Primer sets for amplifying human chromosome STR and InDel loci and their applications

Abstract

The present invention relates to the field of DNA detection technology, and in particular to a primer set and application for amplifying human chromosome STR and InDel loci. The primer set provided by the present invention has been verified by 113 unrelated individuals and can correctly type 25 A-STRs, 28 Y-STRs, 63 InDels and the enamel protein gene. Amelogenin ; The STR and InDel sequences of 8 samples can be detected and typing can be completed within 12 hours; a more complete typing can be obtained with a lower DNA input; the alleles of 9 loci of the secondary sample can be detected in a 1:19 DNA mixed sample; there is no obvious amplification product in the DNA of cats, chickens, cows, dogs, ducks, Escherichia coli, fish, horses, pigs, rabbits, mice, and sheep, and animal and human samples can be clearly distinguished.

Description

Technical Field

[0001] The invention relates to the technical field of DNA detection, in particular to a primer set for amplifying human chromosome STR and InDel gene loci and an application thereof. Background Art

[0002] DNA is the "gold standard" for individual identification and has important application value in the field of personal identification. DNA-based identification mainly distinguishes different individuals by detecting a certain number of genetic markers. The short tandem repeat (STR) repeat unit is usually 2-6 bp. STR typing has certain advantages in the field of forensic identification: (1) Strong identification ability. The polymorphism of a single STR is high. The composite amplification of multiple STRs can increase the genetic information content of a single test, and the cumulative probability can achieve the purpose of identity identification. (2) High species specificity. The primers contained in the current commercial STR kits are all designed for human genomic DNA and have been verified for species specificity.

[0003] According to the different chromosome positions of STR, it can be divided into autosomal STR (A-STR) and sex chromosome STR. Sex chromosome STR includes Y chromosome STR (Y-STR) and X chromosome STR (X-STR). Y-STR is a paternal inheritance characteristic. Except for mutations, all male individuals of the same paternal line have the same Y-STR. Therefore, Y-STR typing has certain practical value in paternal kinship identification and family tracing. However, the STR mutation rate is relatively high, about 10 -3 ; and the amplified fragments are usually longer, such as the AmpFlSTR® Sinofiler™ kit launched by ABI in the United States, the amplicon length of its autosomal STR is 35bp-500 bp, and the detection success rate in degraded samples is low.

[0004] Insertion or deletion (Indel) is a DNA polymorphism caused by the insertion or deletion of DNA fragments. Like SNP, Indel is also a form of genetic variation, mainly including five types: single base pair insertion / deletion, single base pair repeated insertion, multiple base pair repeated insertion, transposon insertion, and random DNA sequence insertion / deletion. Unlike SNP, the binary polymorphism structure of Indel is the insertion or deletion of single or multiple bases. The two alleles show polymorphism of DNA length, most of which differ by 2-4 nucleotides. The mutation rate of Indel is about 10 -8, significantly lower than STR, and the amplified fragment can be less than 100 bp, which is very suitable for degraded samples. The detection method can use the same capillary automatic typing method as STR, but it will not produce slip peaks and has the characteristics of both STR and SNP.

[0005] At present, the most commonly used genetic marker detection method is the short tandem repeat sequence automatic typing technology based on capillary electrophoresis (CE), which calculates the number of repetitions of STR repeat units and the presence or absence of InDel by fluorescent detection of the length of the amplified sequence. For example, ThermoFisher and ANDE in the United States have developed RapidHIT ID and ANDE 6C rapid detection equipment based on microfluidic technology, which can achieve on-site rapid DNA identification within 3 hours. China Bio-Jingdian has also taken the lead in developing a portable fully integrated forensic DNA evidence on-site rapid inspection chip system. However, these detection methods have the following main problems: First, they mainly detect 20-30 STR genetic markers, and have poor applicability to samples with longer amplified fragments; second, the throughput is low, and only 1-5 samples can be detected at a time; third, STR is mainly typed by fragment length, and alleles with the same number of repetitions but different sequences cannot be identified. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a primer set and application for amplifying human chromosome STR and InDel loci. The primer set provided by the present invention can correctly type 25 A-STRs, 28 Y-STRs, 63 InDels and the enamel protein gene. Amelogenin ; The primer set of the present invention can detect the STR and InDel sequences of 8 samples and complete typing within 12 hours; a more complete typing can be obtained at a lower DNA input amount (DNA input amount ≥ 50pg).

