High-throughput sequencing composite amplification system for STR (short tandem repeat) fine typing as well as application and product of high-throughput sequencing composite amplification system

Abstract

The invention belongs to the technical field of forensic medicine, and particularly relates to a high-throughput sequencing composite amplification system for STR fine typing and application and a product of the high-throughput sequencing composite amplification system. The composite amplification system provided by the invention comprises a specific amplification primer combination for simultaneously amplifying 65 autosomal loci, 57 Y chromosome loci, 31 X chromosome loci and Amel loci. The multiplex amplification system is more suitable for being applied to a high-throughput sequencing platform, due to the fact that the lengths of amplicons are close, the enrichment degree and the sequencing depth of all sites tend to be balanced, meanwhile, the proportion of primers for multiplex amplification is further reasonably matched, and the amplification balance of different sites is improved. Due to the fact that the balance of amplification of different sites is high, rich and credible sequencing results can be obtained on the premise that the sequencing data amount is small, the practical significance is that the advantage of low cost of high-throughput sequencing is greatly highlighted, and the cost for obtaining information of a single site is reduced through the design.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic medicine, and in particular relates to a high-throughput sequencing composite amplification system for STR fine typing and its application and products. Background Art

[0002] In the field of forensic evidence, typing tests for sequence polymorphism and length polymorphism of genetic material DNA play an important role in individual identification, paternity testing and even the construction of forensic DNA databases. Short tandem repeats (STRs), also known as microsatellites, refer to continuous repeating DNA units with a length of 2-6 base pairs (bp), which account for at least 3% of the human genome. STRs are highly polymorphic mainly due to the different number of core sequence repetitions. Since they are derived from the slippage-mismatch between DNA chains that are instantly separated during the DNA replication process, most of them are not affected by selection pressure. At present, STRs have been widely used in the field of forensic medicine and have become the "gold standard" for individual identification and paternity testing.

[0003] The second generation of DNA sequencing technology, also known as high-throughput sequencing technology, can rapidly sequence and analyze hundreds of thousands to millions of DNA molecules from hundreds or thousands of samples at a time at a low cost and with an accuracy of more than 99%. With the development and application of high-throughput sequencing technology, the number of STR loci that can be detected in a single experiment can now reach hundreds or even more, and SNP genetic markers can be detected simultaneously to obtain more genetic information.

[0004] However, the genetic information detected by the current individual identification system based on second-generation sequencing is not rich enough, and it is often necessary to combine multiple detection systems to meet the wide application requirements in the forensic field, which limits its application in case detection. In addition, since the existing high-throughput sequencing kits are less compatible with the PCR-CE-based kits, and the established population STR database is basically created based on the PCR-CE platform, it will cause obstacles in the unified comparison and recording of STR data obtained by the two methods. Summary of the invention

[0005] Based on the above-mentioned shortcomings, the present invention provides a high-throughput sequencing composite amplification system for STR refined typing and its application and product. The composite amplification system provided by the present invention includes a specific amplification primer combination for simultaneously amplifying 65 autosomal loci, 57 Y chromosome loci, 31 X chromosome loci and Amel loci. The composite amplification system is more suitable for application on a high-throughput sequencing platform. Since the length of the amplicon is close, the enrichment degree and sequencing depth of each site are more balanced, and the primer ratio of the composite amplification is further reasonably matched to improve the balance of amplification of different sites. Due to the high balance of amplification of different sites, rich and reliable sequencing results can be obtained under the premise of less sequencing data volume. The practical significance is that the low-cost advantage of high-throughput sequencing is greatly highlighted. Through the design of the present invention, the cost of obtaining single site information is reduced.

[0006] The technical solution of the present invention includes:

[0007] In a first aspect, the present invention provides a composite amplification system for high-throughput sequencing of STR refined typing, the composite amplification system comprising a specific amplification primer combination for simultaneously amplifying 65 autosomal loci, 57 Y chromosome loci, 31 X chromosome loci and Amel loci;

[0008] The 65 autosomal loci are D1S1627, D1S1656, D1S1677, D1GATA113, F13B, TPOX, D2S441, D2S1338, D2S1776, D3S1358, D3S1744, D3S3045, D3S4529, D4S2366, D4S2408, FGA, D5S818, CSF1PO, D5S2500, D5S2800, D6S474, D6S477, D6S1 017, SE33, D6S1043, F13A1, D7S820, D7S1517, D7S3048, D8S1132, D8S1179, LPL, D9S925, D9S1122, D10S1248, D10S1435, TH01, D11S2368, D11S4463, vWA, D12S391, D12ATA63, D13S317, D13S325, D14S608, D14S1434, Penta E, D15S659, FES, D16S539, D17S1290, D17S1301, D18S51, D18S535, D18S853, D18S1364, D19S253, D19S433, D20S482, D21S11, D21S1270, D21S2055, Penta D, D22GATA198B05, and D22S1045;

