Primer sets, detection kits, uses thereof, and corresponding detection methods

Abstract

The present disclosure provides a genetic marker with good forensic application value in human genome, and particularly relates to a primer set, a detection kit, uses thereof, and a corresponding detection method.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology and relates to the detection of genetic markers in the human genome that have good forensic applications, specifically involving primer sets, detection kits, their uses, and corresponding detection methods. Background Technology

[0002] Forensic DNA analysis is widely used in all aspects of human forensic identification and is crucial for handling cases, identifying crimes, and identifying victims of accidents or natural disasters. The primary applications of forensic DNA analysis are personal DNA database construction and case handling. The core of a forensic DNA database lies in the microsatellite marker (STR) data it contains. A comprehensive forensic DNA database should include STR typing information from both autosomes and the Y chromosome. STRs are short tandem repeat sequences widely present in human genomic DNA, with repeat units of 2-6 nucleotides. STRs exhibit high stability and polymorphism in repeat units. Utilizing the polymorphism of STR repeat frequencies in a population can effectively provide individual-specific genomic clues, assisting in investigative leads in cases.

[0003] Traditional forensic DNA testing primarily utilizes PCR amplification to amplify target fragments and capillary electrophoresis (CE) to analyze mature STR information. However, the CE method can only obtain information on different STR repeat lengths, not mutation and sequence information, making it difficult to distinguish mixed samples. False peaks may also occur during the analysis, affecting the accuracy of the results. Furthermore, the limited amount of available fluorescence in CE restricts the selection of STR length and number, and standards need to be established for control testing. Its throughput and detection time are also not advantageous. Currently, most mainstream products on the market that detect STR genotyping based on the CE platform generally detect fewer than 30 STRs, with a few detecting more than 30. However, the latest version of the document "Genomic Loci and Data Structures Selected for Forensic DNA Databases GB-T 41009-2021" guides the selection of STR loci, which includes a total of 58 Class A loci and 34 Class B loci. Considering only Class A loci genotyping exceeds the scope that CE technology can cover. Previous products have not been able to adequately cover the Class A loci selected as guided by the new version of "Genomic Loci and Data Structures Selected for Forensic DNA Databases GB-T 41009-2021", or the commonly used Class A loci on autosomes and the Y chromosome.

[0004] Currently, there are various forensic testing platforms and products based on next-generation sequencing (NGS) technology. Commonly used sequencing platforms include Illumina's MiSeq FGx, Thermo Fisher's Ion Torrent PGM and S5. The number of STRs that can be detected using NGS platforms can reach hundreds. While NGS platforms offer advantages such as high throughput, the ability to perform pooled sequencing, the elimination of the need for standards, and the ability to obtain specific sequence and mutation information beyond STR length, which helps improve detection resolution, their sequencing fragment length range is limited, generally around 150 bp and usually not exceeding 300 bp. This reduces detection accuracy, increases detection costs, and / or reduces detection efficiency. Furthermore, the cost of NGS instruments, initial setup costs, and operating costs are high, and most products are supplied by imported manufacturers, which still presents obstacles to their application in forensic research and routine case handling.

[0005] Therefore, it is still necessary to continue exploring forensic testing products that can be applied to the field of forensic medicine with broad coverage, high sensitivity, high accuracy, high efficiency, and low cost. Summary of the Invention

[0006] To address the aforementioned technical challenges, the inventors, through long-term exploration and continuous experimentation, have developed a primer set. By ingeniously designing the primer set, the amplification of the test sample yields a product with broad site coverage, encompassing 48 commonly used sites for forensic DNA library construction and case handling. This includes all 39 class A sites on autosomes and the Y chromosome, 4 class B sites on autosomes, 1 Amelogenin site (a sex identification gene), and 4 InDel sites on the Y chromosome that assist in sex identification, as specified in the latest version of "Selected Loci and Data Structures for Forensic Science DNA Databases GB-T 41009-2021". This significantly improves cumulative individual identification ability and non-paternity exclusion rate, meeting the needs of most forensic DNA analysis application scenarios.

[0007] Furthermore, the inventors have creatively discovered that by optimizing experimental conditions, amplification specificity can be significantly improved, thereby increasing the final effective fragment concentration ratio and library concentration. Therefore, the sensitivity and accuracy of sequencing analysis results can be enhanced.

[0008] Furthermore, autosomal STRs are diploid, requiring accurate STR genotyping results for both alleles during STR typing. Sex chromosomes, however, are haploid, necessitating only accurate haploid STR genotyping results. Therefore, to achieve the same typing effect, the required sequencing depth for haploid sex chromosomes is theoretically half that of diploid autosomes. The inventors have creatively discovered that by precisely adjusting the primer ratio at each locus, the final sequencing depth at each locus can be controlled. For example, if there are two autosomal STR loci and two sex STR loci of equal length, and assuming that 100 reads are needed to analyze the STR information of each haplotype; without primer ratio optimization, if the sequencing of all four loci covers 200 reads each, then each of the two sex STR loci will waste 100 reads, because the sex chromosomes already meet the analysis requirements with 100 reads (waste rate = 200 / 800 = 1 / 4); however, if the primer ratio of each locus is adjusted so that the sequencing of the autosomal STR loci covers 200 reads each, and the sequencing of the sex chromosomes covers 100 reads each, then the aforementioned waste of 1 / 4 reads is avoided. Therefore, to achieve optimal data allocation and application while ensuring data accuracy, and to conserve data to reduce the cost per sample, the primer ratios for 48 loci were tested and adjusted. The optimization aimed to achieve an autosomal STR depth to sex chromosome STR depth ratio of 2:1, while controlling the fluctuation range of sequencing depth differences at each locus. This maximized data utilization and ensured the accuracy of sequencing analysis results while reducing costs. Therefore, the beneficial effects of this disclosure include: broad locus coverage, encompassing 48 loci commonly used by forensic personnel for DNA library construction and case handling, significantly improving cumulative individual identification and non-paternity exclusion rates, and meeting the needs of most forensic DNA analysis application scenarios; high specificity and sensitivity, with high sensitivity and accuracy of sequencing analysis results; an autosomal STR depth to sex chromosome STR depth ratio close to 2:1, with minimal differences in sequencing depth at different loci, resulting in high data utilization, low cost, and high accuracy of sequencing analysis results.