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a primer set for amplifying human chromosome STR and InDel loci. The primer information of the primer set is shown in Table 1.

[0009] The present invention provides an application of the primer set described in the above technical solution in one or more of 1) to 8), specifically: 1) detecting human chromosome STR and InDel loci for individual identification; 2) typing human chromosome STR and InDel loci; 3) distinguishing DNA samples of humans from non-human organisms; 4) typing human chromosome STR and InDel loci in mixed DNA samples; 5) preparing a kit for detecting human chromosome STR and InDel loci; 6) preparing a kit for typing human chromosome STR and InDel loci; 7) preparing a kit for distinguishing DNA samples of humans from non-human organisms; 8) preparing a kit for typing human chromosome STR and InDel loci in mixed DNA samples.

[0010] Preferably, the non-human organism includes animals and / or microorganisms.

[0011] Preferably, the microorganism comprises Escherichia coli.

[0012] The present invention provides a kit for detecting STR and InDel loci of human chromosomes, comprising the primer set described in the above technical solution.

[0013] Preferably, the kit further comprises an enrichment primer pair, wherein the enrichment primer pair comprises primers having nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] The present invention provides a method for detecting STR and InDel loci of human chromosomes by using the primer set described in the above technical solution, comprising the following steps: using the primer set to perform multiple PCR amplification on a DNA sample to be tested to obtain multiple amplification products; sequencing the multiple amplification products to obtain sequencing data; analyzing the sequencing data to obtain a typing result, wherein the analysis method comprises: 1) basecall mode selection: each sample adopts two modes, accurate and super accurate, to generate two fastq files; 2) STR analysis: for the two fastq files generated in step 1), firstly find the allele with the maximum depth of each locus, and then extract the allele with a depth ratio greater than 0.1 to the maximum depth allele, and the allele with a depth ratio greater than 0.3 is considered to be a true allele; 3) InDel analysis: analyzing the two fastq files generated in step 1), including: (1) trim reads; (2) map reads to reference; (3) read mappings; (4) identify known mutations from mappings.

[0015] Preferably, the sequencing instrument includes a MinION sequencer or a GridION sequencer.

[0016] Preferably, based on the DNA content, the input amount of the multiple amplification products during the sequencing is ≥50 pg.

[0017] Beneficial effects:

[0018] The present invention provides a primer set for amplifying human chromosome STR and InDel loci, and the primer information of the primer set is shown in Table 1. The primer set provided by the present invention has been verified by 113 unrelated individuals and can correctly type 25 A-STRs, 28 Y-STRs, 63 InDels and the enamel protein gene Amelogenin ; The STR and InDel sequences of 8 samples can be detected and typing can be completed within 12 hours; a more complete typing can be obtained with a lower DNA input; the alleles of 9 loci of the secondary sample can be detected in a 1:19 DNA mixed sample; there is no obvious amplification product in the DNA of cats, chickens, cows, dogs, ducks, Escherichia coli, fish, horses, pigs, rabbits, mice, and sheep, and animal and human samples can be clearly distinguished.

[0019] Furthermore, using the detection system constructed by the primer set of the present invention, on-site typing of STR can be performed by using the nanopore sequencer MinION, which weighs less than 87g and can work with a laptop computer, expanding the use environment of STR and InDel forensic typing, and can generate sequence information of STR and InDel, thereby improving the identification efficiency of the locus. It is a detection system that can detect low-input DNA and mixed samples in a short time, and can clearly distinguish between animal and human samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The quality inspection library results of Qsep100 fully automatic nucleic acid protein analysis system;

[0022] Figure 2 It is the STR detection of different DNA input amounts in the accurate mode of high-precision base recognition;

[0023] Figure 3 InDel detection of different DNA input amounts in the accurate mode of high-precision base recognition;

[0024] Figure 4 The STR detection results of different DNA input amounts in the super accurate base recognition mode;

[0025] Figure 5 This is the InDel detection status of different DNA input amounts in the super accurate base recognition mode. DETAILED DESCRIPTION

[0026] The present invention provides a primer set for amplifying human chromosome STR and InDel loci. The primer information of the primer set is shown in Table 1.