[0009] The 57 Y chromosome loci are DYS19, DYS385a, DYS385b, DYS388, DYS389 I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS443, DYS444, DYS446, DYS447, DYS448, DYS449, DYS456, DYS458, DYS460, DYS461, DYS481, DYS505, DYS508, DYS510, DYS518, DYS520, DYS522, DYS531, DYS533, DYS534, DYS535 YS546, DYS549, DYS552, DYS557, DYS570, DYS576, DYS587, DYS593, DYS596, DYS612, DYS617, DYS622, DYS626, DYS627, Composed of DYS630, DYS635, DYS643, DYS645, DYS713, DYF387S1a, DYF387S1b, DYF404S1a, DYF404S1b, Y-GATA-H4 and Y-GATA-A10;

[0010] The 31 X chromosome loci are composed of DXS10074, DXS10075, DXS10079, DXS101, DXS10101, DXS10103, DXS10134, DXS10135, DXS10148, DXS10159, DXS10162, DXS10164, DXS6789, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809, DXS6810, DXS7132, DXS7133, DXS7423, DXS7424, DXS8378, DXS981, DXS9902, DXS9907, GATA165B12, GATA172D05, GATA31E08 and HPRTB;

[0011] The Amel locus includes AmelX and AmelY.

[0012] Specifically, the forward primers of the 65 autosomal loci have nucleotide sequences as shown in SEQ ID NO.1-65, and the reverse primers have nucleotide sequences as shown in SEQ ID NO.66-130; or

[0013] The forward primers of the 57 Y chromosome loci have the nucleotide sequences shown in SEQ ID NOs.131-183, and the reverse primers have the nucleotide sequences shown in SEQ ID NOs.184-236; or

[0014] The forward primers for the 31 X chromosome loci have nucleotide sequences as shown in SEQ ID NOs.237-267, and the reverse primers have nucleotide sequences as shown in SEQ ID NOs.237-298; or

[0015] The Amel locus has a nucleotide sequence as shown in SEQ ID NO.299, and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.300.

[0016] Preferably, the forward primer and reverse primer information of the 65 autosomal loci are shown in Table 1, the forward primer and reverse primer information of the 57 Y chromosome loci are shown in Table 2; and the forward primer and reverse primer information of the 31 X chromosome loci are shown in Table 3.

[0017] Specifically, the amount of each primer added in the specific amplification primer combination is shown in Table 5.

[0018] Specifically, the working concentration of the specific amplification primer combination is 1-2 μM. The working concentration of the primers can be adjusted according to the application purpose.

[0019] In a second aspect, the present invention provides the use of the above-mentioned composite amplification system in the preparation of a STR refined typing kit.

[0020] In a third aspect, the present invention provides a kit for STR fine typing, wherein the kit comprises the above-mentioned composite amplification system.

[0021] Specifically, the kit also includes a PCR polymerase mixture, a PCR reaction enhancer, an Index linker sequence, and a PCR product purification reagent.

[0022] Preferably, the Index linker sequence includes IGT-I5 Index and IGT-I7 Index; the working concentrations of IGT-I5 Index and IGT-I7 Index are both 10 μM.

[0023] In a fourth aspect, the present invention provides a method for STR fine typing, wherein the method comprises using the above-mentioned composite amplification system or the above-mentioned kit.

[0024] Specifically, the method comprises the following steps:

[0025] S1, extracting total genomic DNA from samples;

[0026] S2, performing targeted amplification and gene library construction on the total genomic DNA in step S1;

[0027] S3, sequencing the gene library constructed in step S2;

[0028] S4. Process the sequencing data to obtain the test results.

[0029] Preferably, the sample in step S1 includes one or more of blood, blood spots, oral cells, semen, semen stains, bones, hair, saliva, saliva stains, sweat, and amniotic fluid containing fetal cells.

