has stable paternal inheritance characteristics, lacks recombination, and has high polymorphic information content. It has unique application value in the study of human evolutionary origins and population genetics. The study of Y chromosome STR polymorphism is an important means of forensic genomics individual identification and paternity identification, providing biological evidence in paternal tracing, detection of mixed spot male components, and analysis of multi-male individual mixtures.

[0003] At present, the mainstream STR typing technology is based on fluorescent labeled multiplex PCR-capillary electrophoresis (PCR-CE). Commercial Y-STR typing kits mainly include Y23 (Promega), Yfiler Plus (Thermo FisherScientific), Yfiler Platinum (Thermo Fisher Scientific), Microreader TM 40Y Direct ID System (Yuewei Gene), etc. However, most PCR-CE kits in the prior art are limited in the number of Y-STR loci integrated into the composite amplification system due to the limitations of fluorescent labeling and the size of the amplified product fragments, and do not support Y-STR sequence analysis, and cannot reveal the inconsistency of the sequence structure of the repeat region in alleles of the same fragment length.

[0004] Second generation sequencing (SGS), also known as next generation sequencing (NGS), has the advantages of high throughput, integration, and rapidity, and supports comprehensive analysis of STR length and sequence polymorphism. As NGS technology becomes increasingly mature, STR research based on NGS technology is very common in the field of forensic medicine. Compared with PCR-CE technology, NGS technology has the following advantages: ① Low sample input, making it possible to obtain the most genetic information from the least sample, which is conducive to the analysis of trace and degraded samples; ② High accuracy, which can achieve full coverage of the bases of the locus and produce high sequencing depth to ensure the rigor of high-probability and accurate judgment of each allele of the Y-STR locus; ③ Acquisition of nucleotide difference information between Y-STR alleles, differences in core repeat structures or variations in the amplified segments (base mutations or insertions and deletions in flanking sequences), resulting in alleles of equal sequence lengths may be completely different alleles with genetic stability. The sequence polymorphism of Y-STR is a valuable resource for individual identification analysis; ④ Low cost, by adding tags to each sample, multiple samples can be sequenced in parallel, which saves time and reduces sequencing costs; ⑤ More comprehensive data interpretation, the FASTQ file obtained by sequencing, in addition to the allele sequences of each Y-STR locus, also contains non-specific amplification sequences of different Y-STR primers, which can realize the accurate investigation and traceability of primer interference in the amplification system.

[0005] At present, the NGS platforms used in the field of forensic science are mainly Illumina, Thermo Fisher and BGI. The kits suitable for the above platforms mainly include Forenseq TMDNA Signature Prep Kit (Illumina, San Diego, CA, USA), Precison ID Global Filer NGS STR Kit (Thermo Fisher, Waltham, MA, USA), MGIEasy Signature Identification Library Prep Kit (BGI, Shenzhen, China) and Multiplecues Set B Kit (Shenzhen Xiao Gene, Shenzhen, China). The above kits contain 26 Y-STR loci, 2 Y-STR loci, 53 Y-STR loci and 85 Y-STR loci respectively, and the number of Y-STR loci that can be detected is relatively small. At the same time, the kits are expensive, and the supporting data analysis software is only for the kits developed by each company. The parameter settings are relatively fixed, and only the sequencing data of the inherent loci can be analyzed, which is not conducive to the actual application of forensic medicine. In addition, with the introduction of new domestic sequencing platforms, the scalability of second-generation sequencing technology in the field of forensic DNA testing has been continuously increased, and there is also an urgent need to improve the practicality of second-generation sequencing reagents on different sequencing platforms.

[0006] Therefore, it is of certain significance to construct a composite amplification system that is suitable for the current mainstream second-generation sequencing detection platform and open sequencing data analysis software (STRaitRazor, MyFLq, etc.) and can accommodate more Y-STR loci. Summary of the invention

[0007] The purpose of the present invention is to provide a kit for detecting 103 loci based on the second generation sequencing technology and a special primer combination thereof.

[0008] The present invention provides a primer combination, which consists of 188 primers; the primers are single-stranded DNA molecules; the nucleotide sequences of the 188 primers are shown in sequence 1 to sequence 188 in the sequence table.

[0009] The present invention also provides the use of the primer combination in preparing a kit;

[0010] The functions of the kit are as follows (a) or (b) or (c) or (d) or (e) or (f) or (g) or (h):

[0011] (a) Locus STR analysis;

[0012] (b) identification of paternal ancestry;

[0013] (c) individual identification;

[0014] (d) paternity testing;

[0015] (e) racial inference;

[0016] (f) Male pedigree identification;

[0017] (g) Forensic medical examination;

[0018] (h) Identification of kinship.