[0009] In a first aspect of this disclosure, a primer set may be provided comprising primers having sequences as shown in SEQ ID NO:1-SEQ ID NO:100.

[0010] In a second aspect of this disclosure, a detection kit may be provided, comprising the primer set as described herein.

[0011] In a third aspect of this disclosure, the primer sets or test kits described herein may be provided for use in human individual identification and / or forensic DNA database construction and / or suspect pedigree investigation and / or kinship identification and / or identification of degraded samples.

[0012] In a fourth aspect of this disclosure, a detection method may be provided, the detection method comprising the following steps:

[0013] Step 1) Perform a first round of PCR reaction on the sample to be tested using the primer set as described in this disclosure;

[0014] Step 2) Optionally, the product of step 1) is purified;

[0015] Step 3) Perform a second round of PCR reaction on the product of step 1) or step 2) using sequencing primers;

[0016] Step 4) Optionally, the product of step 3) is purified;

[0017] Step 5) Optionally, the product of step 3) or step 4) is subjected to end repair and purification;

[0018] Step 6) Connect the product from step 3), step 4), or step 5) to the sequencing adapter;

[0019] Step 7) Optionally, the product of step 6) is purified;

[0020] Step 8) Sequencing the product of step 6) or step 7).

[0021] The primer set, kit, and detection method disclosed herein can be applied to nanopore sequencing platforms. In addition to possessing the same advantages as next-generation sequencing platforms—high throughput, mixed-sample sequencing capability, no need for reference standards, and the ability to obtain specific sequence and mutation information beyond STR length, which helps improve detection resolution—nanopore sequencing platforms also offer advantages such as unlimited sequencing fragment length, low equipment cost, compact portability, and ease of application in grassroots laboratories and field environments. Their application in forensic genetics has been increasingly explored in recent years, thus broadening the application prospects of the primer set, kit, and detection method disclosed herein in the forensic field. Attached Figure Description

[0022] Figure 1 A schematic diagram of the sequencing library construction process is shown.

[0023] Figure 2The fragment distribution results of the library without sequencing adapters constructed in Example 1 are shown, where the first row from left to right corresponds to samples D01-D03, the second row from left to right corresponds to samples D04-D06, and the third row corresponds to sample D07.

[0024] Figure 3 The site depth distribution map of the library with added sequencing adapters constructed in Example 1 is shown.

[0025] Figure 4 The fragment distribution results of the library without sequencing adapters constructed in Example 2 are shown, where the first row from left to right corresponds to samples F07-F09, and the second row from left to right corresponds to samples F10-F12.

[0026] Figure 5 The site depth distribution map of the library with added sequencing adapters constructed in Example 2 is shown.

[0027] Figure 6 The fragment distribution results of the library without sequencing adapters constructed in Example 3 are shown, where the first row from left to right corresponds to samples A01-A03, and the second row from left to right corresponds to samples A04-A06.

[0028] Figure 7 The site depth distribution map of the library with added sequencing adapters constructed in Example 3 is shown.

[0029] Figure 8 The fragment distribution results of the library without sequencing adapters constructed in Example 4 are shown. The first row from left to right corresponds to samples B01-B03, the second row from left to right corresponds to samples B04-B06, the third row from left to right corresponds to samples B07-B09, and the fourth row corresponds to sample B10.

[0030] Figure 9 The site depth distribution map of the library with added sequencing adapters constructed in Example 4 is shown. Detailed Implementation

[0031] Unless otherwise defined or stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0032] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0033] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0034] It should be noted that the terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the described embodiments can be implemented in orders other than those described. The embodiments described below by example do not represent all embodiments consistent with this application. Rather, they are merely examples consistent with some aspects of this application as detailed in the appended claims.

[0035] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0036] In a first aspect of this disclosure, a primer set may be provided comprising primers having sequences as shown in SEQ ID NO:1-SEQ ID NO:100.

[0037] In some embodiments of this disclosure, the molar ratio of the primers in the primer set is:

[0038]

[0039]

[0040]

[0041] In some embodiments of this disclosure, the molar ratio of the primers in the primer set is:

[0042]

[0043]

[0044]

[0045] In some embodiments of this disclosure, the molar ratio of the primers in the primer set is:

[0046]

[0047]

[0048]

[0049]

[0050] In some embodiments of this disclosure, the molar ratio of the primers in the primer set is:

[0051]

[0052]

[0053]

[0054] or

[0055]

[0056]

[0057]

[0058] In a second aspect of this disclosure, a detection kit may be provided, comprising the primer set as described herein.

[0059] In some embodiments of this disclosure, the detection kit may also include reagents for amplification, including but not limited to DNA polymerase, buffer, dNTP, PCR enhancer, BSA, inorganic salts, molecular weight internal standard, etc.

[0060] In some embodiments of this disclosure, the DNA polymerase may be one or more of Taq DNA polymerase, T4 DNA polymerase, Pfu DNA polymerase, Bst DNA polymerase, and Klenow DNA polymerase.

[0061] In some embodiments of this disclosure, the PCR enhancer may include, but is not limited to, one or more of trehalose, polyethylene glycol at a concentration of less than 5%, dimethyl sulfoxide, and NP40 (ethyl phenyl polyethylene glycol).

[0062] In some embodiments of this disclosure, the inorganic salt may be one or more of MgCl2, KCl, and NaCl, including but not limited to.

