MH locus screening method based on whole genome SNP data and application

Abstract

The invention discloses a whole genome SNP data-based MH locus screening method and application, and the method comprises the following steps: step 1, screening single nucleotide polymorphism SNP loci in thousands of people's genomes to obtain loci meeting set requirements; arranging all possible SNP combinations to obtain a whole genome MH locus library; step 2, removing sites containing InDel from the whole genome MH site library obtained in the step 1; 3, primers are designed according to the sites obtained through screening in the step 2, composite amplification and sequencing are conducted, the sites which do not meet the requirement are removed, and the needed MH sites are obtained; the detection efficiency of the MH composite system obtained by the invention on a trace mixed detection material is superior to that of a conventional STR-CE method, and the MH composite system is high in detection sensitivity and good in detection accuracy and repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of forensic evidence analysis, and specifically to a MH site screening method based on whole genome SNP data and its application. Background Art

[0002] The commonly used genetic marker in forensic medicine is STR (short tandem repeat), which is sufficiently effective in single-source samples for forensic analysis. However, with the improvement of the sensitivity of DNA detection technology, there is an increasing demand for analyzing trace, mixed and other complex samples. The analysis of STR profiles of complex samples faces major challenges.

[0003] In the analysis of mixed DNA samples, forensic experts need to determine whether the target individual's DNA is contained in the map obtained from the on-site evidence after testing, and infer the mixing ratio of the mixed DNA sample based on the peak height of the alleles at each locus, calculate the weight of the map evidence, and split the genotype of the mixed individual. Factors affecting the STR map analysis of mixed DNA samples include: replication slip peaks (Stutter), pull-up peaks, allele coverage, allele insertion (dropin), allele loss (dropout), peak imbalance, peak variability increase, etc. In particular, when the DNA copy number of the sample is low or there is degradation, the PCR amplification effect is affected, the map quality is reduced, and the above problems are more serious, which will interfere with the analyst's accurate interpretation of the STR map, resulting in misjudgment or failure to draw effective analysis conclusions. In addition, when the mixing ratio is large or the DNA content of the minor contributor is low, its genotype is often difficult to detect, thus affecting the interpretation results.

[0004] Microhaplotype (MH) is considered to be one of the most promising new genetic markers in the field of forensic genetics. It is defined as a haploid genetic marker consisting of two or more SNPs (single nucleotide polymorphisms) within a few hundred bp, and each haplotype constitutes an allele. There are a large number of SNPs in the human genome, so MH combinations with short fragment length and high polymorphism can be screened based on the application purpose.

[0005] There are obvious defects in using STR genetic markers for mixed DNA sample analysis, because the generation of stutter cannot be avoided in the process of STR detection. Stutter peaks are caused by DNA replication slippage during PCR, which is usually manifested as a reduction of one repeat unit of STR. In the spectrum, a weak signal peak can be observed at the position of the repeat unit before the STR allele peak. Analysts can reduce the interference of stutter peaks on the analysis by reducing the amount of DNA template, reducing the amount of electrophoresis sample, increasing the analysis threshold, or using peak signal filtering. However, when analyzing mixed DNA samples, the above methods may cause the peak of the smaller DNA contributor to dropout. At the same time, the stutter peak in the spectrum may be mistaken for the allele peak of the minor contributor, thereby interfering with the typing interpretation of the mixed DNA sample. As a genetic marker composed of SNPs, MH does not produce stutter peaks during the typing process, and the typing interpretation of mixed DNA samples is more accurate and simple.

[0006] In addition, the polymorphism of STR genetic markers is fragment length polymorphism, so the number of composite STR loci in the same system is limited. However, when the number of DNA copies of minor contributors in the mixed DNA sample is low, some loci may not be detected, resulting in failure to achieve sufficient typing efficiency. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a MH site screening method based on whole genome SNP data and its application.

[0008] The technical solution adopted by the present invention is:

[0009] A MH locus screening method based on whole genome SNP data comprises the following steps:

[0010] Step 1: Screen single nucleotide polymorphism (SNP) sites in the 1000 Genomes to obtain sites that meet the set requirements; arrange all possible SNP combinations, assemble MH sites and calculate allele frequencies, effective allele numbers Ae, observed heterozygosity Ho, and individual identification probability PD to obtain the genome-wide MH site library;

[0011] Step 2: Remove the sites containing InDels from the whole genome MH library obtained in step 1;

[0012] Step 3: Design primers based on the sites screened in step 2, perform composite amplification and sequencing, remove sites that do not meet the requirements, and obtain the required MH sites.