[0030] The beneficial effects of the present invention are:

[0031] The composite amplification system provided by the present invention comprises a specific amplification primer combination for simultaneously amplifying 65 autosomal loci, 57 Y chromosome loci, 31 X chromosome loci and Amel loci. The composite amplification system is more suitable for application on a high-throughput sequencing platform. Since the length of the amplicon is close, the enrichment degree and sequencing depth of each site are more balanced, and the primer ratio of the composite amplification is further reasonably matched to improve the balance of different site amplifications. Since the balance of different site amplifications is higher, abundant and reliable sequencing results can be obtained under the premise of less sequencing data volume, and the practical significance is that the low-cost advantage of high-throughput sequencing is greatly highlighted, and the cost of obtaining single site information is reduced through the design of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the flow chart of the STR locus multiplex amplification experiment.

[0033] Figure 2 This is a diagram of library fragment length analysis.

[0034] Figure 3 The results are species-specific. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention will be described in detail below. In order to avoid too much unnecessary details, the well-known structures or functions will not be described in detail in the following examples. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs.

[0036] Example 1 Site selection for STR multiplex amplification system

[0037] STR loci were selected as candidate sites for constructing the composite amplification system from multiple commercial kits based on PCR-CE methods and high-throughput sequencing methods, as well as previous scientific research and databases. The frequency of use of each locus in commercial kits was counted to ensure that the commonly used sites in all kits were included in the composite amplification system of this patent to ensure compatibility with existing population databases; at the same time, less commonly used loci and loci that were not subjected to high-throughput sequencing in previous data were taken into consideration and added to the composite amplification system of this patent to achieve "as much as possible" inclusion.

[0038] Information was retrieved through the following websites to collect information on each STR locus, including the STR locus name, the chromosome location of the core repeat region, the core repeat motif, and reported primer information, etc.:

[0039] http: / / trbase.nist.gov / index.html; http: / / genome-asia.ucsc.edu / index.html; http: / / genome-asia.ucsc.edu / index.html; http: / / xdb.qualitype.de / xdb / index.jsf; https: / / www.ncbi.nlm.nih.gov; https: / / www.isogg.org.

[0040] Example 2 Construction of STR locus composite amplification system

[0041] 1. Selection of STR loci in the multiplex amplification system

[0042] The selection of STR loci in the multiplex amplification system must comply with the following requirements: (1) STR has good polymorphism in the population; (2) STR is easy to detect, that is, the core sequence should not be too long, should meet the read length limit of high-throughput sequencing, and the core sequence should not be too complex, otherwise it will be difficult to analyze. (3) The core motif should not be too short or too long, and a locus with a 4 bp sequence as the main component and 3 bp and 5 bp motifs as the auxiliary should be selected.

[0043] 2. Primer construction of STR locus composite amplification system

[0044] Following the above selection criteria, the present invention selects and combines 154 STR loci as the sites included in the composite amplification system, including 65 autosomal loci, 57 Y chromosome loci, 31 X chromosome loci and 1 Amel locus (Amel locus is a gender determination locus, which exists in both men and women, including AmelX and AmelY, and is identified as 1 locus).

[0045] Primer 5 tool is used for primer design. Under the premise of ensuring primer specificity, the length of the amplicon is required to be kept balanced as much as possible to avoid the dominant amplification of the shorter amplicon, so as to achieve a better composite amplification efficiency; at the same time, it is necessary to ensure that the length of each amplicon is within the range of high-throughput sequencing read length. As we all know, the read length of high-throughput sequencing is short (150-400bp). The average amplicon length of the amplification of the present invention is about 280bp, which meets the technical requirements of high-throughput sequencing.

[0046] Table 1 is the primer combination of the autosomal locus in the composite amplification system of the present invention, Table 2 is the primer combination of the Y chromosome locus, Table 3 is the primer combination of the X chromosome locus, and Table 4 is the primer combination of the Amel locus.

[0047] Table 1 Primer combinations for autosomal loci

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] Table 2 Primer combinations for Y chromosome loci

[0054]

[0055]

[0056]

[0057]

[0058] Table 3 Primer combinations for chromosome X loci

[0059]

[0060] Table 4 Primer combinations of Amel locus

[0061]

[0062] 3. Construction of STR locus composite amplification system

[0063] In the composite amplification system, in order to avoid primer competition, inhibit non-specific amplification, reduce the formation of primer dimers, and ensure the balance of amplification of each locus, the working concentration of the composite amplification primer needs to be controlled within the range of 1 μM-2 μM. The initial concentrations of the primers of the autosomal locus, the Y chromosome locus, the X chromosome locus, and the Amel locus are consistent, and the primers are mixed according to the addition amount shown in Table 5 to obtain a specific amplification primer combination. Subsequently, the specific amplification primer combination is diluted to a working concentration (i.e., 1 μM-2 μM), which is referred to as a composite amplification system primer pool in the present invention.