[0063] In some embodiments of this disclosure, the components contained in the test kit may exist independently of each other, or several components may be mixed together in the form of a premix.

[0064] In some embodiments of this disclosure, the test kit may also include reagents and / or materials for DNA fragment purification. In some embodiments of this disclosure, the test kit may also include magnetic beads for DNA fragment purification.

[0065] In a third aspect of this disclosure, the primer sets or test kits described herein may be provided for use in human individual identification and / or forensic DNA database construction and / or suspect pedigree investigation and / or kinship identification and / or identification of degraded samples.

[0066] In a fourth aspect of this disclosure, a detection method may be provided, comprising the following steps:

[0067] Step 1) Perform a first round of PCR reaction on the sample to be tested using the primer set as described in this disclosure;

[0068] Step 2) Optionally, the product from step 1) is purified;

[0069] Step 3) Use sequencing primers to perform a second round of PCR reaction on the product of step 1) or step 2);

[0070] Step 4) Optionally, the product of step 3) is purified;

[0071] Step 5) Optionally, the product of step 3) or step 4) is subjected to end repair and purification;

[0072] Step 6) Ligate the product from step 3), step 4), or step 5) to the sequencing adapter;

[0073] Step 7) Optionally, the product from step 6) is purified;

[0074] Step 8) Sequencing the product from step 6) or step 7).

[0075] In step 1), the sample to be tested can be any material containing the human genome. In some embodiments of this disclosure, in step 1), the sample to be tested can be an environmental sample, blood, body fluid, hair, or other material containing the human genome.

[0076] In some embodiments of this disclosure, in step 1), the amplification program for the first round of PCR reaction is as follows: 92-98℃ for 1-10 min; 97-99℃ for 10 s-1 min; 58-62℃ for 1-10 min; 23-25 ​​cycles; 70-75℃ for 1-10 min.

[0077] In some embodiments of this disclosure, in step 1), the amplification program for the first round of PCR reaction is as follows: 93-97℃ for 2-5 min; 97-99℃ for 10-30 s; 59-61℃ for 2-6 min; 23-25 ​​cycles; 70-74℃ for 2-8 min.

[0078] In some embodiments of this disclosure, in step 1), the amplification program for the first round of PCR reaction is as follows: 94-96℃ for 2-5 min; 97.5-98.5℃ for 10-30 s; 59.5-60.5℃ for 2-6 min; 23-25 ​​cycles; 71-73℃ for 2-8 min.

[0079] In some embodiments of this disclosure, in step 1), the amplification program for the first round of PCR reaction is: 95°C for 2-5 min; 98°C for 15-25 s; 60°C for 3-5 min, 23-25 ​​cycles; 72°C for 4-6 min.

[0080] In some embodiments of this disclosure, in step 1), the amplification program for the first round of PCR reaction is: 95°C for 3 min 30 s; 98°C for 20 s, 60°C for 4 min, 24 cycles; 72°C for 5 min.

[0081] In step 3), the sequencing primers can be any suitable sequencing primers known in the art, preferably primers suitable for nanopore sequencing platforms. In some embodiments of this disclosure, in step 3), the sequencing primers are sequences as shown in any of SEQ ID NO:101-SEQ ID NO:108 and sequences as shown in any of SEQ ID NO:109-SEQ ID NO:116. The sequences of SEQ ID NO:101-SEQ ID NO:116 are shown in the table below:

[0082]

[0083] In some embodiments of this disclosure, in step 3), the amplification program for the second round of PCR reaction is as follows: 92-98℃ for 1-10 min; 97-99℃ for 10 s-1 min; 56-60℃ for 30 s-1 min-30 s; 70-74℃ for 10 s-1 min; 8-10 cycles; 70-75℃ for 1-10 min.

[0084] In some embodiments of this disclosure, in step 3), the amplification program for the second round of PCR reaction is as follows: 93-97℃ for 2-5 min; 97-99℃ for 10-30 s; 57-59℃ for 40-80 s; 71-73℃ for 10-50 s; 8-10 cycles; 70-74℃ for 2-8 min.

[0085] In some embodiments of this disclosure, in step 3), the amplification program for the second round of PCR reaction is as follows: 94-96℃ for 2-5 min; 97.5-98.5℃ for 10-30 s; 57.5-58.5℃ for 50-70 s; 71.5-72.5℃ for 20-40 s; 8-10 cycles; 71-73℃ for 2-8 min.

[0086] In some embodiments of this disclosure, in step 3), the amplification program for the second round of PCR reaction is as follows: 95°C for 2-5 min; 98°C for 15-25 s; 58°C for 55-65 s; 72°C for 25-35 s; 8-10 cycles; 72°C for 4-6 min.

[0087] In some embodiments of this disclosure, in step 3), the amplification program for the second round of PCR reaction is: 95°C for 3 min 30 s; 98°C for 20 s, 58°C for 1 min, 72°C for 30 s, for 9 cycles; 72°C for 5 min.

[0088] In some embodiments of this disclosure, step 5) can be performed. End repair can correct the sticky ends of a DNA fragment into blunt ends, and then an adenine (A) tail can be added to the 3' end to facilitate subsequent adapter ligation. Methods for end repair are well known to those skilled in the art and can be selected according to actual needs. In some embodiments of this disclosure, step 5) can be omitted.

[0089] In step 6), the sequencing adapter can be any sequencing adapter suitable for high-throughput sequencing platforms, preferably any sequencing adapter suitable for nanopore sequencing platforms, such as, but not limited to, the adapters described in Chinese patent applications CN202111398053.X and CN202210111742.6.

[0090] In step 6), the DNA fragment can be ligated to the sequencing adapter using any method known in the art. In some embodiments of this disclosure, in step 6), the ligation is performed using enzymatic ligation or chemical ligation. In some embodiments of this disclosure, in step 6), the ligation is performed using enzymatic ligation, and step 7) is then performed. In some embodiments of this disclosure, in step 6), the ligation is performed using chemical ligation, and step 7) is not performed.