[0013] Furthermore, the single nucleotide polymorphism (SNP) sites screened in step 1 satisfy the Hardy-Weinberg equilibrium.

[0014] Furthermore, after removing the sites containing InDels in step 2, the following screening is also included:

[0015] MH length ≤ 200 bp, Ae value ≥ 4 in the 1000 Genomes CHS population;

[0016] When there are overlapping regions in MH, the site with the largest Ae and the shortest fragment length in the overlapping MH is retained;

[0017] MH sequences do not include polymeric or repetitive structures and have a GC content of less than 60%.

[0018] Furthermore, the specific method for removing the sites that do not meet the requirements in step 3 is as follows:

[0019] Sites that were not detected in all sequencing results, detected total sequencing reads less than the set value, and heterozygous balance less than the set threshold were removed.

[0020] Further, the MH loci in step 3 are as follows: CHSCHR012014346719, CHSCHR032004074132, CHSCHR032011490391, CHSCHR032012011089, CHSCHR041002503464, CHSCHR041003461891, CHSCHR041009957783, CHSCHR052005992852, CHSCHR061000278501, CHSCHR061004090362, CHSCHR061004099994, CHSCHR061004108240, CHSCHR061004110364, CHSCHR061004138149, CHSCHR061004305239, CHSCHR071002265948, CHSCHR071005677591, CHSCHR071006205424, CHSCHR071007964753, CHSCHR072002164574, CHSCHR072002653007, CHSCHR081000372932, CHSCHR081001455485, CHSCHR081001463479, CHSCHR081001637924, CHSCHR081001657262, CHSCHR09_011304904, CHSCHR10_000619714, CHSCHR10_000738833, CHSCHR10_001661751, CHSCHR10_002335898, CHSCHR10_002375898, CHSCHR10_004376287, CHSCHR10_010597190, CHSCHR11_008241477, CHSCHR12_008249836, CHSCHR12_011375274, CHSCHR12_015964172, CHSCHR13_002354479, CHSCHR13_003459887, CHSCHR13_010245881, CHSCHR14_000988093, CHSCHR14_001491531, CHSCHR15_001328077, CHSCHR15_009526446, CHSCHR16_001324794, CHSCHR16_007622404, CHSCHR17_003732905, CHSCHR18_000194309, CHSCHR18_004292355, CHSCHR19_001869151,CHSCHR21_005168059, CHSCHR21_005204036, CHSCHR21_005458456, CHSCHR22_001533470, CHSCHR22_001751875. ,

[0021] Furthermore, the primers in step 3 are shown as SEQ ID NOs. 1 to 120.

[0022] An application of an MH site, wherein the MH site is used for DNA sample typing detection.

[0023] Furthermore, the DNA sample is a mixed DNA sample.

[0024] The beneficial effects of the present invention are:

[0025] (1) The present invention screens MH from an MH site library assembled based on the whole genome sequencing data of 2,504 unrelated individuals from the 1000 Genomes. The site library provides a large number of MH sites with different fragment lengths and different polymorphism levels in the whole genome, covering 90.56% of the human genome sequence. According to the actual needs of forensic mixed DNA sample analysis, we screened and deduplicated from the site library, and obtained all MH sites with good polymorphism (Ae>4), short fragment length (<200bp), conforming to Hardy-Weinberg equilibrium, and capable of stable composite amplification and sequencing in the CH population, and constructed a new composite system containing 56 MH sites. The detection efficiency of this system for trace mixed samples is better than that of conventional STR-CE methods, and it improves the typing ability for difficult samples.

[0026] (2) The MH system obtained by the present invention is suitable for all types of forensic DNA sample detection, especially trace and mixed DNA samples; the detection results are stable and highly sensitive. Compared with the conventional STR system, it has better polymorphism, shorter fragment length, and no stutter product.

[0027] (3) The MH system obtained by the present invention is suitable for large-scale parallel sequencing technology, has high one-time detection efficiency, obtains more genetic information than the conventional CE-STR platform, has good detection accuracy and repeatability, and is suitable for forensic individual identification and the inspection of mixed samples.

[0028] (4) The MH system obtained by the present invention has good responsiveness of the allele sequencing reads detected to the mixing ratio of the mixed DNA sample and can be used to infer the mixing ratio of the mixed DNA sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The test results of the samples in Example 2 of the present invention, A is the average allele sequencing depth DoC of 56 MH sites, B is the average allele coverage ratio ACR of 56 MH sites, and C is a schematic diagram of the relationship between DoC and ACR in the test results of the samples in Example 2 of the present invention.

