Str primer set, kit and method for detecting polyploidy, upd and maternal contamination in a sample

Abstract

The application belongs to the field of gene detection, and provides an STR primer group, a kit and a method for detecting polyploidy, UPD and maternal contamination in a sample. The primer group comprises a first primer group, the first primer group comprises 178 pairs of primers, the nucleotide sequences of the 178 pairs of primers are shown in SEQ ID NO. 1-SEQ ID NO. 356, the kit comprises an amplification reaction solution, dNTP, DMSO, nuclease-free water and the above-mentioned STR primer group. The primer group and the kit of the application can simultaneously detect polyploidy, chromosome-level and whole genome-level uniparental disomy and maternal contamination of a sample. Moreover, the primer group and the kit can be configured for Cnv-seq or PGT-A, and can effectively solve the problem that the two technologies cannot detect polyploidy, uniparental disomy and maternal contamination.

Description

Technical Field

[0001] This invention belongs to the field of gene detection and relates to STR genetic marker technology, which has good application value in the human genome. Specifically, it relates to an STR primer set, kit and method for detecting polyploidy, UPD and maternal contamination in a sample. Background Technology

[0002] High-throughput sequencing-based genome copy number variation sequencing (CNV-seq) can detect chromosomal number abnormalities, large deletions / duplications, and pathogenic copy number variations, and has wide applications in prenatal diagnosis, assisted reproduction, and auxiliary diagnosis of pediatric genetic diseases. However, polyploidy, uniparental diploidy, and maternal cell contamination in samples cannot be detected by CNV-seq, requiring the use of short tandem repeats (STRs) to compensate for the limitations of CNV-seq.

[0003] Preimplantation genetic testing (PGT-A) is a genetic test performed on the aneuploidy of embryonic chromosomes before implantation during assisted reproduction. This technology can guide the selection of high-quality embryos with euploid chromosomes for transfer. However, it also faces the challenge of not being able to directly detect polyploidy or uniparental diploidy at the chromosome level. Therefore, it is necessary to perform short tandem repeat (STR) analysis to overcome these limitations.

[0004] Currently, STR locus detection primarily employs PCR amplification followed by electrophoretic separation, or, when constructing a multi-locus fluorescently labeled multiplex amplification detection system, multicolor fluorescent dyes are typically used. The principle is that the allele lengths of loci labeled with the same fluorescent dye must not overlap, while the allele lengths of loci labeled with different fluorescent dyes can overlap. This allows for the differentiation of different loci based on fluorescence color and allele size regions. However, due to limitations in electrophoretic resolution and the types of fluorescent dyes available, this approach can only detect a limited number of STR loci at a time, making it impossible to simultaneously detect hundreds of STR loci and effectively detect uniparental diploids at the chromosome level.

[0005] Therefore, there is a need for a method that can simultaneously overcome the shortcomings of CNV-seq and PGT-A in detecting polyploidy, UPD (uniparental diploidy), and maternal contamination in samples. Summary of the Invention

[0006] To address the limitations of CNV-seq and PGT-A in detecting polyploidy, UPD (uniparental diploid), and maternal contamination in samples, this invention discloses an STR primer set, kit, and method for detecting polyploidy, UPD, and maternal contamination in samples.

[0007] The first aspect of the present invention provides a STR primer set for detecting polyploidy, UPD and maternal contamination in a sample. The primer set includes a first primer set comprising 178 pairs of primers. The nucleotide sequences of the forward primers are shown in SEQ ID NO.1-SEQ ID NO.178, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.179-SEQ ID NO.356.

[0008] In an improved embodiment, the primer set further includes a second primer set comprising 10 pairs of primers, wherein the nucleotide sequences of the forward primers are shown in SEQ ID NO.357-SEQ ID NO.366, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.367-SEQ ID NO.376.

[0009] A second aspect of the present invention provides a method for designing the STR primer set described in the first aspect above, the method comprising:

[0010] In the STR database, all STR loci on the 23 pairs of human chromosomes were analyzed. STR loci containing polynucleotides were removed; STR loci with a polymorphism information content (PIC) > 0.2 were retained; STR loci with repeating unit bases of 3, 4, 5, and 6 were retained; STR loci with a total length greater than 150 bp were removed; STR loci with a GC content greater than 60% within a 200 bp range upstream and downstream were removed; and STR loci with homology in the human genome within a 200 bp range upstream and downstream were removed, resulting in 178 STR loci. Primers were designed for each of these STR loci to obtain the first primer set.