[0027] Existing studies have attempted to use nanopores to type STR and SNP, but only a small number of standards were tested, and the reliability and practicality of the method were not verified according to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM). In the latest literature, the system constructed by Luo can correctly type only 9 A-STRs (autosomes STRs) out of 50 samples, making it difficult to achieve effective individual identification [Luo Y, Zhang J, Ni M, Mei Z, Ye Q, Guo B, Fang L, Feng D, Wang L, Yan J, Wang G. Pilot validation of on-field STR typing and human identity testing by MinION nanopore sequencing [J]. ELECTROPHORESIS, 2024, 45 (9–10): 885–896.]. The primer set provided by the present invention has been verified by 113 unrelated individuals and can correctly type 25 A-STRs, 28 Y-STRs, 63 InDels and enamel protein genes. Amelogenin ; The STR and InDel sequences of 8 samples can be detected and typing can be completed within 12 hours; a more complete typing can be obtained with a lower DNA input; the alleles of 9 loci of the secondary sample can be detected in a 1:19 DNA mixed sample; there is no obvious amplification product in the DNA of cats, chickens, cows, dogs, ducks, Escherichia coli, fish, horses, pigs, rabbits, mice, and sheep, and animal and human samples can be clearly distinguished.

[0028] Based on the above advantages, the present invention provides the application of the primer set described in the above technical scheme in one or more of 1) to 8), specifically: 1) detecting human chromosome STR and InDel loci for individual identification; 2) typing human chromosome STR and InDel loci; 3) distinguishing DNA samples of humans and non-human organisms; 4) typing human chromosome STR and InDel loci in mixed DNA samples; 5) preparing a kit for detecting human chromosome STR and InDel loci; 6) preparing a kit for typing human chromosome STR and InDel loci; 7) preparing a kit for distinguishing DNA samples of humans and non-human organisms; 8) preparing a kit for typing human chromosome STR and InDel loci in mixed DNA samples.

[0029] As one embodiment, the non-human organism includes animals and / or microorganisms; the microorganism includes Escherichia coli.

[0030] Based on the above advantages, the present invention provides a kit for detecting human chromosome STR and InDel loci, comprising the primer set described in the above technical solution. As an embodiment, the kit also contains an enrichment primer pair; the enrichment primer pair includes primers with nucleotide sequences such as SEQ ID NO.1 and SEQ ID NO.2. The enrichment primer pair provided by the present invention can enrich the amplification product obtained by amplification of the primer set.

[0031] Based on the above advantages, the present invention provides a method for detecting human chromosome STR and InDel loci using the primer set described in the above technical solution, comprising the following steps: using the primer set to perform multiple PCR amplification on the DNA sample to be tested to obtain multiple amplification products; sequencing the multiple amplification products to obtain sequencing data; analyzing the sequencing data to obtain typing results, and the analysis method comprises:

[0032] 1) Basecall mode selection: Each sample uses the accurate and super accurate modes to generate two fastq files; 2) STR analysis: For the two fastq files generated in step 1), first find the allele with the maximum depth of each locus, then extract the allele with a depth ratio greater than 0.1 to the maximum depth allele. The allele with a depth ratio greater than 0.3 is considered to be the true allele; 3) InDel analysis: Analyze the two fastq files generated in step 1), including: (1) trimreads; (2) map reads to reference; (3) read mappings; (4) identify known mutations from mappings.

[0033] As an embodiment, the sequencing instrument is a MinION sequencer or a GridION sequencer.

[0034] As an embodiment, the input amount of the multiple amplification products during the sequencing is ≥50 pg based on the DNA content.