[0030] Figure 2 Schematic diagram of the relationship between the actual proportion of allele sequencing reads in the total sequencing reads of the entire site in Example 3 and the expected proportion of the amount of each allele DNA in the total DNA amount of the site calculated based on the known mixing ratio. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] A MH locus screening method based on whole genome SNP data comprises the following steps:

[0033] Step 1: Based on the VCF file of the whole genome sequencing results of 2504 unrelated individuals in the 1000 Genomes Project, all SNP sites that meet the criteria are obtained. The criteria are as follows: ① Autosomal SNP; ② Satisfy Hardy-Weinberg equilibrium, p value greater than 0.05. ③ No restriction on the minimum allele frequency (MAF). Under the condition that the distance between the first and last SNPs constituting MH is less than or equal to 350bp, all possible SNP combinations are arranged to form MH sites. The fragment length, number of SNPs and physical location of SNPs of all MH sites are counted, and the allele frequency, effective allele number Ae, observed heterozygosity Ho, and individual identification probability PD of MH in different populations of the 1000 Genomes Project are calculated to construct a whole genome MH library.

[0034] Step 2: In the whole genome MH library obtained in step 1, based on the actual needs of forensic mixed DNA sample analysis, write an R script to further screen and remove duplicates. First, remove the sites containing InDel;

[0035] The MH sites that met the requirements were further screened according to the following criteria:

[0036] MH length ≤ 200 bp, Ae value ≥ 4 in the 1000 Genomes CHS population.

[0037] MH deduplication, in each region only retains the sites with the largest Ae and the shortest fragment length in all overlapping MHs in the 1000 Genomes CHS population.

[0038] The Hardy-Weinberg equilibrium was met in the CHS population, and there was no linkage disequilibrium.

[0039] The sequence does not contain poly or repetitive structures, and the GC content is less than 60%.

[0040] Step 3: Design primers based on the sites screened in step 2, perform composite amplification and sequencing, remove sites that do not meet the requirements, and obtain the required MH sites.

[0041] Composite amplification and sequencing excluded MH sites that could not be stably detected (not detected in all sequencing results), had too low total sequencing reads (reads were all less than 300), and had significant differences in heterozygous allele sequencing reads (heterozygous balance < 0.3). 56 MH sites that could be stably amplified and sequenced were retained to form the MH composite system.

[0042] The screened site information is shown in Table 1:

[0043] Table 1. Information of the selected sites

[0044]

[0045]

[0046]

[0047]

[0048] The primer sequence information corresponding to the loci and the concentration of each pair of primers in the primer premix are shown in Table 2. In Table 2 and below, SEQ ID NO.1 to SEQ ID NO.120 are DNA sequence numbers.

[0049] Table 2. Front and rear primers corresponding to the MH site and the final concentration of each primer pair

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] The present invention will be further described below in conjunction with specific drawings and specific embodiments.

[0056] The MH locus is used for typing of mixed DNA samples. The detection process is as follows:

[0057] S1: DNA extraction: DNA from blood samples was extracted using the QIAamp DNA Blood Mini Kit and the Qubit TM The dsDNA HS Assay Kit quantifies the extracted DNA and then dilutes it to 1ng / μL. There are no restrictions on the human DNA extraction method and biological sample type, and DNA can also be extracted from serum, saliva, tissue, etc.

[0058] S2: DNA samples were amplified by PCR using Qiagen Multiplex PCR kit, which was prepared according to the following system: Multiplex Mix 12.5μL, primer premix 2.5μL, Q-solution 2.5μL, ddH2O 6.5μL, sample DNA 1μL. PCR reaction program: 95℃ pre-denaturation for 5min; 95℃30s, 65℃90s, 72℃30s, annealing temperature was reduced by 1℃ for each cycle, 10 cycles; 95℃30s, 55℃90s, 72℃30s, 25 cycles; 68℃ extension for 10min.

[0059] S3: Purify the PCR product using Qiagen MinElute PCR Purification Kit or magnetic beads.

[0060] S4: The PCR product obtained in step S3 is used Ultra TM Sequencing libraries were prepared using a DNA library preparation kit (NEB, USA), and indexes were added to the sequences of each sample. The sample DNA was end-polished, A-tailed, and connected to full-length adapters for Illumina sequencing. Subsequently, the library was purified using the Agencourt AMPure XP PCR Purification Kit to remove unconnected adapters, primers, and other impurities. The library DNA size distribution was analyzed using the Agilent 5400 system, and the library was quantified using qPCR (1.5 nM) to ensure that the quality and concentration of the library met the sequencing requirements. Finally, sequencing was performed using the PE150 strategy on the Illumina platform.