[0091] The purification in steps 2) and / or 4) and / or 5) and / or 7) can be performed using any method known in the art for purifying DNA fragments. In some embodiments of this disclosure, the purification in steps 2) and / or 4) and / or 7) is performed using magnetic beads.

[0092] In some embodiments of this disclosure, in step 8), sequencing is performed using a high-throughput sequencing platform. The high-throughput sequencing platform can be any platform capable of sequencing the products of step 6) or step 7). In some embodiments of this disclosure, the high-throughput sequencing platform includes single-molecule real-time sequencing platforms and nanopore sequencing platforms.

[0093] The various embodiments and preferences described above with respect to the primer set of this disclosure can be combined with each other (as long as they are not inherently contradictory to each other), and are equally applicable to the kits, uses and detection methods of this disclosure (as long as they are not inherently contradictory to each other), and vice versa. All embodiments formed by such combinations are considered part of this application disclosure.

[0094] The technical solutions of this disclosure will be illustrated more clearly and explicitly below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this disclosure. The scope of protection of this disclosure is defined only by the claims.

[0095] Example

[0096] Library Structure

[0097] Sequencing library construction process as follows Figure 1As shown, after the target fragment undergoes a first round of PCR amplification and purification, and a second round of PCR amplification and purification, a library without sequencing adapters is obtained after PCR amplification, which can be used for quality control. Then, by adding sequencing adapters, a library with added sequencing adapters is obtained for sequencing. Since the principle of nanopore sequencing is mainly based on the motor proteins on the sequencing adapters controlling the base sequence to pass through the nanopore in a certain pattern, causing changes in the electrical signal, the corresponding base sequence can be obtained by analyzing the changes in the electrical signal. The sequencing signal is generated when the motor proteins begin to function; therefore, the base sequence at the 3' end of the motor proteins, the PCR primer sequence, and the sequencing adapter sequence at the 3' end of the library will all be sequenced. Therefore, in addition to the amplified target DNA fragment, this sequencing method will also detect the second-round PCR primer sequence and part of the sequencing adapter sequence. Based on the designed interval length and primer length, the final data effectiveness rate, i.e., the amplified interval length divided by the total sequencing length, should be approximately 51%.

[0098] Primers and reagents

[0099] The specific information and sources of the primers and reagents used in the examples are as follows:

[0100]

[0101]

[0102] General experimental procedure

[0103] Step 1) First round of PCR reaction

[0104] The system for the first round of PCR reaction is as follows:

[0105]

[0106]

[0107] [1] The concentration of gDNA was obtained from the quantification results of Qubit (Life Technologies); when using blood card samples, the blood card size was 1 mm, 1-2 wells / reaction tube, and direct amplification was performed.

[0108] [2] The primer set consists of primers with the sequences shown in SEQ ID NO:1-100 as shown in the table below.

[0109]

[0110]

[0111] Step 2) Purification

[0112] 1. Add 27 μL of room temperature equilibrated DNA clean beads to 30 μL of PCR product (i.e., the product from step 1), and gently mix 20 times with a pipette.

[0113] 2. After incubating at room temperature for 5 minutes, place the PCR tube on a DynaMag-96Side magnetic rack for 3 minutes;

[0114] 3. Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, and gently pipette and mix 20 times.

[0115] 4. After incubating at room temperature for 5 minutes, place the PCR tube on a DynaMag-96Side magnetic rack for 3 minutes;

[0116] 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 seconds;

[0117] 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 seconds and then completely remove the supernatant. Use a 10 μL pipette to remove any residual ethanol solution at the bottom.

[0118] 7. Let stand at room temperature for 3 minutes to allow the residual ethanol to evaporate completely;

[0119] 8. Remove the PCR tube from the magnetic rack, add 24 μL of water without nuclease, gently pipette to resuspend the magnetic beads, avoiding the formation of air bubbles, and let stand at room temperature for 2 minutes;

[0120] 9. Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0121] 10. Use a pipette to draw 13.5 μL of supernatant and transfer it to a new 200 μL PCR tube. The supernatant in the tube is the purified first PCR reaction product.

[0122] Step 3) Second round of PCR reaction

[0123] 1. Reaction system

[0124] reagents Volume (μL) The product of step 2) 13.5 Enhancer buffer M 2.5 <![CDATA[ddH2O]]> 2 SEQ ID NO:101(10μM) 1 SEQ ID NO:109(10μM) 1 Polymerase Mix 10

[0125] 2. Reaction conditions

[0126] Running the PCR instrument program:

[0127]

[0128] Step 4) Purification

[0129] 1. Add 27 μL of room temperature equilibrated DNA cleanbeads to 30 μL of PCR reaction system (i.e., the product of step 3), and gently mix 20 times with a pipette.

[0130] 2. After incubating at room temperature for 5 minutes, place the PCR tube on a DynaMag-96Side magnetic rack for 3 minutes;

[0131] 3. Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, and gently pipette and mix 20 times.

[0132] 4. After incubating at room temperature for 5 minutes, place the PCR tube on a DynaMag-96Side magnetic rack for 3 minutes;

[0133] 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 seconds;

[0134] 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 seconds and then completely remove the supernatant. Use a 10 μL pipette to remove any residual ethanol solution at the bottom.

[0135] 7. Let stand at room temperature for 3 minutes to allow the residual ethanol to evaporate completely;

[0136] 8. Remove the centrifuge tube from the magnetic rack, add 24 μL of water without nuclease, gently pipette and mix 20 times, resuspend the magnetic beads to avoid generating air bubbles, and let stand at room temperature for 2 minutes;

[0137] 9. Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0138] 10. Use a pipette to draw 20 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the purified second PCR reaction product, i.e., the library without sequencing adapters.