[0035] The method provided by the present invention is a detection method that can detect low-input DNA and mixed samples in a short time. On-site typing of STR can be performed by relying on the nanopore sequencer MinION. The instrument weighs less than 87g and can work with the help of a laptop computer, thus expanding the use environment of STR and InDel forensic typing, and can generate sequence information of STR and InDel, improve the identification efficiency of gene loci, and can clearly distinguish between animal and human samples.

[0036] To further illustrate the present invention, the primer set and application for amplifying human chromosome STR and InDel loci provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] 1. Locus screening.

[0039] 1. STR screening.

[0040] A-STR markers were screened using the Hirsch Human STRtyper-32G Amplification Fluorescence Detection Kit and the ForenSeq™ DNA Signature Prep Kit; Y-STR markers were screened using the Hirsch SureID® PathFinder Plus Kit and the ForenSeq™ DNA Signature Prep Kit.

[0041] 2. InDel screening.

[0042] Based on the dbSNP database (https: / / www.ncbi.nlm.nih.gov / snp / ), human GRCh38 was used as the reference genome for genetic marker screening, and VCFtools and plink software were used to screen out InDel loci with high identification efficiency. The specific screening criteria are as follows:

[0043] (1) Screen out binary polymorphic insertion / deletion sequences located in the intronic regions of autosomes with a global minor allele frequency (GMAF) between 0.4 and 0.5; (2) Screen out loci that satisfy the Hardy-Weinberg equilibrium (HWE) law and set P HWE >0.05, and heterozygosity (H)>0.4; (3) Screen out those that meet the linkage equilibrium test (LE), set r 2 <0.01; (4) Screening out loci with a population differentiation index (Fst) <0.06 among different populations (East Asia, South Asia, Europe, America, and Africa); (5) Screening out InDel loci with a fragment length >1bp; (6) Screening out InDel loci on the same chromosome with a physical distance ≥10MB.

[0044] Based on the genetic markers screened above, during the design of multiple primers, according to factors such as primer specificity, primer length, annealing temperature and primer structure design, a composite primer set containing 53 STRs, 63 InDels and Amelogenin was finally constructed (see Table 1).

[0045] 2. Materials and methods.

[0046] Table 1 Loci and primer information included in the A331 primer set

[0047]

[0048] Note: The position information of the front (back) primer in Table 1 is the physical position of the front (back) primer on the chromosome in the human genome database (GRCh38); the primer molar ratio is the molar ratio of different primer pairs in the A331 primer set; the nucleotide sequence of the front primer is the sequence corresponding to the position information in Table 1, and the nucleotide sequence of the back primer is the reverse complementary sequence of the sequence corresponding to the position information in Table 1.

[0049] Table 2 Main reagents and instruments

[0050]

[0051] (A) Generation and quality control of PCR products (5 h for 8 samples).

[0052] 1. The first round of PCR reaction.

[0053] Table 3 First round PCR reaction system

[0054]

[0055] In Table 3, x represents the added volume of sample DNA. When the DNA sample is an FTA blood collection card smear sample, 2 blood cards with a diameter of 1 mm are added, and x is considered to be 0. When the sample DNA is gDNA, the starting amount is 1ng-5ng / reaction tube, and it can also be other constant DNA products.

[0056] After all the components in Table 3 were mixed evenly, the following reaction was performed: 95 °C, 3.5 min; 98 °C, 20 s, 60 °C, 4 min, 24 cycles; 72 °C, 5 min.

[0057] 2. First round of magnetic bead purification: (1) Add 30 μl of AMPure XP magnetic beads equilibrated at room temperature to 30 μl of PCR product, and mix gently by pipetting 20 times; (2) After incubation at room temperature for 5 min, place the PCR tube on a magnetic rack for 3 min; (3) Completely remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μl of YF buffer B to the tube, and mix gently by pipetting 20 times; (4) After incubation at room temperature for 5 min, place the PCR tube on a magnetic rack for 3 min; (5) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μl of 80% ethanol solution to the tube, and let it stand for 30 s; (6) Remove the supernatant, keep the PCR tube on the magnetic rack, add 180 μl of 80% ethanol solution to the tube, let it stand for 30 s, and then completely remove the supernatant; (7) Let it stand at room temperature for 3 min to allow the residual ethanol to evaporate completely; (8) Remove the PCR tube from the magnetic rack, add 24 μl of nuclease-free water, and gently pipette to resuspend the magnetic beads to avoid creating bubbles. Let it stand at room temperature for 2 min; (9) Place the PCR tube back on the magnetic rack and let it stand for 3 min; (10) Use a pipette to aspirate 13.5 μl of the supernatant and transfer it to a new 200 μl PCR tube. The supernatant in the tube is the amplified multiple PCR product.