[0011] In an improved embodiment, the design method further includes extracting STR loci with sequencing depth ≤ sequencing depth threshold from 178 STR loci as low-depth STR loci, and designing primers for each of the low-depth STR loci to obtain a second primer set.

[0012] A third aspect of the present invention provides a kit for detecting polyploidy, UPD, and maternal contamination in a sample. The kit includes an amplification reaction solution, dNTPs as an enhancer, DMSO as an enhancer, nuclease-free water, and the STR primer set described in the first aspect above. The amplification reaction solution includes hot-start polymerase, Tris buffer, KCl, MgCl2, (NH4)2SO4, DNTP, betaine, Triton, BSA, Tween, and glycerol.

[0013] A fourth aspect of the present invention provides a method for detecting polyploidy, UPD, and maternal contamination in a sample, comprising:

[0014] S1. Obtain gDNA or whole genome amplification product of the sample to be tested, wherein, when judging maternal contamination, the sample to be tested includes offspring samples and offspring mother samples, and when judging polyploidy and UPD, the sample to be tested is an offspring sample.

[0015] S2. The gDNA or the whole genome amplification product is subjected to multiplex targeted amplification and purification using the primer set described in the first aspect above or the kit described in the second aspect above. A DNA library is constructed from the purified product after multiplex targeted amplification and sequencing is performed to obtain sequencing data.

[0016] S3. Based on the sequencing data, determine the polyploidy, UPD, and maternal contamination of the sample to be tested.

[0017] Compared with existing technologies, the beneficial effects achieved by at least one of the above-mentioned technical solutions adopted in the embodiments of this specification include at least the following: the STR primer set, kit, and detection method for detecting polyploidy, UPD, and maternal contamination in samples of the present invention can realize the ploidy identification of gDNA samples and the identification of maternal contamination. Simultaneously, it can analyze the triploid and uniparental diploid status of whole-genome amplification products. The present invention is simple to operate, compatible with CNV-Seq and PGT-A, and can overcome the deficiency of CNV-seq in detecting polyploidy and maternal contamination. Furthermore, the high-throughput STR detection scheme can also overcome the limitations of PCR-capillary electrophoresis or fluorescent PCR-capillary electrophoresis methods, which are hampered by dyes and specialized instruments, and can simultaneously detect hundreds of STR loci. Detailed Implementation

[0018] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features of the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0019] One embodiment of the present invention provides an STR primer set for detecting polyploidy, UPD, and maternal contamination in a sample. This primer set is designed based on all STR loci on the 23 pairs of human chromosomes from the STR database, through the following process:

[0020] 1. Remove STR loci containing polynucleotide STR sites;

[0021] 2. STR loci with polymorphic information content (PIC) > 0.2 are retained; STR loci with repeating base numbers of 3, 4, 5, and 6 are retained.

[0022] 3. Remove STR loci with a total length greater than 150 bp;

[0023] 4. Remove STR loci with GC content greater than 60% within a 200 bp range upstream and downstream;

[0024] 5. Remove STR loci whose upstream and downstream sequences are homologous to STR sites in the human genome within 200 bp.

[0025] The above operations yield 178 STR loci. Primers are designed for each STR locus to obtain the first primer set, which includes 178 primer pairs. The nucleotide sequences of the forward primers are shown in SEQ ID NO.1-SEQ ID NO.178, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.179-SEQ ID NO.356.