[0064] Table 5 Specific amplification primer combination components

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Example 3 STR gene locus complex amplification system kit description

[0071] 1. Composite amplification kit of the present invention

[0072] The composite amplification kit of the present invention comprises the components described in Table 6:

[0073] Table 6 Components of the Multiplex Amplification Kit

[0074] Reagents use Primer pool T1 Mixed primer pool for multiplex amplification system IGT-EM808 Polymerase Mixture PCR polymerase mix PCR buffer Mix (1N) PCR reaction substrate Mix Enhancer buffer M PCR Enhancer YF buffer B Organic solvent cleaning agent (used in magnetic bead purification step) IGT-I5 Index (10 μM) Used for sample specific adapter ligation reaction (A5 end) IGT-I7 Index (10 μM) Specific adapter ligation reaction for samples (A7 end)

[0075] Among them, Enhancer buffer M and YF buffer B need to be stored at 4°C, and the other components need to be stored at -20°C. In addition, users need to prepare anhydrous ethanol, enzyme-free water, purified magnetic beads and other reagents as well as conventional experimental instruments and consumables. Figure 1 This is the process of STR locus multiplex amplification experiment.

[0076] The IGT-I7 Index and IGT-I5 Index sequences are shown in Table 7, and 1 μL is added to each sequence.

[0077] Table 7 IGT-I7 Index, IGT-I5 Index sequence

[0078]

[0079]

[0080]

[0081] 2. Library construction experimental process

[0082] 2.1 First round of multiplex PCR reaction

[0083] The first round of multiplex PCR reaction system and reaction conditions are shown in Tables 8-9.

[0084] Table 8 Multiplex PCR reaction system

[0085] Reagents Volume (μL) <![CDATA[ddH2O]]> 9-x Enhancer buffer NB (1N) 3.5 Enhancer buffer M 2.5 Primer pool T1 5 Genomic DNA x IGT-EM808 polymerase mixture 10

[0086] Table 9 Multiplex PCR reaction conditions

[0087]

[0088] To avoid affecting the effects of the reagents, the above reagents should be promptly packaged, properly protected, and avoid repeated freezing and thawing. The above operations should be performed on an ice box.

[0089] 2.2 Magnetic bead purification products

[0090] (1) Add 27 μL of AMPure XP beads equilibrated at room temperature to 30 μL of PCR product and mix gently by pipetting 20 times;

[0091] (2) After incubation at room temperature for 5 min, the PCR tube was placed on a magnetic stand for 3 min;

[0092] (3) Completely remove the supernatant, remove the PCR tube from the magnetic stand, add 50 μL YF buffer B to the tube, and mix gently by pipetting 20 times;

[0093] (4) After incubation at room temperature for 5 min, place the PCR tube on a magnetic stand for 3 min;

[0094] (5) Remove the supernatant, place the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution to the tube, and let it stand for 30 seconds. Note: The 80% ethanol solution must be freshly prepared.

[0095] (6) Remove the supernatant, place 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 (it is recommended to use a 10 μL pipette to remove the residual ethanol solution at the bottom);

[0096] (7) Let stand at room temperature for 3 min to allow the residual ethanol to evaporate completely;

[0097] (8) Remove the PCR tube from the magnetic stand, add 16 μL of Nuclease-free water, gently pipette to resuspend the magnetic beads to avoid bubbles, and let stand at room temperature for 2 min;

[0098] (9) Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0099] (10) Use a pipette to take 13.5 μL of the supernatant, which is the product of the composite PCR amplification, and add the product to the second round of linker sequence PCR reaction system.

[0100] 2.3 Second round of adapter sequence PCR reaction

[0101] The second round of linker sequence PCR reaction system and reaction conditions are shown in Tables 10-11.

[0102] Table 10 Adapter sequence PCR reaction system

[0103] Reagents Volume (μL) PCR product mixture 13.5 <![CDATA[ddH2O]]> 2 Enhancer buffer M 2.5 IGT-I5 Index(10μM) 1 IGT-I7 Index(10μM) 1 IGT-EM808polymerase mixture 10

[0104] Table 11 PCR reaction conditions for linker sequences

[0105]

[0106]

[0107] 2.4 Second round of magnetic bead purification products

[0108] (1) Add 27 μL of AMPure XP beads equilibrated at room temperature to 30 μL of PCR product and mix gently by pipetting 20 times;

[0109] (2) After incubation at room temperature for 5 min, the PCR tube was placed on a magnetic stand for 3 min;

[0110] (3) Completely remove the supernatant, remove the PCR tube from the magnetic stand, add 50 μL YF buffer B to the tube, and mix gently by pipetting 20 times;

[0111] (4) After incubation at room temperature for 5 min, place the PCR tube on a magnetic stand for 3 min;

[0112] (5) Remove the supernatant, place the PCR tube on the magnetic rack, add 180 μL of 80% ethanol solution to the tube, and let it stand for 30 seconds. Note: The 80% ethanol solution must be freshly prepared.