[0019] The present invention also provides the application of the primer combination in paternal ancestry identification, male family identification, kinship identification or parentage identification.

[0020] The present invention also provides application of the primer combination in forensic identification, individual recognition or race inference.

[0021] The present invention also provides application of the primer combination in gene locus STR analysis.

[0022] The present invention also provides a kit, comprising any one of the above primer combinations.

[0023] The kit may also include other PCR amplification reagents.

[0024] Exemplarily, the PCR amplification reagent may be a DNA polymerase.

[0025] Exemplarily, the PCR amplification reagent may be a premix of a PCR amplification system, such as 2×Master Mix.

[0026] 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.

[0027] The present invention also protects the use of the kit in paternal ancestry identification, male family identification, kinship identification or parentage identification.

[0028] The present invention also protects the use of the kit in forensic identification, individual recognition or ethnicity inference.

[0029] The present invention also protects the use of the kit in gene locus STR analysis.

[0030] The primer combination is used for locus STR analysis.

[0031] The kit is used for locus STR analysis.

[0032] The loci are sex determination loci and Y-STR loci.

[0033] The locus STR analysis refers to the identification of sex-determining loci and Y-STR loci.

[0034] The primer combinations are used to identify sex-determining loci and Y-STR loci.

[0035] The kit is used to identify sex-determining loci and Y-STR loci.

[0036] The sex determination locus is: Amelogenin locus.

[0037] The Y-STR loci are: DYS19 locus, DYS385a / b locus, DYF387S1a / b locus, DYS388 locus, DYS389-I locus, DYS389-II locus, DYS390 locus, DYS391 locus, DYS392 locus, DYS393 locus, DYF399S1a / b / c locus, DYF404S1a / b locus, DYS426 locus, DYS435 locus, DYS437 locus, DYS438 locus, DYS439 locus, DYS443 locus, DYS444 locus, DYS447 locus, DYS448 locus, DYS450 locus, DYS451 locus, DYS452 locus, DYS453 locus, DYS454 locus, DYS455 locus, DYS456 locus, DYS457 locus, DYS458 locus, DYS459 locus, DYS460 locus, DYS461 locus, DYS462 locus, DYS463 locus, DYS464 locus, DYS465 locus, DYS466 locus, DYS467 locus, DYS468 locus, DYS469 locus, DYS470 locus, DYS471 locus, DYS472 locus, DYS473 locus, DYS474 locus, DYS475 locus, DYS476 locus, DYS477 locus, DYS478 locus, DYS479 locus, DYS480 locus, DYS481 locus, DYS locus DYS449, locus DYS450, locus DYS453, locus DYS454, locus DYS455, locus DYS456, locus DYS458, locus DYS459a / b, locus DYS460, locus DYS461, locus DYS472, locus DYS476, locus DYS481, locus DYS485, locus DYS502, locus DYS504, locus DYS505, locus DYS508, locus DYS510, locus DYS511, locus DYS512, locus DYS513, locus DYS518, locus DYS520, locus DYS521 locus DYS525, locus DYS527a / b, locus DYS530, locus DYS531, locus DYS533, locus DYS534, locus DYS538, locus DYS541, locus DYS549, locus DYS552, locus DYS556, locus DYS557, locus DYS561, locus DYS565, locus DYS568, locus DYS570, locus DYS571, locus DYS572, locus DYS573, locus DYS576, locus DYS578, locus DYS585, locus DYS587, locus DYS590 , DYS593 locus, DYS596 locus, DYS612 locus, DYS613 locus, DYS616 locus, DYS617 locus, DYS622 locus, DYS626 locus, DYS627 locus, DYS630 locus, DYS635 locus, DYS638 locus, DYS640 locus, DYS641 locus, DYS643 locus, DYS644 locus, DYS645 locus, DYS710 locus, DYS712 locus, DYS720 locus, DYS722 locus, DYS724 locus, Y-GATA-A10 locus and Y-GATA-H4 locus.

[0038] The nucleotide sequences of each locus in the hg38 human reference genome are shown in column 3 of Table 2 .