[0058] 3. In the second round of PCR reaction, the primer pair is MultipSeq® UDI Primer, and the specific sequence is as follows:

[0059] Upstream primer (SEQ ID NO.1):

[0060] 5'-ctctcagtacgtcagcagttcgttgttgttcaactccttggctcacagaacgacatggctacgatccgactt-3';

[0061] Downstream primer (SEQ ID NO.2):

[0062] 5'-gcatggcgaccttatcagaacaacaacgttgtcttcctaagaccgcttggcctccgactt-3'.

[0063] Table 4 Second round PCR reaction system

[0064]

[0065] Note: PCR product mixture is the multiplex PCR product purified in the previous step.

[0066] After all the components in Table 4 were mixed evenly, the following reaction was performed: 95°C, 3.5 min; 9 cycles (98°C, 20 s; 58°C, 1 min; 72°C, 30 s); 72°C, 5 min.

[0067] 4. The second round of magnetic bead purification is the same as the first round of magnetic bead purification to obtain a multiplex PCR library.

[0068] 5. Library quantification: Take 1 μl of library and use Qubit® 3.0 Fluorometer (Qubit dsDNA HS AssayKit) to measure the library concentration and record the library concentration.

[0069] 6. Library quality detection: Take 1 μl of library sample and use Qsep100 automatic nucleic acid protein analysis system to measure the length and purity of library fragments. The target fragments of normal library are as follows: Figure 1 As shown. Figure 1 It can be seen that the target fragments of the normal library are mainly concentrated around 286 bp, and the fragments are concentrated between 150-500 bp.

[0070] (ii) From PCR reaction products to sequencing.

[0071] 1. Terminal repair.

[0072] Table 5 End repair reaction system

[0073]

[0074] (1) After mixing all the components in Table 5, perform the following reactions: 20℃, 5min; 65℃, 5min. (2) Add 15μl of resuspended AMPure XP magnetic beads (AXP) to the above DNA end repair reaction system, flick the test tube to mix thoroughly, and incubate at room temperature for 5min. (3) Centrifuge the sample briefly and place it on a magnetic rack to allow the magnetic beads and liquid phase to separate. Keep the centrifuge tube on the magnetic rack and remove the supernatant with a pipette. (4) Keep the test tube on the magnetic rack and wash the magnetic beads with 200μl of freshly prepared 80% ethanol. Remove the ethanol with a pipette and discard. (5) Repeat step (4) above. (6) Centrifuge the centrifuge tube briefly and place it on the magnetic rack. Remove the residual ethanol with a pipette. Allow the magnetic beads to air dry for 30 seconds. (7) Remove the centrifuge tube from the magnetic rack and resuspend the magnetic beads in 10μl of nuclease-free water. Centrifuge briefly and then incubate at room temperature for 2 minutes. (8) Place the centrifuge tube on a magnetic rack until the magnetic beads and liquid phase are separated and the eluate is clear and colorless. Transfer 10 μl of the eluate to a new 1.5 ml Eppendorf DNA LoBind tube. The supernatant in the tube is the end-repair product.

[0075] 2. Barcode connection.