[0139] Quantitative

[0140] Take 1 μL of the product from step 4) (i.e., the library without sequencing adapters), and use... The concentration was determined using a 3.0 Fluorometer (Qubit dsDNA HS Assay Kit).

[0141] Quality Inspection

[0142] Take 1 μL of the product from step 4 (i.e., the library without sequencing adapters) and use the Qsep100 fully automated nucleic acid and protein analysis system to measure the fragment length and purity. The target fragment distribution range of about 280-420 bp is considered acceptable.

[0143] Step 5) End repair and purification

[0144] 1. Take 300 fmol of the above amplification product and prepare the terminal repair reaction according to the following system:

[0145] reagents Volume (μL) The product of step 4) y water 47-y DNA Repair Buffer (DRB) 7 Terminal repair enzyme (EPM) 3 DNA Repair Enzymes (DRM) 3

[0146] 2. Gently tap the tube wall to mix, centrifuge briefly, then place in a PCR instrument. Incubate at 30°C for 10 min, then at 65°C for 10 min (with the hot lid set to 75°C), and finally store at 4°C.

[0147] 3. Purification of end-repair products:

[0148] (1) Vortex the room-temperature equilibrated DNA Clean Beads for 30 seconds to mix them thoroughly.

[0149] (2) Add 60 μL of DNA Clean Beads (1:1 ratio) to the PCR tube reaction product, gently tap the tube wall to mix, and let stand at room temperature for 5 min.

[0150] (3) Place the centrifuge tube on the magnetic rack and wait for the solution to become clear (about 2-3 minutes), then remove the supernatant.

[0151] (4) Add 200 μL of freshly prepared 80% ethanol, wait 30 seconds, and then aspirate the supernatant.

[0152] (5) Repeat step (4) once.

[0153] ⑹ Completely remove any remaining liquid from the tube, open the cap and let it stand until the surface of the magnetic bead is no longer shiny (3-5 minutes).

[0154] (7) Add 63 μL of nuclease-free water to resuspend the magnetic beads and let stand at room temperature for 3 min.

[0155] (8) Place the PCR tube on a magnetic rack and wait for the magnetic beads to be completely adsorbed before transferring 60 μL of supernatant into a new 1.5 mL centrifuge tube.

[0156] Step 6) Connect the sequencing adapter

[0157] Prepare the linkage reaction according to the following system:

[0158] reagents Volume (μL) The product of step 5) 60 Sequencing adapter (SAC) 5 4x Ligation Buffer (LRB) 25 DNA ligase (DLE) 10

[0159] Gently tap the tube wall to mix, then centrifuge briefly and let stand at room temperature for 10 minutes.

[0160] Step 7) Purification

[0161] (1) Add 40 μL (1:0.4 ratio) of magnetic beads that have been equilibrated at room temperature to the product of step 6), gently tap the tube wall to mix, and let stand at room temperature for 5 min.

[0162] (2) Place the centrifuge tube on the magnetic rack and wait for the magnetic beads to be completely attracted before removing the supernatant.

[0163] (3) Remove the centrifuge tube from the magnetic rack, add 200 μL of short fragment washing buffer (SWB) equilibrated at room temperature, gently tap the tube wall to resuspend the magnetic beads, and then place the centrifuge tube back on the magnetic rack. After the magnetic beads are completely adsorbed, remove the supernatant.

[0164] (4) Repeat the above steps once.

[0165] (5) Aspirate as much residual liquid as possible from the centrifuge tube, then resuspend the magnetic beads in 15 μL of elution buffer (AEB) and elute for 5 min at room temperature.

[0166] (6) Place the centrifuge tube on the magnetic rack and wait for the magnetic beads to be completely attracted before transferring the supernatant to a new centrifuge tube.

[0167] Quantitative

[0168] Take 1 μL of the product from step 7) (i.e., the library with added sequencing adapters), and use... Library concentration was determined using a 3.0 Fluorometer (Qubit dsDNA HS Assay Kit).

[0169] Step 8) Sequencing

[0170] A library containing 80 fmol of sequencing adapters was sequenced on the QNome-3841 platform to obtain real-time sequencing results for all 48 loci in the mixed sample, followed by genotyping. The specific sequencing procedures and steps are as follows:

[0171] 1. Thaw the PRA (Potential Connector), SRB (Sequencing Reaction Buffer), and PRB at room temperature;

[0172] 2. Vortex or invert the container to mix the PRA, SRB, and PRB, then briefly centrifuge and set aside.

[0173] 3. Take 20 μL of the thawed conduction linker (PRA) and add it to 980 μL of conduction buffer (PRB), vortex to mix, and then centrifuge briefly;

[0174] 4. Add 200 μL of the mixture obtained in step 3 to the sequencing chip (avoid introducing air bubbles) and let stand for 15 min;

[0175] 5. Take a new centrifuge tube and mix according to the table below:

[0176] reagents Volume (μL) Sequencing reaction buffer (SRB) 100 The product of step 7) 80 fmol Water without nuclease Up to 200 μL

[0177] 6. Add 200 μL of the mixed sample obtained in step 5 into the sequencing chip, let it stand for 15 minutes, and then prepare for sequencing.

[0178] Example 1

[0179] Following the steps in the "General Experimental Procedure," in step 1), the following PCR reaction procedure is used:

[0180]

[0181]

[0182] All primers in the primer set are stored at the same concentration of 1 μM. The total volume of the 100 primers in the table below is 5 μL, and the volume ratios and molar ratios are as follows:

[0183]

[0184]

[0185]

[0186] The sample information and the library information obtained according to Example 1 without sequencing adapters are shown below. The male saliva, female saliva, and blood smear samples were obtained from employees of Beijing Qitan Technology Co., Ltd. The saliva gDNA was extracted using the Tiangen Magnetic Bead Extraction Kit DP705-01, and the blood smear was directly amplified without extraction.