[0026] Among them, 178 STR basis loci are included: D1S1656, GATA23G09, TTTA063, GATA26G09, AAT259, AAT252, Human_STR_99081 / F13B, AATA011, TPOX, D2S441, GAAT1A 5, GATA8F07, GATA176C01, GATA8H05, GATA194A05, AAT203, D3S1358, ATCT053, GATA146D07, ATC4D07, AAT071, ATA22E01, GATA22G05, ATC T018, GATA24H01, GATA2F11, ATA26B08, GATA8A05, ATT077P, FGA, ATA20G07, GATA63C02, GATA51A07, D5S818, CSF1PO, AAT013, SE30, ATC0 33, GATA11B08, D6S1043, GATA23F08, ATA6C09P, D6S1009, G15833, D6S494, TATT019, TAT028, D7S820, ATA55A05, TATG002, D7S1818, D7S18 20, D7S3052, ATT023, Human_STR_1395783, GATA156H01, AAT076, D8S1179, LPL, MFD455-AAT052, GATA165A11, GATA89A11, ATA42G04, ATC C001, ATA24F10, TTA007, D10S1248, TH01, ATA25D12, GATA46A12, AAT228, D11S2362, D11S4465, D11S1981, D11S1392, D11S2363, D11S4952 , D11S1998, AAT262, ATA73C05, GATA63D12, GATA5H03, vWA, ATA5A09, GATA11C08, D13S317, AGAT113Z, D13S1493, D13S801, D13S1824, D13 S796, D14S1434, ATA70B03, GGAA30H04, D14S1280, D14S121, D14S748, D14S125, D14S588, D14S617, D14S1426, TAT032z, GATA85D02, PentaE. D15S1513, D15S822, D15S1232, D15S659, D15S643, D15S1507, D15S816, D15S1515, ATA 67B07, GATA151C03, D16S539, GAAT2C03, GATA25A04, ATA58A02, AGAT060, D18S51, D18S86 2. D18S878, D18S391, D18S865, D18S1371, D19S433, TGA012, AAT257, GATA81E09, TATT031 , D20S482, D20S604, D20S1145, D20S607, D20S1085, D20S469, D20S1082, GGAA3C07, Penta D. D21S1432, D21S1409, D21S1442, D21S226, D21S1413, D21S11, AGAT120, UT7136, D22S1045, Human_STR_1547748, DXS7132, DXS10074, DXS8378, HPRTB, DXS981, DXS10135, DYS19, DYS391 , DYS439, DYS438, DYS437, DYS392, DYS393, DYS456, DYS458, DYS388, DYS426, Y-GATA-H4, DYS 435, DYS436, DYS462, DYS549, DYS533, DYS570, DYS643, DYS434, DYS460, DYS461 and Y-GATA-A10.

[0027] Furthermore, when designing primers for the screened STR loci, STR loci with lower sequencing depths were extracted based on sequencing depth thresholds. These STR loci included GATA8F07, GATA89A11, DXS10074, YGATAA10, and DYS19. These five STR loci were defined as low-depth STR loci. Several additional primer pairs were designed for each low-depth STR locus to increase its sequencing depth. This can be understood as designing primers for each low-depth STR locus to form a second primer set, consisting of 10 primer pairs. The nucleotide sequences of the forward primers are shown in SEQ ID NO.357-SEQ ID NO.366, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.367-SEQ ID NO.376. Combining the 10 primer pairs from the second primer set with the 178 primer pairs from the first primer set yields 188 primer pairs. These 188 primer pairs constitute a primer composition with relatively uniform amplification.

[0028] A second embodiment of the present invention provides a kit comprising the primer set shown in the above embodiment, and further comprising: amplification reaction solution, dNTP enhancer, DMSO enhancer, and nuclease-free water. The amplification reaction solution comprises high-fidelity hot-start polymerase, 100-200 mM Tris buffer, 100-200 mM KCl, 3.75-7.5 mM MgCl2, 50-100 mM (NH4)2SO4, 50-200 μm DNTP, 1000-1500 mM betaine, 0.19%-0.38% Triton (v / v), 2-3 mg / ml BSA, 5%-15% Tween (v / v), and 2.5%-7.5% glycerol (v / v). It should be noted that the enhancer dNTP and DNTP have the same composition. The difference is that DNTP used in conventional PCR amplification has a concentration range, and too high a concentration of dNTP will affect the amplification efficiency. However, since multiplex PCR is used, only an extra part needs to be added in the first round to promote the multiplex PCR reaction. Therefore, this part is named DNTP separately for distinction.

[0029] A third embodiment of the present invention provides a method for detecting polyploidy, UPD, and maternal contamination in a sample, comprising:

[0030] S1. Obtain the gDNA or whole genome amplification product of the sample to be tested. When determining maternal contamination, the sample to be tested includes both offspring samples and offspring-mother samples; when determining polyploidy and UPD, the sample to be tested is the offspring sample. The sample to be tested can be any one of peripheral blood, saliva, hair follicles, and blastocyst cells. Blastocyst cells can be derived from aborted tissue or non-embryonic tissue from in vitro cultured embryos. gDNA is extracted from peripheral blood, saliva, or hair follicles, while the whole genome amplification product is obtained by WGA amplification of blastocyst cells.