[0076] Table 6 Barcode ligation reaction system

[0077]

[0078] (1) Mix all the components in Table 6 with the end-repair product and incubate at room temperature for 20 minutes. (2) Add 2 μl EDTA to each tube, mix by pipetting, and then centrifuge briefly. (3) In a clean 1.5 ml Eppendorf DNA LoBind centrifuge tube, mix the barcoded DNA samples. (4) Add 0.8× AMPure XP magnetic beads (AXP) to the mixed reaction system, purify the samples, mix by pipetting, and incubate at room temperature for 10 minutes. (5) Centrifuge the samples briefly and place them on the magnetic rack for 5 minutes. After the magnetic beads and liquid phase are separated, aspirate the supernatant. (6) Keep the test tube on the magnetic rack and wash the magnetic beads with 700 μl of freshly prepared 80% ethanol. Use a pipette to aspirate the ethanol and discard it. (7) Repeat the above step (6). (8) Centrifuge the tube briefly and place it on a magnetic rack. Use a pipette to remove the residual ethanol and let the magnetic beads dry in the air for about 30 seconds. (9) Remove the tube from the magnetic rack, resuspend the magnetic beads in 35 μl of nuclease-free water, and incubate at 37°C for 10 minutes. Flick the tube for 10 seconds every two minutes to stir the sample and promote DNA elution. (10) Leave the tube on the magnetic rack until the magnetic beads and liquid phase are separated and the eluate is clear and colorless. Transfer the 35 μl eluate to a new 1.5 ml Eppendorf DNA LoBind tube, which contains the DNA library after barcoding.

[0079] 3. Sequencing adapter connection.

[0080] Table 7 Sequencing adapter ligation reaction system

[0081]

[0082] (1) Mix all the components in Table 7 and incubate at room temperature for 20 minutes. (2) Add 0.8× AMPure XP magnetic beads to the reaction system, pipette to mix, and incubate at room temperature for 10 minutes. (3) Centrifuge the sample briefly and place it on a magnetic rack to allow the magnetic beads to separate from the liquid phase. Keep the centrifuge tube on the magnetic rack and remove the supernatant with a pipette. (4) Wash the magnetic beads with 125μl short fragment buffer (SFB). Gently flick the centrifuge tube to mix the magnetic beads, centrifuge the centrifuge tube briefly, put it back on the magnetic rack, leave it to separate from the magnetic beads and the liquid phase, and remove the supernatant with a pipette. (5) Repeat step (4) above. (6) Centrifuge the centrifuge tube briefly and place it on a magnetic rack. Remove the remaining supernatant with a pipette. (7) Remove the centrifuge tube from the magnetic rack. Resuspend the magnetic beads in 15μl elution buffer (EB). (8) Centrifuge briefly and incubate at 37°C for 10 min. Flick the tube for 10 seconds every two minutes to agitate the sample and promote DNA elution. (9) Place the centrifuge tube on a magnetic rack for at least one minute until the beads and liquid phase are separated and the eluate is clear and colorless. Transfer this 15 μl eluate to a new 1.5 ml Eppendorf DNA LoBind tube, which contains the DNA library after ligation of sequencing adapters.

[0083] 4. Chip processing and computerization.

[0084] The loading library mixture system is: 12μl DNA library connected to the sequencing adapter, 37.5μl sequencing buffer (SB) and 25.5μl library particles (LIB, amplification-free barcode kit-96 V14). After all the components of the loading library mixture system are mixed evenly, add them dropwise to the pretreated R10.4.1 chip. Place the chip in the MinION sequencer and select the sequencing time according to the required data volume. Each sample requires an average of 500M data volume, and the data volume of 8 samples can be generated in about 2 hours.

[0085] (III) Sequencing data analysis (8 samples took about 2 hours).

[0086] 1. Basecall mode selection: The basecall software selected for this system is Dorado 7.2.13. Each sample uses two modes, accurate and super accurate, to generate two fastq files.

[0087] 2. STR analysis: This system uses STRait Razor 3.0 to analyze two fastqs. For the allsequences.txt file generated by fastq, first find the allele with the maximum depth of each locus, then extract the allele with a depth ratio greater than 0.1 to the maximum depth allele, and the depth ratio greater than 0.3 is considered to be the true allele.

[0088] 3. InDel analysis: This system uses CLC Genomics Workbench 24 software from QIAGEN, USA, to analyze two fastqs, including: (1) trim reads; (2) map reads to reference; (3) read mappings; (4) identify known mutations from mappings.