[0113] (6) Remove the supernatant, place 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 (it is recommended to use a 10 μL pipette to remove the residual ethanol solution at the bottom);

[0114] (7) Let stand at room temperature for 3 min to allow the residual ethanol to evaporate completely;

[0115] (8) Remove the PCR tube from the magnetic stand, add 23 μL of Nuclease-free water, gently pipette to resuspend the magnetic beads to avoid bubbles, and let stand at room temperature for 2 min;

[0116] (9) Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0117] (10) Use a pipette to aspirate 20 μL of the supernatant, which is the constructed composite amplification library. Transfer the library to a new PCR tube for proper storage and future use.

[0118] After the above amplification and purification steps, the final sample library that can be used for the machine is obtained. The library length fragment analysis is performed using the Qseq 400 fully automated nucleic acid protein analysis system. The results are as follows: Figure 2 .

[0119] The fragment length distribution of this library is relatively concentrated, and the signal peak appears in the range of 440-450bp.

[0120] Experimental Example 1 Determination of Template Amplification Capacity

[0121] Forensic female DNA standard 9947A (Origene, FD200005) and forensic male DNA standard 9948 (Origene, FD200006) were used as library construction DNA, and DNA input gradients (5ng, 2ng, 1ng, 0.5ng, 0.25ng, 0.125ng, 0.063ng, 0.032ng) were set respectively. The high-throughput sequencing library was constructed according to the method described in Example 3, and 2 repeated experiments were set for each concentration gradient of the two standard samples. After library quantification, machine operation, etc., the template amplification ability of the STR locus composite amplification system kit of the present invention was sequenced and data analyzed. The measurement results are shown in Tables 12 and 13.

[0122] Table 12 Detection rate of the 9948 standard product input gradient loci

[0123]

[0124] Table 13 Detection rate of the input gradient loci of standard 9947A

[0125]

[0126] In the table, "Au-STR" represents autosomal loci; "Y-STR" represents Y chromosome loci;

[0127] "X-STR" stands for the X chromosome locus. The measurement results show that the STR locus composite amplification system kit provided by the present invention still has a strong template amplification ability when the amount of DNA input for library construction is extremely low (as shown in the example, when 0.032ng). After sequencing and subsequent data analysis, when a low amount of template is used for library construction, it still has a relatively good detection rate. It should be noted that the composite amplification system of the present invention includes 154 loci, of which 1 Amel locus plays a role in gender recognition and has no polymorphism in the population. Therefore, in the analysis of the experimental example, only 153 STR loci are analyzed and counted.

[0128] Experimental Example 2 Determination of resistance to adverse PCR environment

[0129] 5 ng of forensic female DNA standard 9947A (Origene, FD200005) and 5 ng of forensic male DNA standard 9948 (Origene, FD200006) were used as the library construction DNA, and different concentration gradients of PCR inhibitors were added to the amplification system: ① humic acid (concentration gradient of 1000 ng / μL, 500 ng / μL, 200 ng / μL, 150 ng / μL, 100 ng / μL, 50 ng / μL, 20 ng / μL); ② tannic acid (concentration gradient of 500 ng / μL, 200 ng / μL, 150ng / μL, 100ng / μL, 50ng / μL, 20ng / μL, 10ng / μL); ③ heme (concentration gradient is 200μM, 150μM, 100μM, 50μM, 20μM, 10μM); ④ bilirubin (concentration gradient is 500μM, 200μM, 150μM, 100μM, 50μM, 20μM, 10μM) The resistance of the STR locus composite amplification system kit of the present invention to the adverse environment of PCR was investigated, and 2 repeated experiments were set for each of the 4 PCR inhibitor concentration gradients of each standard DNA. The resistance to humic acid is shown in Tables 14 and 15, the resistance to tannic acid is shown in Tables 16 and 17, the resistance to heme is shown in Tables 18 and 19, and the resistance to bilirubin is shown in Tables 20 and 21.