[0187]

[0188] Libraries without sequencing adapters were quality checked using the Qsep100 fully automated nucleic acid and protein analysis system. The system revealed numerous non-specifically amplified short fragments in library D02 (a 1 ng blood mixture sample) and libraries D03, D04, and D05 (saliva samples), which could affect the final effective fragment concentration. Fragment distribution results are shown below. Figure 2 As shown in the figure. Furthermore, the library concentrations obtained from D02 / D03 / D04 / D05 were relatively low; the average library concentration obtained from samples with a starting amount of 1 ng of gDNA was 2.54 ng / μL. The average library concentration obtained from a 1 mm blood smear sample was 17.85 ng / μL. Further adjustments to the experiment are needed to enhance amplification specificity and the output library concentration.

[0189] The data quality control analysis results of the above libraries after ligation with sequencing adapters and sequencing analysis are shown in the table below. Except for female samples (female sample D05 lacks a Y chromosome, therefore it cannot cover the locus region on the Y chromosome, resulting in lower coverage than other samples), the primer interval coverage of other male samples was almost 100%, indicating that the primers can cover the target region well. Considering only non-saliva samples D01 / D02 / D03 / D06 / D07 (saliva samples may have degradation and other factors leading to different DNA quality states, which is not conducive to comparison of multiple experiments and is only used as a reference), the average primer interval data effectiveness was 44.91%, slightly worse than expected.

[0190]

[0191] Furthermore, using the above data, the depth of each sequencing site is divided by the average depth of each sample to obtain the normalized depth of each site in each sample = original depth of each site / average depth of each sample, as shown in the table below. Figure 3 As shown, the differences in depth at different sites fluctuate to some extent. It is necessary to control the range of fluctuation as much as possible to improve data utilization and ensure the accuracy of analysis results.

[0192]

[0193]

[0194] After normalization, for non-female samples D01-D04 and D06-D07, the normalized average depth of autosomal STRs was calculated to be 1.15, and the normalized average depth of sex chromosome STRs and loci was 0.65. That is, the ratio of autosomal locus depth to sex chromosome locus depth is 1.76:1, which is somewhat different from the expected ratio of 2:1. The autosomal depth is slightly lower, and further optimization of the depth of some loci is needed to make the ratio closer to the expectation.

[0195] The results of Example 1 show that it is necessary to continue to improve library concentration and amplification specificity, to make the depth of different types of sites reach the expected range as much as possible, to control the difference in depth of different sites, to improve data efficiency, and to improve the accuracy of sequencing analysis results.

[0196] Example 2:

[0197] Following the steps in the "General Experimental Procedure," in step 1), the following PCR reaction procedure is used:

[0198]

[0199] All primers in the primer set were stored at the same concentration of 1 μM. The total volume of the 100 primers in the table below is 5 μL. To optimize the final sequencing depth at each site to achieve the expected range, the volume ratio and molar ratio were adjusted as follows:

[0200]

[0201]

[0202]

[0203] The sample and the library information without sequencing adapters obtained according to Example 2 are shown below. The sample source and the extraction and / or amplification of gDNA are the same as described in Example 1:

[0204]

[0205] Libraries without sequencing adapters were quality checked using the Qsep100 fully automated nucleic acid and protein analysis system. The system revealed a certain proportion of non-specifically amplified short fragments in 1 ng blood mixture and saliva sample libraries F08 / F09 / F10. Fragment distribution results are shown below. Figure 4 As shown. The average library concentration obtained reached 5.9 ng / μL, which was 2.32 times higher than that of Example 1, but the concentration was still low; the average library concentration of blood smear samples F11 / F12 also reached 45.2 ng / μL, which was 2.53 times higher than that of Example 1.

[0206] After ligating the above libraries with sequencing adapters and performing sequencing analysis, the data quality control analysis results are shown in the table below. It can be seen that the data efficacy of primer intervals for non-saliva samples F07 / F08 / F11 / F12 has been slightly improved, averaging about 46.11%. However, the primer interval coverage of male saliva sample F09 is poor (corresponding to the highest proportion of short fragments and the worst data efficacy, which may be related to sample quality or accidental factors in experimental operation).

[0207]

[0208] Furthermore, using the above data, the depth of each sequencing site is divided by the average depth of each sample to obtain the normalized depth of each site in each sample = original depth of each site / average depth of each sample, as shown in the table below. Figure 5 As shown, the depth differences at different sites are improved compared to Example 1, but some fluctuations still exist. It is necessary to control the range of fluctuations as much as possible to improve data efficiency and ensure the accuracy of analysis.

[0209]

[0210]

[0211] After normalization, for non-female samples F07-F09 and F11-F12, the normalized average depth of autosomal STRs was calculated to be 2.66, and the normalized average depth of sex chromosome STRs and loci was 1.17. That is, the ratio of autosomal locus depth to sex chromosome locus depth is 2.28:1, which is close to the expected ratio of 2:1. Further optimization of the depth of some loci is needed to make the ratio closer to the expectation.

[0212] The results of Example 2 show that although there is an improvement compared to Example 1, it is still necessary to further increase the library concentration and amplification specificity, make the depth of different types of sites reach the expected range as much as possible, control the difference in depth of different sites, improve data efficiency, and improve the accuracy of sequencing analysis results.