[0089] 4. The results generated by the samples are compared with the sequencing results generated by the verified BGI sequencer.

[0090] 3. Results

[0091] 1. Consistency and repeatability.

[0092] The 9948 standard and 2800M standard were sequenced and analyzed, and the typing results are shown in Table 8. The results are completely consistent with the typing results of the BGI sequencer.

[0093] The 9948 standards were sequenced three times, with average depths of 2276×, 1194×, and 1974× in the accurate mode and 2883×, 1800×, and 2627× in the super mode. The three typing results were completely consistent.

[0094] Table 8 Typing results of 9948 standard and 2800M standard

[0095]

[0096] Note: “-” in Table 8 stands for Deletion, indicating that this sequence is missing.

[0097] (B) Routine sample testing.

[0098] 113 unrelated individuals were typed, including 73 males and 40 females. The results are shown in Table 9.

[0099] As shown in Table 9, 50 STRs and 62 InDels can be correctly typed in the accurate mode; 51 STRs and 63 InDels can be correctly typed in the superaccurate mode; the combination of the accurate mode and the super accurate mode can correctly type 53 STRs and 64 InDels, including 25 A-STRs, which is greater than the latest literature data [Luo Y, Zhang J, Ni M, Mei Z, Ye Q, Guo B, Fang L, Feng D, Wang L, Yan J, Wang G. Pilot validation of on-field STR typing and human identity testing by MinION nanopore sequencing[J]. ELECTROPHORESIS, 2024, 45(9–10): 885–896.].

[0100] Table 9 Loci correctly typed in high-precision base calling mode and ultra-precision base calling mode

[0101]

[0102] (iii) Sensitivity.

[0103] The 9948 standard stock solution was diluted to different concentration gradients, and 5 ng, 1 ng, 500 pg, 200 pg, 100 pg, 50 pg, and the libraries were constructed and sequenced three times in parallel according to this system, and were named 9948-5ng-1, 9948-5ng-2, 9948-5ng-3, 9948-1ng-1, 9948-1ng-2, 9948-1ng-3, 9948-500pg-1, 9948-500pg-2, 9948-500pg-3, 9948-200pg-1, 9948-200pg-2, 9948-200pg-3, 9948-100pg-1, 9948-100pg-2, 9948-100pg-3, 9948-50pg-1, 9948-50pg-2, and 9948-50pg-3. The typing results are as follows Figure 2~Figure 5 shown.

[0104] The results showed that when the DNA input was greater than or equal to 200 pg, all except rs11471707 were correctly typed. When the DNA input was 100 pg, D12S391 and rs11471707 were incorrectly typed in the accurate mode, and D6S477 was not detected. In the superaccurate mode, D12S391, rs3028599, rs144581522, rs34529638, and rs11471707 were incorrectly typed, among which rs3028599 and rs144581522 could be correctly typed in the accurate mode. When the DNA input was 50 pg, 8 typing errors and 2 non-detections occurred in the accurate mode; 9 typing errors and 1 non-detection occurred in the super accurate mode. The above errors were all related to allele loss or low amplification number. Taking into account the actual situation of forensic medicine, two identical typing results will be used as the final result. Therefore, when the DNA input amount is greater than or equal to 50pg, all loci can be detected. Except for rs11471707, all typings are correct, with a detection rate of 100% and a typing accuracy of 99.15%.

[0105] (iv) Mixed samples.

[0106] 9948 and 2800M were mixed at a molar ratio of 19:1, 9:1, 4.5:1, 3:1, 1:1, 1:3, 1:4.5, 1:9, and 1:19, respectively, and basecalling was performed using the super accurate mode. When performing STR analysis, the allele with the maximum depth of each locus was first found, and all alleles with a depth ratio greater than 0.1 to the maximum depth were considered to be correct alleles. InDel analysis was set according to the sequencing data analysis in Materials and Methods.