[0039] In the primer combination, according to the primer sequence, the molar ratios of the primers are as follows:

[0040] 0.100：0.100：0.230：0.230：0.070：0.070：0.150：0.150：0.165：0.165：0.150：0.150：0.050：0.050：0.100：0.100：0.400：0.400：0.100：0.100：0.075：0.075：0.050：0.050：0.060：0.060：0.200：0.200：0.050：0.050：0.050：0.050：0.350：0.350：0.100：0.100：0.070：0.070：0.400：0.400：0.060：0.060：0.400：0.400：0.230：0.230：0.160：0.160：0.180：0.180：0.200：0.200：0.050：0.050：0.120：0.120：0.075：0.075：0.100：0.100：0.100：0.100：0.200：0.200：0.050：0.050：0.120：0.120：0.250：0.250：0.050：0.050：0.115：0.115：0.150：0.150：0.100：0.100：0.125：0.125：0.100：0.100：0.250：0.250：0.050：0.050：0.200：0.200：0.060：0.060：0.050：0.050：0.200：0.200：0.080：0.080：0.080：0.080：0.180：0.180：0.080：0.080：0.125：0.125：0.080：0.080：0.160：0.160：0.120：0.120：0.115：0.115：0.230：0.230：0.100：0.100：0.230：0.230：0.230：0.230：0.200：0.200：0.100：0.100：0.300：0.300：0.300：0.300：0.200：0.200：0.230：0.230：0.080：0.080：0.150：0.150：0.100：0.100：0.050：0.050：0.090：0.090：0.120：0.120：0.100：0.100：0.180：0.180：0.230：0.230：0.200：0.200：0.100：0.100：0.200：0.200：0.250：0.250：0.100：0.100：0.080：0.080：0.300：0.300：0.230：0.230：0.115：0.115：0.080：0.080：0.165：0.165：0.075：0.075：0.100：0.100：0.100：0.100：0.100：0.100：0.200：0.200：0.250：0.250：0.050：0.050：0.180：0.180。.

[0041] In the primer combination, according to the primer sequence, the working concentration of each primer is as follows (the unit is μM): 0.100, 0.100, 0.230, 0.230, 0.070, 0.070, 0.150, 0.150, 0.165, 0.165, 0.150, 0.150, 0.050, 0.050, 0.100, 0.100, 0.400, 0.400, 0.100, 0.100, 0.075, 0.075, 0.050, 0.050, 0.060, 0.060, 0.200, 0.200, 0.050, 0.050, 0.050, 0.050, 0.350, 0.350, 0.100, 0. 100, 0.070, 0.070, 0.400, 0.400, 0.060, 0.060, 0.400, 0.400, 0.230, 0.230, 0.160, 0.160, 0.180, 0.180, 0.200, 0.200, 0.050, 0.050, 0.120, 0.120, 0 .075, 0.075, 0.100, 0.100, 0.100, 0.100, 0.200, 0.200, 0.050, 0.050, 0.120, 0.120, 0.250, 0.250, 0.050, 0.050, 0.115, 0.115, 0.150, 0.150, 0.100, 0.100, 0.125, 0.125, 0.100, 0.100, 0.250, 0.250, 0.050, 0.050, 0.200, 0.200, 0.060, 0.060, 0.050, 0.050, 0.200, 0.200, 0.080, 0.080, 0.080, 0.080 , 0.180, 0.180, 0.080, 0.080, 0.125, 0.125, 0.080, 0.080, 0.160, 0.160, 0.120, 0.120, 0.115, 0.115, 0.230, 0.230, 0.100, 0.100, 0.230, 0.230, 0.23 0, 0.230, 0.200, 0.200, 0.100, 0.100, 0.300, 0.300, 0.300, 0.200, 0.200, 0.230, 0.230, 0.080, 0.080, 0.150, 0.150, 0.100, 0.100, 0.050, 0.0 50, 0.090, 0.090, 0.120, 0.120, 0.100, 0.100, 0.180, 0.180, 0.230, 0.230, 0.200, 0.200, 0.100, 0.100, 0.200, 0.200, 0.250, 0.250, 0.100, 0.100, 0.0.080, 0.080, 0.300, 0.300, 0.230, 0.230, 0.115, 0.115, 0.080, 0.080, 0.165, 0.165, 0.075, 0.075, 0.100, 0.100, 0.100, 0.100, 0.100, 0.100, 0.200, 0.200, 0.250, 0.250, 0.050, 0.050, 0.180, 0.180. .

[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 the STR typing of each locus.

[0047] Paternal ancestry was identified based on STR typing of the loci.