[0061] S5: The original fluorescent image files obtained from the Illumina platform sequencing were converted into short reads data through base calling and recorded in FASTQ format, which contains sequence information and sequencing quality information. Fastp (version 0.23.1) was then used to perform quality control on the raw data to remove poor quality data and ensure the accuracy and reliability of subsequent analysis, including the removal of adapter contamination, excessive uncertain bases, and data with too high a proportion of low-quality bases. Finally, the laboratory's own Python script was used to obtain the typing of each MH site from the data.

[0062] Example 1

[0063] The sensitivity of the MH composite system obtained by the present invention was verified according to the above detection method.

[0064] The typing of NA12878 standard DNA with sample amounts of 5ng, 1ng, 0.5ng, 0.25ng, 0.125ng, 0.0625ng, 0.03125ng, and 0.015625ng was performed, and each sample was repeated three times.

[0065] The results show that when the concentration is higher than 0.03125ng, 56 MH typing can be fully detected, and the detection rate is 100%. When the sample concentration is 0.03125ng, the number of allele dropouts in the three repeated samples is 2, 1, and 6, respectively. When the sample concentration is 0.015625ng, the number of allele dropouts in the three repeated samples is 8, 11, and 12, respectively. Allele dropouts are heterozygous alleles. The detection rates of samples at each concentration are shown in Table 3:

[0066] Table 3. Detection rate of MH system at different sample concentrations

[0067]

[0068] Detection rate = (total number of loci × 2 - total number of dropout alleles in three replicate samples at this concentration) / (total number of loci × 2)

[0069] Example 2

[0070] The MH system obtained by the present invention was used to perform typing tests on blood samples of 100 Southwestern Han individuals, and the forensic parameters and sequencing performance of all 56 MH loci were calculated. The forensic parameters include heterozygosity Ho, individual identification probability DP, polymorphic information content PIC, and effective allele number Ae, as shown in Table 4.

[0071] Table 4. Ae value, observed heterozygosity Ho, polymorphic information content PIC and recognition probability DP

[0072]

[0073]

[0074] As can be seen from Table 4, the ranges of Ho, DP, PIC, and Ae are: 0.695-0.886 (mean 0.784), 0.872-0.954 (mean 0.905), 0.540-0.855 (mean 0.740), and 2.173-6.605 (mean 4.294), respectively. This indicates that the MH system of the present invention has good polymorphism in the southwestern Han population.

[0075] The average depth of coverage (DoC) of the 56 MH loci in the test results of 100 samples ranged from 4800.73 to 86784.41, with a mean of 25589.75. Figure 1 The average allele coverage ratio (ACR) ranged from 0.784 to 0.928, with a mean of 0.890. Figure 1 There was no correlation between Doc and ACR (linear correlation coefficient R2<0.01, Figure 1 C) The results show that the average allele sequencing depth and allele coverage ratio of 56 loci in the MH system of the present invention are both high, and the sequencing performance is good.

[0076] Example 3

[0077] Prepare two-person and three-person mixed DNA samples. The total amount of sample DNA in the first group of samples with a mixing ratio of 1:1, 1:2, 1:5, 10:1 and 15:1 was 1 ng, of which the amount of DNA from the minor contributors ranged from 0.5 ng to 0.0625 ng, and each sample was repeated three times; the second group of samples with a mixing ratio of 1:1, 1:2, 1:4, 1:5, 1:1:1, 1:2:1 and 1:4:1, of which the amount of DNA from the minor contributors was 1 ng, and each sample was repeated twice. The mixed DNA samples were typed according to the above detection method.

[0078] When the DNA amount of the minor contributors is above 0.09 ng, the complete typing of all minor contributors can be detected. The actual proportion of allele sequencing reads in the total sequencing reads of the entire site has a good linear relationship with the expected proportion of each allele DNA amount in the total DNA amount of the site calculated based on the known mixing ratio (linear correlation coefficient R2 = 0.95), as shown in Figure 2. Figure 2This indicates that the MH system obtained by the present invention has good reactivity to the ratio of different alleles in the mixed DNA sample and can be used to infer the mixing ratio of the mixed DNA sample.