[0031] S2. Using the primer set described in the first aspect or the kit described in the second aspect, perform multiplex targeted amplification and purification of the gDNA or the whole genome amplification product. Construct a DNA library from the purified product after multiplex targeted amplification and perform sequencing to obtain sequencing data.

[0032] S3. Based on the sequencing data, determine the polyploidy, UPD, and maternal contamination of the sample to be tested, including:

[0033] S31. Align the test data with the reference genome sequence fragment of the STR locus corresponding to each primer in the primer set, calculate the copy number and read number of each STR locus, calculate the proportion of reads with a ratio of 1:1:1 in all the STR loci, and determine the polyploidy of the test sample according to the threshold and the proportion.

[0034] Specifically, the sequencing data can be used to locate the target region by aligning it with the reference genomic sequence fragment used to design primers (i.e., the sequence of the STR locus). The copy number and corresponding read count of each genomic repeat unit after alignment are then calculated. STR loci are classified into two types: homozygous loci, which contain only one repeat unit, and heterozygous loci, which contain two repeat units. The corresponding read ratios are (1:1, 1:1:1, 2:1). Loci with a ratio of 1:1:1 are rare in normal diploid samples but common in polyploid samples. Therefore, the read ratio can be used to determine the proportion of 1:1:1 loci to ascertain whether the sample being tested is polyploid.

[0035] The threshold settings for polyploid determination can be found in Table 1 below:

[0036] Table 1: Thresholds and ploidy categories for polyploid determination

[0037]

[0038] It should be noted that in the last item of Table 1 above, when the ratio of reads cannot be determined, it is not considered when making a plurality determination.

[0039] S32. The UPD determination of the test sample is performed based on the heterozygosity of the STR loci. Specifically, for uniparental diploid (UPD) samples, since all homologous chromosomes come from the same parent, uniparental diploid at the chromosome level and uniparental diploid at the whole genome level lack heterozygosity, making all loci homozygous. Therefore, the heterozygosity of the STR loci on the chromosome is used to analyze whether the sample is uniparental diploid.

[0040] S33. Calculate the proportion of maternal contamination in the sample based on the calculated proportion of maternally specific loci in the sequencing data. Specifically, when maternal contamination is present in the sample, maternally specific loci will be present in the STR loci of the sequencing data. The proportion of maternally contaminant in the sample can be determined by calculating the proportion of maternally specific loci.

[0041] This invention provides a detailed description of the primer set, reagent kit, and detection method described above through the following examples:

[0042] Example 1: Screening STR loci and designing primers for each STR locus.

[0043] The screening of STR loci was based on the genomic version hg19. Specific primers were designed according to the STR loci. Genomic DNA was used as the detection target. A large number of experiments were conducted to screen, optimize and verify the primers. Finally, 188 pairs of primers with high amplification efficiency and good specificity were selected. The nucleotide sequences of the 188 pairs of primers are shown in SEQ ID NO.1-SEQ ID NO.376.

[0044] Example 2: Construct a DNA library and perform sequencing to obtain sequencing data. Analyze the sequencing data to obtain the polyploid results of the sample.

[0045] (1) In this example, a triploid sample is used as the test sample. The gDNA sample of the triploid sample is obtained and the concentration of the gDNA sample is detected. The first round PCR reaction system and program, as well as the second round PCR reaction system and program are set up. The first round PCR reaction system is shown in Table 2 below, and the reaction program is shown in Table 3 below; the second round PCR reaction system is shown in Table 4 below, and the reaction program is shown in Table 5 below.

[0046] Table 2: First-round PCR reaction system

[0047]

[0048] The concentration of gDNA samples was quantified using Qubit (Thermo Fisher).

[0049] Table 3: First-round PCR reaction procedure

[0050]

[0051] For the first round of PCR, place the PCR tube containing the reaction reagents into the PCR instrument and set the PCR program according to the table above for the first round of amplification. After amplification, perform magnetic bead purification: vortex to mix, briefly centrifuge, add 27 μl of AMPure XP magnetic beads equilibrated at room temperature to 30 μl of PCR product, and mix by pipetting or vortexing to purify. After purification, wash the product with 24 μl of nuclease-free water.