[0130] Table 14 Detection rate of humic acid concentration gradient loci of standard 9948

[0131] Humic acid concentration (ng / μL) 20 50 100 150 200 500 1000 Au-STR detection rate 100％ 100％ 100％ 100％ 93.84％ 38.46％ 0％ Y-STR detection rate 100％ 100％ 100％ 100％ 92.98％ 57.89％ 0％ X-STR detection rate 100％ 100％ 100％ 100％ 96.77％ 64.52％ 0％ 153 STR total detection rate 100％ 100％ 100％ 100％ 94.12％ 50.98％ 0％

[0132] Table 15 Detection rate of humic acid concentration gradient loci of standard 9947A

[0133] Humic acid concentration (ng / μL) 20 50 100 150 200 500 1000 Au-STR detection rate 100％ 100％ 98.46％ 98.46％ 98.46％ 23.08％ 3.07％ X-STR detection 100％ 100％ 100％ 100％ 100％ 41.94％ 16.13％ Total detection rate of 96 STRs 100％ 100％ 98.96％ 98.96％ 98.96％ 29.17％ 7.29％

[0134] The results in Tables 14 and 15 show that when the humic acid concentration in the amplification system reaches 200 ng / μL, an excellent detection rate can still be achieved when a small amount of DNA sample (5 ng) is used for library construction.

[0135] Table 16 Detection rate of tannic acid concentration gradient loci in standard 9948

[0136]

[0137] Table 17 Detection rate of tannic acid concentration gradient loci in standard 9947A

[0138]

[0139]

[0140] The results in Tables 16 and 17 show that when the tannic acid concentration in the amplification system reaches 200 ng / μL, an excellent detection rate can still be achieved when a small amount of DNA sample (5 ng) is used for library construction.

[0141] Table 18 Detection rate of heme concentration gradient loci of standard 9948

[0142] Heme concentration (μM) 10 20 50 100 150 200 Au-STR detection rate 100％ 100％ 96.92％ 79.36％ 21.54％ 0％ Y-STR detection rate 82.46％ 82.46％ 77.19％ 47.37％ 12.28％ 0％ X-STR detection rate 96.77％ 96.77％ 90.32％ 48.39％ 16.13％ 0％ 153 STR total detection rate 92.81％ 92.81％ 88.24％ 60.13％ 16.99％ 0％

[0143] Table 19 Detection rate of heme concentration gradient loci of standard 9947A

[0144] Heme concentration (μM) 10 20 50 100 150 200 Au-STR detection rate 100％ 95.38％ 95.38％ 84.61％ 52.31％ 15.38％ X-STR detection rate 100％ 100％ 100％ 93.55％ 25.81％ 16.13％ Total detection rate of 96 STRs 100％ 96.88％ 96.88％ 87.50％ 43.75％ 15.63％

[0145] The results in Tables 18 and 19 show that when the heme concentration in the amplification system reaches 100 μM, the number of loci detected is still considerable when a smaller amount of DNA sample (5 ng) is used to construct the library.

[0146] Table 20 Detection rate of bilirubin concentration gradient loci of standard 9948

[0147] Bilirubin concentration (μM) 10 20 50 100 150 200 500 Au-STR detection rate 96.92％ 93.85％ 92.31％ 90.77％ 95.38％ 87.69％ 76.92％ Y-STR detection rate 94.74％ 94.74％ 85.96％ 91.23％ 89.47％ 78.95％ 75.44％ X-STR detection rate 87.10％ 83.87％ 83.87％ 83.87％ 87.10％ 74.19％ 61.29％ 153 STR total detection rate 94.12％ 92.16％ 88.24％ 89.54％ 91.50％ 81.70％ 73.20％

[0148] Table 21 Detection rate of bilirubin concentration gradient loci of standard 9947A

[0149] Bilirubin concentration (μM) 10 20 50 100 150 200 500 Au-STR detection rate 95.38％ 95.38％ 93.85％ 83.07％ 92.31％ 87.69％ 58.46％ X-STR detection rate 83.87％ 77.42％ 70.97％ 77.42％ 77.42％ 74.19％ 38.71％ Total detection rate of 96 STRs 91.66％ 89.58％ 86.46％ 81.25％ 87.50％ 83.33％ 52.08％

[0150] The results in Tables 20 and 21 show that when the bilirubin concentration in the amplification system reaches 200 μM, a relatively good detection rate can still be achieved when a smaller amount of DNA sample (5 ng) is used for library construction.