[0048] Individual identification is performed based on STR typing of the locus.

[0049] Parentage identification was performed based on STR typing of the locus.

[0050] Ethnicity is inferred based on STR typing of the locus.

[0051] Male pedigree identification was performed based on STR typing of the locus.

[0052] Forensic identification is performed based on STR typing of the locus.

[0053] The relationship was identified based on STR typing of the loci.

[0054] Specifically, the sequencing may be next generation sequencing.

[0055] Specifically, the sequencing may be high-throughput sequencing.

[0056] 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 1.

[0057] Specifically, the reaction procedure of the multiplex PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 2 min, 72°C for 2 min, 22 cycles; 72°C for 5 min; and hold at 4°C.

[0058] Specifically, the DNA library was prepared using TruSeq DNA PCR-Free HT Library Prep Kit according to the instructions.

[0059] Specifically, the sequencing is carried out using Reagent Kit v3-600 cycles Sequencing reagents in MiSeqFGx TM Forensic genomic analysis is performed systematically.

[0060] Specifically, the data analysis method includes the following steps:

[0061] After sequencing, MiSeq FGx TM The forensic genome analysis system automatically generates fastq files, and the fastq files are analyzed using STRaitRazor3.0 software;

[0062] Before running the STRaitRazor 3.0 software, enter the configuration files of 103 loci in the Excel table, with each part separated by the Tab key; ① locus name; ② Y; 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;

[0063] After the 103 gene loci gene configuration files were entered, the STRaitRazor 3.0 software was run to generate the allsequences.txt file; this file provides the following five parts of information: ① locus name; ② genotyping; ③ amplicon base number; ④ amplicon sequence information; ⑤ sequencing depth;

[0064] The core repeat structure of the sequences of 102 Y-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 "Sequence Polymorphism STR Naming Rules" (GB / T 44393-2024).

[0065] Compared with the STR typing kit of the current mainstream second-generation sequencing detection platform, the identification target of the primer combination provided by the present invention includes 102 Y-STR loci, which can provide more Y chromosome genetic information and have higher resolution in the practice of fine differentiation of male pedigrees. The maximum amplicon length of all Y-STR loci in the composite amplification system provided by the present invention is less than 300bp, which is conducive to the analysis of degraded samples. Experiments have shown that the STR typing of 103 loci in the positive control 2800M is detected by the primer combination provided by the present invention, and the typing results are accurate. The present invention has important practical application value.

[0066] The invention has great application and promotion value for forensic identification and kinship identification. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present invention, rather than 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 according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0068] Example 1: Design of primer combinations for detecting 103 loci based on next-generation sequencing technology

[0069] 1. Screening of 103 loci

[0070] The inventors of the present invention included a large number of candidate Y-STR loci by consulting and analyzing the prior art, and then finally screened and obtained 103 loci based on strategies such as sequencing data evaluation, non-specific amplification screening, and amplification primer optimization. The 103 loci consist of sex-determining loci and 102 Y-STR loci.

[0071] The sex determination locus is: Amelogenin locus.

[0072] The 102 Y-STR loci are: DYS19 locus, DYS385a / b locus (two-copy locus), DYF387S1a / b locus (two-copy locus), DYS388 locus, DYS389-I locus, DYS389-II locus, DYS390 locus, DYS391 locus, DYS392 locus, DYS393 locus, DYF399S1a / b / c locus (three-copy locus), DYF404S1a / b locus (two-copy locus), DYS426 locus, DYS435 locus, DYS437 locus, DYS438 locus, DYS439 locus, DYS440 locus, DYS441 locus, DYS442 locus, DYS4439 locus, DYS4444 locus. 3 locus, DYS444 locus, DYS447 locus, DYS448 locus, DYS449 locus, DYS450 locus, DYS453 locus, DYS454 locus, DYS455 locus, DYS456 locus, DYS458 locus, DYS459a / b locus (two-copy locus), DYS460 locus, DYS461 locus, DYS472 locus, DYS476 locus, DYS481 locus, DYS485 locus, DYS502 locus, DYS504 locus, DYS505 locus, DYS508 locus, DYS510 locus, DYS511 locus, DYS locus DYS512, DYS513, DYS518, DYS520, DYS522, DYS525, DYS527a / b (two-copy locus), DYS530, DYS531, DYS533, DYS534, DYS538, DYS541, DYS549, DYS552, DYS556, DYS557, DYS561, DYS565, DYS568, DYS570, DYS571, DYS572, DYS573, DYS574 locus DYS576, locus DYS578, locus DYS585, locus DYS587, locus DYS590, locus DYS593, locus DYS596, locus DYS612, locus DYS613, locus DYS616, locus DYS617, locus DYS622, locus DYS626, locus DYS627, locus DYS630, locus DYS635, locus DYS638, locus DYS640, locus DYS641, locus DYS643, locus DYS644, locus DYS645, locus DYS710, locus DYS712, locus DYS720,DYS722 locus, DYS724 locus (two-copy locus), Y-GATA-A10 locus, Y-GATA-H4 locus. Two-copy loci are counted as 2 loci, and three-copy loci are counted as 3 loci, so there are a total of 102 loci.