[0052] Table 4: Second-round PCR reaction system

[0053]

[0054] Table 5: Second Round PCR Reaction Procedure

[0055]

[0056] For the second round of PCR, place the PCR tube containing the reaction reagents into the PCR instrument and set the PCR program according to the table above for the second round of amplification. After the second round of amplification, perform magnetic bead purification: briefly centrifuge the PCR tube, add 36 μl of AMPure XP magnetic beads equilibrated at room temperature to 30 μl of PCR product, purify the product, wash with 24 μl of nuclease-free water, and transfer 20 μl of supernatant to a new PCR tube.

[0057] (2) DNA library quantification and quality control

[0058] Firstly, the concentration of the product after two amplifications and purifications in (1) was detected using a Qubit Fluorometer. The normal range was 5 ng / ul to 50 ng / ul, and its concentration was mainly related to the quality of the template.

[0059] Secondly, 10 μl of library samples (concentration between 1-2 ng / uL) were taken and the fragment length and purity of the library were measured using the Qsep100 fully automated nucleic acid and protein analysis system. The fragment distribution range of a normal library is around 350 bp.

[0060] (3) Sequencing of DNA libraries

[0061] The sequencing was performed according to the instructions for the Miseq DX sequencing reagent. The raw sequencing data was in FastQ format. Short fragments smaller than 50 bp were removed from the processed data. Then, the genotypes of each locus were analyzed using GangSTR software.

[0062] (4) Statistical analysis of the proportion of 1:1:1 genotypes to determine whether the sample is polyploid. The results of some genome detection are shown in Table 6 below.

[0063] Table 6: Results of partial loci detection in triploid samples

[0064]

[0065] According to the statistical method in Table 6 above, a total of 141 loci were detected in this sample. Among them, 13 loci could not be determined, 19 were 1:1:1 loci and 2 were 2:1 loci, totaling 66 loci, accounting for 51% of the loci that could be determined. The proportion of 1:1:1 loci and 2:1 loci in normal diploid samples does not exceed 15%. Therefore, this sample was determined to be a triploid sample.

[0066] Example 3: Using a uniparental diploid chromosome 6 as the sample to be tested, a DNA library was constructed and sequenced according to the library construction and sequencing steps in Example 2 above. Sequencing data was obtained, and the uniparental diploidity of the sample was obtained by analyzing the sequencing data. The results are shown in Table 7 below;

[0067] Table 7: Results of uniparental diploidy detection of chromosome 6

[0068]

[0069] According to Table 7 above, all STR loci on chromosome 6 of this sample are homozygous and lack heterozygous loci. Therefore, this sample is determined to be a uniparental diploid on chromosome 6.

[0070] Example 4: Using peripheral blood, saliva, and hair follicles of a mother and her child as samples, gDNA was extracted from each sample. The maternal gDNA and the child's gDNA were diluted to the same concentration and mixed at a volume ratio of 25% and 30%. Following the library construction and sequencing steps in Example 2 above, a DNA library was constructed and sequencing data was obtained. The sequencing data was analyzed to obtain the results of maternal contamination of the samples.

[0071] The proportion of pollution originating from the mother source is determined by the following formula:

[0072] The final contamination rate is the average of the contamination rates of all sites. The calculation results are shown in Tables 8 and 9 below: Tables 8 and 9 only show the usable sites out of 178 sites, listing cases of 30% contamination and 25% contamination. The method of this invention can detect 20% contamination.

[0073] Table 8: Detection results of maternal source pollution (30%)

[0074]

[0075] Table 9: Detection results of maternal source pollution (25%)

[0076]

[0077] Example 5: Using a triploid gDNA sample as the test sample, it was diluted to 18 pg and then amplified using an MDA whole genome amplification kit. The kit of this invention was used to construct a library and sequence the amplified whole genome products to obtain sequencing data. The sequencing data was analyzed to obtain the sample detection results. Some site results are shown in Table 10 below:

[0078] Table 10: Detection results of ploid gDNA samples

[0079]

[0080] Based on the statistical calculations performed on the 10 pairs of loci in Table 10 above, there are a total of 144 non-homozygous loci in this sample, of which 20 are 1:1:1 loci, accounting for 13.8%. The proportion of 1:1:1 loci in normal diploid samples is less than 10%, therefore this sample is judged to be triploid.