[0151] The test results show that the STR gene locus composite amplification system kit provided by the present invention has strong resistance to PCR adverse environment. When a high concentration of the above PCR inhibitor exists in the composite amplification system, a relatively good detection rate can still be achieved.

[0152] Experimental Example 3 Species-specific assay

[0153] QIAampDNA Investigation Kit (50) (QIAGEN, ID: 56504) was used to extract DNA samples of cattle, sheep, rabbit, fish, dog, cat, pig, chicken, duck, mouse and human according to the instructions. Using DNA of different species as templates, the kit of the present invention was used to construct a high-throughput sequencing library with reference to the method described in Example 3. The total input volume of DNA of each sample was 20-30 ng. The library construction results were detected by 1% agarose gel electrophoresis. The species-specific test results are shown in Figure 3According to the gel image, a band appears at about 420 bp in human samples, which is consistent with the expected library length. This proves the population specificity of the composite amplification system.

[0154] After sequencing the libraries of each species and analyzing the data, the following Table 22 was obtained. This table shows the number of loci that can be detected in samples of each species when different minimum signal thresholds are used in data analysis. When the detection threshold is 30 reads, STR loci can be detected in all 10 animal samples; when the detection threshold is 60 reads, only 3 animal samples can detect STR loci; when the detection threshold is 100 reads, no loci are detected in all animal samples. The loci detected at low detection thresholds are caused by nonspecific amplification or signal noise. When performing normal sequencing result analysis, the detection threshold is set to 50-100 reads.

[0155] Table 22 Species-specific assay results

[0156]

[0157] From the above analysis results, it can be seen that this composite amplification system has strong species specificity. It is difficult to detect STR loci in the above ten common animals, while excellent detection results can be obtained for human samples at various detection thresholds.

[0158] Example 4 Forensic parameters

[0159] 106 unrelated individual samples (53 male samples and 53 female samples) were randomly recruited from the Han population in northern China, and their venous blood samples were drawn. Whole blood DNA was extracted using the QIAampDNA Investigation Kit (50) kit (QIAGEN, ID: 56504) according to the operating instructions. After high-throughput sequencing library construction, library quantification, pool mixing and other operations were performed with reference to the steps described in Example 3, high-throughput sequencing was performed using the Illumina platform. The sequencing files of each sample were analyzed for data analysis to obtain STR locus typing, and Matlab and Excel tools were used for data aggregation and statistics. Finally, each site was analyzed for forensic parameters based on STR length polymorphism and STR sequence polymorphism. The results are shown in Tables 23-25.

[0160] Table 23 Autosomal Forensic Parameters

[0161]

[0162]

[0163]

[0164] Table 24 Y chromosome forensic parameters

[0165]

[0166]

[0167] Table 25 X chromosome forensic parameters

[0168]

[0169]

[0170] The forensic parameter calculation method mentioned above mainly refers to the method concept provided in ① Technical Specifications for Forensic Identification (SF / Z JD0105010-2018) "Forensic Parameter Calculation Specifications for Autosomal STR Loci", and also refers to the ideas and methods provided in ② Technical Specifications for Forensic Identification (SF / Z JD0105012-2018) "Technical Specifications for Individual Identification". Due to the huge amount of calculation, the forensic parameter calculation was completed by software or online program: Powerstats was used to complete the forensic parameter calculation of autosomal STR, Arlequin was used to calculate the forensic parameter calculation of Y chromosome STR, and StatsXv2.0 was used to complete the forensic parameter calculation of X chromosome STR.

[0171] Calculated by forensic parameters, autosomal efficacy: cumulative individual identification probability: 1-4.62153E-70 (based on length polymorphism), 1-8.98372E-76 (based on sequence polymorphism); Y chromosome efficacy: cumulative individual identification probability: 1-6.65868E-31; X chromosome efficacy: cumulative individual identification probability: 1-1.22919E-20 (male samples), 1-1.4244E-34 (female samples).

[0172] When calculating the forensic parameters of autosomal STR, the individual identification ability is improved by about 514,000 times when considering sequence polymorphism compared to only considering length polymorphism. Through high-throughput technology and analysis methods, while paying attention to length polymorphism and sequence polymorphism, the evidentiary power of some sites is further enhanced, providing more biological genetic information, and highlighting the advantages of high-throughput sequencing technology.

[0173] The above values ​​far exceed the cumulative personal identification probability required by the current standards (required to be 1-1.0E-4). The composite amplification system of the patent of the present invention is fully capable of meeting the requirements of forensic identification practice.