[0079] The system of the present invention combines 56 MH sites, the fragment length is within 200bp, the polymorphism is good, the Ae value is greater than 4 in the 1000 Genome CHS population, the Ae average is 4.294 in the Southwest Han population, and it is not interfered by the Stutter peak. The detection efficiency of trace mixed samples is better than the conventional STR-CE method, and the typing ability of difficult samples is further improved. The MH complex system has high detection sensitivity, and allele dropout will occur only when the input DNA amount is as low as 0.03125ng, which is suitable for the inspection of forensic trace physical evidence samples.

[0080] The MH composite system obtained by the present invention is suitable for large-scale parallel sequencing technology, has high one-time detection efficiency, and obtains more genetic information than the conventional CE-STR platform. The detection accuracy and repeatability are good, and it is suitable for forensic individual identification and the inspection of mixed samples. The detected allele sequencing readings have good reactivity to the mixing ratio of the mixed DNA sample, and can be used to infer the mixing ratio of the mixed DNA sample.

Claims

1. A MH site screening method based on whole genome SNP data, characterized in that: The following steps are involved: Step 1: Screen single nucleotide polymorphism (SNP) sites in the 1000 Genomes to obtain sites that meet the set requirements; arrange all possible SNP combinations, assemble MH sites and calculate allele frequencies, effective allele numbers Ae, observed heterozygosity Ho, and individual identification probability PD to obtain the genome-wide MH site library; Step 2: Remove the sites containing InDel from the whole genome MH site library obtained in step 1; Step 3: Design primers based on the sites screened in step 2, perform composite amplification and sequencing, remove sites that do not meet the requirements, and obtain the required MH sites.

2. The MH site screening method based on whole genome SNP data according to claim 1, characterized in that: In the step 1, the single nucleotide polymorphism (SNP) site is screened to satisfy the Hardy-Weinberg equilibrium.

3. The MH site screening method based on whole genome SNP data according to claim 1, characterized in that: After removing the site containing InDel in step 2, Includes the following filters: MH length ≤ 200 bp, Ae value ≥ 4 in the 1000 Genomes CHS population; When there are overlapping regions in MH, the site with the largest Ae and the shortest fragment length in the overlapping MH is retained; MH sequences do not include polymeric or repetitive structures and have a GC content of less than 60%.

4. The MH site screening method based on whole genome SNP data according to claim 1, characterized in that: The specific method for removing the sites that do not meet the requirements in step 3 is as follows: Sites that were not detected in all sequencing results, detected total sequencing reads less than the set value, and heterozygous balance less than the set threshold were removed.

5. The MH site screening method based on whole genome SNP data according to claim 1, characterized in that: The MH loci in step 3 are as follows: CHSCHR012014346719, CHSCHR032004074132, CHSCHR032011490391, CHSCHR032012011089, CHSCHR041002503464, CHSCHR041003461891, CHSCHR041009957783, CHSCHR052005992852, CHSCHR061000278501, CHSCHR061004090362, CHSCHR061004099994, CHSCHR061004108240, CHSCHR061004110364, CHSCHR061004138149, CHSCHR061004305239, CHSCHR071002265948, CHSCHR071005677591, CHSCHR071006205424, CHSCHR071007964753, CHSCHR072002164574, CHSCHR072002653007, CHSCHR081000372932, CHSCHR081001455485, CHSCHR081001463479, CHSCHR081001637924, CHSCHR081001657262, CHSCHR09_011304904, CHSCHR10_000619714, CHSCHR10_000738833, CHSCHR10_001661751, CHSCHR10_002335898, CHSCHR10_002375898, CHSCHR10_004376287, CHSCHR10_010597190, CHSCHR11_008241477, CHSCHR12_008249836, CHSCHR12_011375274, CHSCHR12_015964172, CHSCHR13_002354479, CHSCHR13_003459887, CHSCHR13_010245881, CHSCHR14_000988093, CHSCHR14_001491531, CHSCHR15_001328077, CHSCHR15_009526446, CHSCHR16_001324794, CHSCHR16_007622404, CHSCHR17_003732905, CHSCHR18_000194309, CHSCHR18_004292355, CHSCHR19_001869151, CHSCHR21_005168059,CHSCHR21_005204036、CHSCHR21_005458456、CHSCHR22_001533470、CHSCHR22_001751875。、 6. The MH site screening method based on whole genome SNP data according to claim 5, characterized in that: The primers in step 3 are shown as SEQ ID NOs. 1 to 120.

7. Use of the MH site obtained by the screening method according to any one of claims 1 to 6, characterized in that: The MH site is used for DNA sample typing detection.

8. The use of a MH site according to claim 7, characterized in that: The DNA sample is a mixed DNA sample.

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