[0081] The embodiments of this invention achieve the following technical effects: The primer set, kit, and analysis method of this invention can simultaneously detect 178 STR loci. Compared with conventional PCR methods, it has higher throughput and more signal values, thus being more reliable and accurate. Furthermore, this method can detect polyploidy in samples, as well as uniparental diploidy at the chromosome level and whole-genome level, and maternal contamination. Due to throughput limitations, PCR methods cannot detect chromosome-level UPDs, only whole-genome-level UPDs.

[0082] Meanwhile, the primer set and kit of the present invention, when used in conjunction with Cnv-seq or PGT-A, can effectively solve the problem that these two technologies cannot detect polyploids and maternal contamination from uniparental diploids.

[0083] Obviously, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the embodiments of the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer set for detecting polyploidy, UPD, and maternal contamination in a sample, characterized in that, The primer set includes a first primer set, which includes 178 pairs of primers. The nucleotide sequences of the forward primers are shown in SEQ ID NO.1-SEQ ID NO.178, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.179-SEQ ID NO.

356.

2. The STR primer set according to claim 1, characterized in that, The primer set further includes a second primer set, which comprises 10 pairs of primers. The nucleotide sequences of the forward primers are shown in SEQ ID NO.357-SEQ ID NO.366, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.367-SEQ ID NO.

376.

3. A method for designing an STR primer set as described in claim 1 or 2, characterized in that, The design method includes: In the STR database, all STR loci on the 23 pairs of human chromosomes were analyzed. STR loci containing polynucleotides were removed; STR loci with a polymorphism information content (PIC) > 0.2 were retained; STR loci with repeating unit bases of 3, 4, 5, and 6 were retained; STR loci with a total length greater than 150 bp were removed; STR loci with a GC content greater than 60% within a 200 bp range upstream and downstream were removed; and STR loci with homology in the human genome within a 200 bp range upstream and downstream were removed, resulting in 178 STR loci. Primers were designed for each of these STR loci to obtain the first primer set.

4. The method for designing STR primer sets according to claim 3, characterized in that, Including 178 STR base loci: D1S1656, GATA23G09, TTTA063, GATA26G09, AAT259, AAT252, Human_STR_99081 / F13B, AATA011, TPOX, D2S441, GAAT1A5, G ATA8F07, GATA176C01, GATA8H05, GATA194A05, AAT203, D3S1358, ATCT053, GATA146D07, ATC4D07, AAT071, ATA22E01, GATA22G05, ATCT01 8, GATA24H01, GATA2F11, ATA26B08, GATA8A05, ATT077P, FGA, ATA20G07, GATA63C02, GATA51A07, D5S818, CSF1PO, AAT013, SE30, ATC033, GATA11B08, D6S1043, GATA23F08, ATA6C09P, D6S1009, G15833, D6S494, TATT019, TAT028, D7S820, ATA55A05, TATG002, D7S1818, D7S1820 , D7S3052, ATT023, Human_STR_1395783, GATA156H01, AAT076, D8S1179, LPL, MFD455-AAT052, GATA165A11, GATA89A11, ATA42G04, ATCC0 01, ATA24F10, TTA007, D10S1248, TH01, ATA25D12, GATA46A12, AAT228, D11S2362, D11S4465, D11S1981, D11S1392, D11S2363, D11S4952, D11S1998, AAT262, ATA73C05, GATA63D12, GATA5H03, vWA, ATA5A09, GATA11C08, D13S317, AGAT113Z, D13S1493, D13S801, D13S1824, D13S 796, D14S1434, ATA70B03, GGAA30H04, D14S1280, D14S121, D14S748, D14S125, D14S588, D14S617, D14S1426, TAT032z, GATA85D02, PentaE, D15S1513, D15S822, D15S1232, D15S659, D15S643, D15S1507, D15S816, D15S1515, ATA67B07, GATA151C03, D16S539, GAAT2C03, GATA25A04, ATA58A02, AGAT060, D18S51, D18S862, D18S878, D18S391, D18S865, D18S1371, D19S433, TGA012, AAT257, GATA81E09, TATT031, D20S482, D20S604, D20S1145, D20S607, D20S1085, D20S469, D20S1082, GGAA3C07, Penta D, D21S1432, D21S1409, D21S1442, D21S226, D21S1413, D21S11, AGAT120, UT7136, D22S1045, Human_STR_1547748, DXS7132, DXS10074, DXS8378, HPRTB, DXS981, DXS10135, DYS19, DYS391, DYS439, DYS438, DYS437, DYS392, DYS393, DYS456, DYS458, DYS388, DYS426, Y-GATA-H4, DYS435, DYS436, DYS462, DYS549, DYS533, DYS570, DYS643, DYS434, DYS460, DYS461 and Y-GATA-A10.