[0174] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A composite amplification system for high-throughput sequencing of STR refined typing, characterized in that: The composite amplification system includes a specific amplification primer combination for simultaneously amplifying 65 autosomal loci, 57 Y chromosome loci, 31 X chromosome loci and Amel loci; The 65 autosomal loci are D1S1627, D1S1656, D1S1677, D1GATA113, F13B, TPOX, D2S441, D2S1338, D2S1776, D3S1358, D3S1744, D3S3045, D3S4529, D4S2366, D4S2408, FGA, D5S818, CSF1PO, D5S2500, D5S2800, D6S474, D6S477, D6S1 017, SE33, D6S1043, F13A1, D7S820, D7S1517, D7S3048, D8S1132, D8S1179, LPL, D9S925, D9S1122, D10S1248, D10S1435, TH01, D11S2368, D11S4463, vWA, D12S391, D12ATA63, D13S317, D13S325, D14S608, D14S1434, Penta E, D15S659, FES, D16S539, D17S1290, D17S1301, D18S51, D18S535, D18S853, D18S1364, D19S253, D19S433, D20S482, D21S11, D21S1270, D21S2055, Penta D, D22GATA198B05, and D22S1045; The 57 Y chromosome loci are DYS19, DYS385a, DYS385b, DYS388, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS443, DYS444, DYS446, DYS447, DYS448, DYS449, DYS456, DYS458, DYS460, DYS461, DYS481, DYS505, DYS508, DYS510, DYS518, DYS520, DYS522, DYS531, DYS533, DYS544 6. DYS549, DYS552, DYS557, DYS570, DYS576, DYS587, DYS593, DYS596, DYS612, DYS617, DYS622, DYS626, DYS627, DY Composed of S630, DYS635, DYS643, DYS645, DYS713, DYF387S1a, DYF387S1b, DYF404S1a, DYF404S1b, Y-GATA-H4 and Y-GATA-A10; The 31 X chromosome loci are composed of DXS10074, DXS10075, DXS10079, DXS101, DXS10101, DXS10103, DXS10134, DXS10135, DXS10148, DXS10159, DXS10162, DXS10164, DXS6789, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809, DXS6810, DXS7132, DXS7133, DXS7423, DXS7424, DXS8378, DXS981, DXS9902, DXS9907, GATA165B12, GATA172D05, GATA31E08 and HPRTB; The Amel locus includes AmelX and AmelY.

2. The composite amplification system according to claim 1, characterized in that: The forward primers of the 65 autosomal loci have nucleotide sequences as shown in SEQ ID NO.1-65, and the reverse primers have nucleotide sequences as shown in SEQ ID NO.66-130; or The forward primers of the 57 Y chromosome loci have the nucleotide sequences shown in SEQ ID NOs.131-183, and the reverse primers have the nucleotide sequences shown in SEQ ID NOs.184-236; or The forward primers for the 31 X chromosome loci have nucleotide sequences as shown in SEQ ID NOs.237-267, and the reverse primers have nucleotide sequences as shown in SEQ ID NOs.237-298; or The Amel locus has a nucleotide sequence as shown in SEQ ID NO.299, and the reverse primer has a nucleotide sequence as shown in SEQ ID NO.

300.

3. The composite amplification system according to claim 2, characterized in that: The amount of each primer added in the specific amplification primer combination is as follows:

4. The composite amplification system according to claim 2, characterized in that: The working concentration of the specific amplification primer combination is 1 μM-2 μM.

5. Use of the composite amplification system according to any one of claims 1 to 4 in the preparation of a STR refined typing kit.

6. A kit for STR fine typing, characterized in that: The kit comprises the composite amplification system according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that The kit also includes a PCR polymerase mixture, a PCR reaction enhancer, an Index linker sequence, and a PCR product purification reagent.

8. The kit according to claim 7, characterized in that The Index linker sequences include IGT-I5 Index and IGT-I7 Index; the working concentrations of IGT-I5 Index and IGT-I7 Index are both 10 μM.

9. A method for STR fine typing, characterized in that: The method comprises using the composite amplification system according to any one of claims 1 to 4 or the kit according to any one of claims 6 to 8, and the method comprises the following steps: S1, extracting total genomic DNA from samples; S2, performing targeted amplification and gene library construction on the total genomic DNA in step S1; S3, sequencing the gene library constructed in step S2; S4. Process the sequencing data to obtain the test results.

10. The method according to claim 9, characterized in that The sample described in step S1 includes one or more of blood, blood spots, oral cells, semen, semen stains, bones, hair, saliva, saliva stains, sweat, and amniotic fluid containing fetal cells.

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