[0213] Example 3:

[0214] Following the steps in the "General Experimental Procedure," in step 1), the following PCR reaction procedure is used:

[0215]

[0216] All primers in the primer set are stored at the same concentration of 1 μM. The total volume of the 100 primers in the table below is 5 μL, and the volume ratios and molar ratios are as follows:

[0217]

[0218]

[0219]

[0220] The sample and the library information without sequencing adapters obtained according to Example 3 are shown below. The sample source and the extraction and / or amplification of gDNA are the same as described in Example 1:

[0221]

[0222]

[0223] Based on the concentration values ​​of the un-sequencing adapter libraries measured by Qubit and the fragment length distribution detected by Qsep100, it can be seen that the A02 / A03 / A04 library concentration in 1 ng blood mixture and saliva samples increased to an average of 13.91 ng / μL, a 5.47-fold increase compared to Example 1. The A05 / A06 library concentration in 1 mm blood smear samples increased to an average of 57.2 ng / μL, a 3.2-fold increase compared to Example 1. The Qsep100 detection results show that the proportion of non-specifically amplified short fragments from the A02 / A03 / A04 samples starting at 1 ng decreased significantly compared to Examples 1 and 2. The fragment distribution results are as follows. Figure 6 As shown.

[0224] The sequencing data quality control analysis results after the above library was ligated with sequencing adapters and sequenced are shown below. It can be seen that the efficiency of non-saliva sample A01 / A02 / A05 / A05 data has been improved, with an average of 48.3%, which is more than 3 percentage points higher than Example 1.

[0225] Document Number Data volume after quality control (Mb) Total number of segments read Data efficiency Average depth of primer interval Primer region coverage A01 289.52 664635 48.62％ 11098.78 100.00％ A02 439 968065 49.19％ 17026.76 100.00％ A03 423.48 921471 48.22％ 16100.82 97.80％ A04 342.32 747937 48.35％ 13048.88 71.45％ A05 318.02 716682 47.14％ 11820.66 100.00％ A06 213.25 479694 48.30％ 8121.53 100.00％

[0226] Furthermore, using the above data, the depth of each sequencing site is divided by the average depth of each sample to obtain the normalized depth of each site in each sample = original depth of each site / average depth of each sample, as shown in the table below. Figure 7 As shown, the depth differences at different sites are improved compared to Example 1, but some fluctuations still exist. It is necessary to control the range of fluctuations as much as possible to improve data efficiency and ensure the accuracy of analysis results.

[0227]

[0228]

[0229] After normalization, for non-female samples A01-A03 and A05-A06, the normalized average depth of autosomal STRs was calculated to be 1.51, and the normalized average depth of sex chromosome STRs and loci was 0.53. That is, the autosomal locus depth: sex chromosome locus depth = 2.85:1, which is somewhat different from the designed ratio of 2:1. The autosomal depth is too high, and the depth of some loci needs to be further optimized to make the ratio closer to the expectation.

[0230] The results of Example 3 show that the primer specificity and library concentration are good, but it is necessary to make the depth of different types of sites reach the expected range as much as possible, control the difference in depth of different sites, improve the data efficiency, and ensure the accuracy of sequencing analysis results.

[0231] Example 4:

[0232] Follow the steps in the "General Experimental Procedure", wherein in step 1), the same PCR reaction procedure as in Example 3 is used.

[0233] All primers in the primer set are stored at the same concentration of 1 μM. The total volume of the 100 primers in the table below is 5 μL, and the volume ratios and molar ratios are as follows:

[0234]

[0235]

[0236]

[0237] The sample and the library information without sequencing adapters obtained according to Example 4 are shown below. The positive standard is a 2800M sample purchased from Promega. The sample source and the extraction and / or amplification of gDNA are the same as described in Example 1.

[0238]

[0239] The concentration values ​​of the libraries without sequencing adapters were measured using Qubit and quality controlled using the Qsep100 fully automated nucleic acid and protein analysis system. The results showed that the average library concentration of 1 ng samples of non-positive standards (B02 / B03 / B04) was 11.11 ng / μL, similar to Example 3. The average library concentration of blood smear samples (B05 / B06) was 68.2 ng / μL, slightly better than Example 3. The concentration of positive standard libraries starting at 1 ng exceeded 40 ng / μL. Fragment distribution showed that the non-specific short fragments of 1 ng samples of non-positive standards (B02 / B03 / B04) were significantly lower than in Examples 1 and 2, similar to Example 3. The overall fragment distribution results are as follows: Figure 8 As shown.

[0240] After the above library was ligated with sequencing adapters and sequenced, the sequencing data quality control analysis results are shown below. All data indicators performed well, and the effective rate of non-saliva sample data reached an average of 50.15%, which is nearly 6 percentage points higher than that of Example 1.

[0241] Document Number Data volume after quality control Total number of segments read Primer interval data efficiency Average depth of primer interval Primer region coverage B01 230.7(1) 498096 50.74％ 9229.74 100.00％ B02 395 866289 51.69％ 16097.18 100.00％ B03 224.67 526135 49.76％ 8814.44 100.00％ B04 211.09 483453 47.47％ 7900.56 53.77％ B05 210.6 463857 51.02％ 8471.22 100.00％ B06 674.57 1475745 50.20％ 26699.44 100.00％ B07 445.95 983316 49.73％ 17486.47 100.00％ B08 1016.82 2291896 51.93％ 41637.11 100.00％ B09 901.3 1996020 48.02％ 34127.51 100.00％ B10 279.87 590650 47.89％ 10568.2 100.00％

[0242] For the above data, the depth of each site obtained from sequencing is divided by the average depth of each sample to obtain the normalized depth of each site in each sample = original depth of each site / average depth of each sample, as shown in the table below. Figure 9 As shown, the difference in depth at different sites has been significantly improved, which is in line with expectations and helps to obtain higher data efficiency and ensure the accuracy of data analysis.

[0243]

[0244]

[0245] The normalized depth distribution map is shown below. The ratio of autosomal locus depth to sex chromosome locus depth basically meets the designed requirement of 2:1. After normalization, for non-female samples B01-B03 and B05-B10, the normalized average depth of autosomal STRs is calculated to be 1.39, and the normalized average depth of sex chromosome STRs and loci is 0.64, that is, the ratio of autosomal locus depth to sex chromosome locus depth is 2.17:1, which is close to the expected ratio distribution, and the aforementioned quality control data show good performance.