5. The method for designing STR primer sets according to claim 4, characterized in that, The design method further includes: STR loci with sequencing depth ≤ sequencing depth threshold were extracted from 178 STR loci as low-depth STR loci. Primers were designed for each of the low-depth STR loci to obtain a second primer set, wherein the low-depth STR loci include GATA8F07, GATA89A11, DXS10074, YGATAA10, and DYS19.

6. A reagent kit, characterized in that, The amplification reaction solution includes an amplification reaction solution, dNTPs as an enhancer, DMSO as an enhancer, nuclease-free water, and the STR primer set as described in claim 1 or 2, wherein the amplification reaction solution includes a hot-start polymerase, Tris buffer, KCl, MgCl2, (NH4)2SO4, DNTP, betaine, Triton, BSA, Tween, and glycerol.

7. A method for detecting polyploidy, UPD, and maternal contamination in samples for non-disease diagnosis or non-treatment purposes, characterized in that, include: Obtain gDNA or whole genome amplification product of the sample to be tested. When determining maternal contamination, the sample to be tested includes offspring samples and offspring mother samples. When determining polyploidy and UPD, the sample to be tested is an offspring sample. The gDNA or the whole genome amplification product is subjected to multiple targeted amplification and purification using the primer set as described in claim 1 or 2 or the kit as described in claim 6. A DNA library is constructed from the purified product after multiple targeted amplification and sequencing is performed to obtain sequencing data. Based on the sequencing data, the sample to be tested is assessed for polyploidy, UPD, and maternal contamination, including: The test data is compared with the reference genome sequence fragment of the STR locus corresponding to each primer in the primer set, the number of reads of each STR locus is calculated, the proportion of reads with a ratio of 1:1:1 in all the STR loci is calculated, and the polyploidy of the test sample is determined according to the threshold and the proportion. UPD was performed on the test samples based on the heterozygosity of the STR loci. The proportion of maternal contamination in the sample to be tested is calculated based on the proportion of maternally specific loci in the sequencing data.

8. The method for detecting polyploidy, UPD, and maternal contamination in samples for non-disease diagnosis or non-treatment purposes according to claim 7, characterized in that, The test sample can be any one of peripheral blood, saliva, hair follicle, and blastocyst cells.

9. The method for detecting polyploidy, UPD, and maternal contamination in samples for non-disease diagnosis or non-treatment purposes according to claim 7, characterized in that, Multiplex targeted amplification involves two rounds of PCR reactions. The first round of PCR reaction system includes: 40-50 ng of gDNA or whole genome amplification product, 5 μL of primer set, 10 μL of amplification reaction solution, 3.5 μL of dNTP enhancer and 2.5 μL of DMSO enhancer, and the system volume is made up to 30 μL with nuclease water. The second round of PCR reaction system includes: 13.5 μL of the first round of PCR amplification product, 2.5 μL of DMSO enhancer, 2 μL of nuclease water, 2 μL of universal adapter, and 10 μL of amplification reaction solution.

10. The method for detecting polyploidy, UPD, and maternal contamination in samples for non-disease diagnosis or non-treatment purposes according to claim 9, characterized in that, The first round of PCR amplification program was as follows: 95℃ for 3-4 min; 15-20 cycles, 98℃ for 20 s, 60℃ for 2-5 min; 72℃ for 5-10 min, and hold at 16℃. The second round of PCR amplification program is as follows: 95℃ for 3-4 min; 6-15 cycles, 98℃ for 20 s, 58℃ for 1-3 min; 72℃ for 30 s-2 min, and hold at 16℃.

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