[0073] 2. Obtaining Primer Combinations

[0074] 1. Artificially design and synthesize primers for amplifying each locus (the amplification length is required to be less than 300 bp), and then perform PCR amplification to obtain specific amplification products for each locus.

[0075] 2. Comprehensively consider the amplification conditions of each locus and select an appropriate amplification program for composite amplification. Since there are many composite loci and the mutual interference between primers is complex, it is necessary to conduct primer interference investigation, find out the loci with interference, and redesign and synthesize primers.

[0076] 3. During the initial composite amplification, the concentration of each primer in the reaction system was 0.05 μM. The primer concentration was then adjusted according to the sequencing data to obtain the optimal concentration of each primer in the composite amplification system (multiple PCR amplification system).

[0077] After the above steps, a primer combination is obtained. The primer combination consists of 94 primer pairs (188 primers) for detecting the above-mentioned sex loci and 102 Y-STR loci. One primer pair is used to detect the Amelogenin locus. One primer pair is used to detect the DYS389-I locus and the DYS389-II locus. One primer pair is used to detect two-copy loci or three-copy loci. One primer pair is used to detect the remaining loci. The names of each locus, the names of the primers corresponding to the amplified loci, the nucleotide sequences of the primers, and the numbers of the primers in the sequence table are shown in columns 1, 2, 3, and 4 in Table 1, respectively. The optimal concentration of each primer when performing multiple PCR amplification is shown in column 5 in Table 1. Primers with odd-numbered names are forward primers, and primers with even-numbered names are reverse primers.

[0078] The length of the amplified product of the primers corresponding to each locus in the hg38 human reference genome (for information on the hg38 human reference genome, see the website http: / / hgdownload.soe.ucsc.edu / goldenPath / hg38 / bigZips / hg38.2bit) and the nucleotide sequence of the amplified product are shown in Table 2.

[0079] Table 1

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Table 2

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Example 2: Detection of STR typing of positive control 2800M using the primer combination designed in Example 1 and its application

[0097] Test sample: 2800M. 2800M is a positive control DNA (single-source male human genomic DNA) for Human STR Analysis, Promega, product catalog number DD7101.

[0098] 1. Multiplex PCR amplification using primer combinations and DNA purification

[0099] 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.

[0100] 2. Take the template solution prepared in step 1 and use the primer combination (i.e. the primer combination obtained in Example 1, consisting of 188 primers, the sequences of which are shown in Table 1) to perform multiplex PCR amplification to obtain PCR amplification products.

[0101] The reaction system is 20 μL, consisting of 10 μL 2×Master Mix, 1 μL template solution prepared in step 1, primer combination, and nuclease-free water. The concentration of each primer in this reaction system is shown in column 5 of Table 1.

[0102] 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,

[0103] 400 mM dNTP, 0.2 U / μL Taq DNA polymerase (Roche product, catalog number 11147633103), and the balance is water.

[0104] Reaction program: 95°C for 5 min; 95°C for 30 s, 60°C for 2 min, 72°C for 2 min, 22 cycles; 72°C for 5 min; maintain at 4°C.

[0105] 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.

[0106] 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.

[0107] Qubit TM dsDNA quantification kit: Thermo Fisher Scientific, product catalog number is Q32854.

[0108] 3.0 Fluorometer: Thermo Fisher Scientific.

[0109] 2. Library Preparation

[0110] 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.

[0111] 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.

[0112] 3. Sample test

[0113] 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).

[0114] Reagent Kit v3-600 cycles sequencing reagents: Illumina, product catalog number is MS-102-3003.