[0246] The STR loci involved in the 2800M positive standard samples were genotyped with the common STR loci of the 48 loci designed in this patent. All loci yielded consistent results with the standard (the order of the numbers in the genotyping results is irrelevant), as shown in the table below:

[0247]

[0248]

[0249] The results of Example 4 show that the primer specificity and library concentration are good, the depth of different types of sites reaches the expected range, the difference in depth between different sites is small, the data validity is high, and the accuracy of sequencing analysis results is high.

Claims

1. A primer set comprising primers having sequences as shown in SEQ ID NO: 1-SEQ ID NO: 100, in, In the primer set, the molar ratio of each primer is: 。 2. The primer set as described in claim 1, wherein, In the primer set, the molar ratio of each primer is: 。 3. The primer set as described in claim 1, wherein, In the primer set, the molar ratio of each primer is: 。 4. The primer set as claimed in claim 1, wherein, In the primer set, the molar ratio of each primer is: 。 5. The primer set as claimed in claim 1, wherein, In the primer set, the molar ratio of each primer is: 。 6. A detection kit comprising a primer set as described in any one of claims 1-5.

7. The use of the primer set as described in any one of claims 1-5 or the detection kit as described in claim 6 in human individual identification and / or forensic DNA database construction and / or suspect pedigree investigation and / or kinship identification and / or identification of degraded samples.

8. A detection method, the detection method comprising the following steps: Step 1) Perform a first round of PCR reaction on the sample to be tested using the primer set as described in any one of claims 1-5; Step 2) Perform a second round of PCR reaction on the product of step 1) using sequencing primers; Step 3) Ligate the product from step 2) to the sequencing adapter; Step 4) Sequencing the product from step 3).

9. The detection method according to claim 8, wherein in step 1), the amplification program of the first round of PCR reaction is: 92-98℃ for 1-10 min; 97-99℃ for 10 s-1 min; 58-62℃ for 1-10 min; 23-25 ​​cycles; 70-75℃ for 1-10 min.

10. The detection method according to claim 8, wherein in step 1), the amplification program of the first round of PCR reaction is: 93-97℃ for 2-5 min; 97-99℃ for 10-30 s; 59-61℃ for 2-6 min; 23-25 ​​cycles; 70-74℃ for 2-8 min.

11. The detection method according to claim 8, wherein in step 1), the amplification program of the first round of PCR reaction is: 94-96℃ for 2-5 min; 97.5-98.5℃ for 10-30 s; 59.5-60.5℃ for 2-6 min; 23-25 ​​cycles; 71-73℃ for 2-8 min.

12. The detection method according to claim 8, wherein in step 1), the amplification program of the first round of PCR reaction is: 95℃ for 2-5 min; 98℃ for 15-25 s; 60℃ for 3-5 min, 23-25 ​​cycles; 72℃ for 4-6 min.

13. The detection method according to claim 8, wherein in step 1), the amplification program of the first round of PCR reaction is: 95℃ for 3 min 30 s; 98℃ for 20 s, 60℃ for 4 min, 24 cycles; 72℃ for 5 min.

14. The detection method according to any one of claims 8-13, wherein in step 2), the sequencing primers are sequences shown in any one of SEQ ID NO: 101-SEQ ID NO: 108 and sequences shown in any one of SEQ ID NO: 109-SEQ ID NO:

116.

15. The detection method according to any one of claims 8-13, wherein in step 2), the amplification program of the second round of PCR reaction is: 92-98℃ for 1-10 min; 97-99℃ for 10 s-1 min; 56-60℃ for 30 s-1 min-30 s; 70-74℃ for 10 s-1 min, 8-10 cycles; 70-75℃ for 1-10 min.

16. The detection method according to any one of claims 8-13, wherein in step 2), the amplification program of the second round of PCR reaction is: 93-97℃ for 2-5 min; 97-99℃ for 10-30 s; 57-59℃ for 40-80 s; 71-73℃ for 10-50 s; 8-10 cycles; 70-74℃ for 2-8 min.

17. The detection method according to any one of claims 8-13, wherein in step 2), the amplification program of the second round of PCR reaction is: 94-96℃ for 2-5 min; 97.5-98.5℃ for 10-30 s, 57.5-58.5℃ for 50-70 s, 71.5-72.5℃ for 20-40 s, 8-10 cycles; 71-73℃ for 2-8 min.

18. The detection method according to any one of claims 8-13, wherein in step 2), the amplification program of the second round of PCR reaction is: 95℃ for 2-5 min; 98℃ for 15-25 s, 58℃ for 55-65 s, 72℃ for 25-35 s, 8-10 cycles; 72℃ for 4-6 min.

19. The detection method according to any one of claims 8-13, wherein in step 2), the amplification program of the second round of PCR reaction is: 95℃ for 3 min 30 s; 98℃ for 20 s, 58℃ for 1 min, 72℃ for 30 s, 9 cycles; 72℃ for 5 min.

20. The detection method according to any one of claims 8-13, wherein in step 3), the ligation is performed using an enzymatic ligation method or a chemical ligation method.

21. The detection method according to any one of claims 8-13, wherein the detection method further comprises: The products of step 1) and / or step 2) and / or step 3) are purified; and / or End repair is performed on the product of step 2).

22. The detection method of claim 21, wherein the purification in step 1) and / or step 2) and / or step 3) is performed using magnetic beads.

23. The detection method according to any one of claims 8-13, wherein in step 4), sequencing is performed using a high-throughput sequencing platform.

24. The detection method as described in claim 23, wherein the high-throughput sequencing platform includes a single-molecule real-time sequencing platform and a nanopore sequencing platform.

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