[0107] Table 10 lists 58 loci that were differently typed between 9948 and 2800M and had no typing errors. It can be observed that when the DNA mixing ratio reached 1:4.5, this system could successfully identify the genotypes of the major and minor contributors, and when the mixing ratio reached 1:19, TPOX, D4S2366, CSF1PO, D8S1179, D10S1435, TH01, D19S253, DYS437, and Y-GATA-H4 could also detect the alleles of the minor contributors.

[0108] Table 10 Loci that can be correctly detected in super accurate base calling mode with different proportions of mixed samples

[0109]

[0110] Note: Bold results mean that all 9948 and 2800M alleles were detected; non-bold results mean that some alleles were lost; "-" means Deletion, indicating that this sequence is in a missing state.

[0111] (V) Species specificity.

[0112] The above method was used to detect samples from cats, chickens, cattle, dogs, ducks, E. coli, fish, horses, pigs, rabbits, mice, sheep and humans, and the results are shown in Table 11. As can be seen from Table 11, the method provided by the present invention has almost no detection in cats, chickens, cattle, dogs, ducks, E. coli, fish, horses, pigs, rabbits, mice and sheep, the average depth of each locus is 7.5, and the maximum total depth does not exceed 2000×, which is easy to distinguish from human samples.

[0113] Table 11 Number of sites and total depth detected by this system in animal and human samples

[0114]

[0115] Note: Sequencing depth greater than 20 is considered a call.

[0116] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer set for amplifying human chromosome STR and InDel loci, characterized in that: The primer information of the primer set is as follows: The primer position information is the physical position of the primer on the chromosome of the GRCh38 human genome database, the nucleotide sequence of the front primer is the sequence corresponding to the front primer position information, and the nucleotide sequence of the rear primer is the reverse complementary sequence of the sequence corresponding to the rear primer position information.

2. The primer set according to claim 1, characterized in that The primer information of the primer set is as follows: The primer molar ratio is the molar ratio of different primer pairs in the primer set.

3. The use of the primer set according to claim 1 or 2 in one or more of 1) to 8), specifically: 1) Detect human chromosome STR and InDel loci for individual identification; 2) Typing of human chromosome STR and InDel loci; 3) Distinguish DNA samples from humans and non-human organisms; 4) Typing of human chromosome STR and InDel loci in mixed DNA samples; 5) Prepare a kit for detecting human chromosome STR and InDel loci; 6) Prepare a kit for typing human chromosome STR and InDel loci; 7) A kit for preparing DNA samples for distinguishing between humans and non-human organisms; 8) Prepare a kit for typing human chromosome STR and InDel loci in mixed DNA samples.

4. The use according to claim 3, characterized in that: The non-human organisms include animals and / or microorganisms.

5. The use according to claim 4, characterized in that: The microorganisms include Escherichia coli.

6. A kit for detecting human chromosome STR and InDel loci, characterized in that: Comprising the primer set according to claim 1 or 2.

7. The kit according to claim 6, characterized in that The kit also includes an enrichment primer pair, which includes primers with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.

2.

8. A method for detecting human chromosome STR and InDel loci using the primer set of claim 1 or 2, characterized in that: The following steps are involved: Performing multiple PCR amplification on the DNA sample to be tested using the primer set to obtain multiple amplification products; Sequencing the multiple amplification products to obtain sequencing data; The sequencing data is analyzed to obtain typing results, wherein the analysis method comprises: 1) Basecall mode selection: Each sample uses two modes, accurate and super accurate, to generate two fastq files; 2) STR analysis: For the two fastq files generated in step 1), first find the allele with the maximum depth for each locus, then extract the allele with a depth ratio greater than 0.1 to the maximum depth allele. Alleles with a depth ratio greater than 0.3 are considered to be true alleles; 3) InDel analysis: Analyze the two fastq files generated in step 1), including: (1) trim reads; (2) map reads to reference; (3) read mappings; (4) identify known mutations from mappings.

9. The method according to claim 8, characterized in that The sequencing instrument includes a MinION sequencer or a GridION sequencer.

10. The method according to claim 8 or 9, characterized in that: Calculated by DNA content, the input amount of the multiple amplification product during the sequencing is ≥50 pg.

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