[0115] 4. Data Analysis

[0116] After sequencing, MiSeq FGx TMThe 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, the configuration files of the 103 loci were entered in the excel table. The locus configuration files are as follows: ① locus name; ② Y; 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. The 103 loci were entered in sequence according to this format. After the 103 loci gene configuration files were entered, the STRaitRazor 3.0 software was run 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 the amplicon sequence information; ⑤ sequencing depth. The core repeat structure of the sequences of 102 Y-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 "Sequence Polymorphism STR Naming Rules" (GB / T 44393-2024).

[0117] The results of the Amelogenin locus are: X, Y. The genotyping results of other loci are shown in Table 3. 2800M obtained a complete STR typing, which can fully meet the requirements of forensic STR testing.

[0118] Table 3

[0119]

[0120]

[0121]

[0122] Note: N represents any nucleotide, and its subscript number represents the number of nucleotides (N and the number following it represent a base sequence, and the number following 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.

[0123] Example 3: Accuracy Verification

[0124] Test samples: 2800M or 9948. 2800M is the positive control DNA (Single-source male human genomic DNA) for Human STR Analysis, Promega Company, product catalog number DD7101. 9948 (Control DNA 9948) is the male positive control DNA, Qiagen Company, product catalog number 386041.

[0125] Take 1 ng of the test sample and use Microreader TM 40Y Direct ID System (product of Beijing ReadGene Technology Co., Ltd., product batch number L / N: 40YD30F201012S21P30K) was used according to the instructions (the steps included: PCR amplification and capillary electrophoresis detection in sequence) to obtain the allele typing of each locus (the results correspond to CE in Table 4).

[0126] Take 1 ng of the test sample and perform the operation according to the steps of Example 2 (the results correspond to the NGS in Table 4).

[0127] The results for the Amelogenin locus were: X, Y. The genotyping results for other loci are shown in Table 4.

[0128] The results showed that the typing results obtained according to the steps of Example 2 were consistent with those of Microreader TM The typing results of the overlapping loci in the 40Y Direct IDSystem in the positive controls 2800M and 9948 were completely consistent.

[0129] Table 4

[0130]

[0131] Note: CE is the typing result of capillary electrophoresis detection, and NGS is the typing result of second-generation sequencing detection.

[0132] Example 4, comparative examples of primer combinations

[0133] The primer combination prepared in Example 1 was obtained by the inventors of the present invention after a large number of experiments, mainly including repeated adjustments of primers for amplifying each locus. Due to the large number of complex loci and the complex mutual inhibition between primers, the primers used to amplify each locus in the kit cannot be replaced.

[0134] Control primer combination A and control primer combination B were prepared by replacing some primers in the primer combination in Table 1 of Example 1. The replaced primers are shown in Table 5 (unrecorded primers indicate no replacement). Among them, the primers of DYS385a / b, DYS392, DYS447, DYS449, DYS518, DYS534, DYS570, DYS617, DYS644 and DYS722 are different.

[0135] The method is basically the same as Example 2. The concentration of each primer in the reaction system is 0.2 μM.

[0136] Table 5

[0137]

[0138] Note: “—” indicates that the primer combination does not contain the primer pair for this locus.

[0139] The test results are shown in Table 6. The results show that after the primers in the primer combination prepared in Example 1 were replaced, part of the loci were suppressed and the suppression conditions of the control primer combination A and the control primer combination B were different.

[0140] Table 6

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] Note: N represents any nucleotide, and its subscript number represents the number of nucleotides (N and the number following it represent a base sequence, and the number following 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 was obtained.

[0147] The above results show that the primer combination prepared in Example 1 is irreplaceable. The inventors of the present invention obtained the primer combination and optimized the primer concentration after a lot of experimental exploration.

[0148] 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 188 primers; the primers are single-stranded DNA molecules; the nucleotide sequences of the 188 primers are shown in sequence 1 to sequence 188 in the sequence listing.

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) or (g) or (h): (a) Locus STR analysis; (b) identification of paternal ancestry; (c) individual identification; (d) paternity testing; (e) racial inference; (f) Male pedigree identification; (g) Forensic medical examination; (h) Identification of kinship.

3. Use of the primer combination of claim 1 in paternal ancestry identification, male family identification, kinship identification, or parentage identification.

4. Use of the primer combination of claim 1 in forensic identification, individual recognition or ethnicity inference.

5. Use of the primer combination according to claim 1 in locus STR 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 paternal ancestry identification, male family identification, kinship identification or parentage identification.

9. Use of the kit according to claim 6 in forensic identification, individual recognition or ethnicity inference.

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

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