A police dog 10K liquid chip and a design method, application and kit thereof

Abstract

The present application relates to the technical field of molecular detection, and specifically discloses a police dog 10K liquid chip, a design method, application and kit thereof, wherein the genotyping sites of the police dog 10K liquid chip include 10463 SNP sites; the physical positions of the 10463 SNP sites are determined based on the whole genome sequence alignment of the canine reference genome CanFam 3.1, wherein the 10463 SNP sites are shown in Table 1. The 10463 SNP sites of the present application are obtained by screening the DNA samples of police dogs, and the 10463 SNP sites can be used as the basis for genotyping the police dogs; the liquid chip designed according to the 10463 SNP sites can quickly realize the genotyping of target SNP sites, and the obtained genotyping data can be used for breed identification of the police dogs and analysis of the diversity of the police dog population.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, specifically to a 10K liquid phase chip for police dogs, its design method, applications, and reagent kit. Background Technology

[0002] Genetic diversity in police dogs is crucial for the health and adaptability of the breed. A police dog population with rich genetic diversity is better able to adapt to different working environments and mission requirements.

[0003] Molecular techniques can accurately assess genetic diversity within and between police dog breeds. If closely related individuals are found, this could lead to inbreeding depression, resulting in decreased fertility and weakened immunity. Based on these findings, subsequent breeding programs can be adjusted by introducing police dogs from other regions with more distant bloodlines, thereby effectively improving the genetic diversity of the population and laying the foundation for breeding better working police dogs.

[0004] Single nucleotide polymorphisms (SNPs), as the latest generation of molecular markers, have been widely used in medical diagnostics, agricultural breeding, and other fields due to their advantages such as large number, wide distribution, rich polymorphism, and ease of detection. With the rapid development of molecular biology and related disciplines, more and more species genomes have been sequenced, leading to the emergence of high-throughput SNP detection technologies to meet the ever-growing demand for genome-level SNP detection. Currently, high-throughput SNP detection technologies mainly employ two strategies: solid-phase microarrays and sequencing. Solid-phase microarrays are based on the complementary hybridization of probes and DNA sequences, and genotyping is performed through the fluorescent signals of the markers. They offer advantages such as high genotyping accuracy and short cycle time, but also have disadvantages such as high cost per genotype and difficulty in customization.

[0005] Targeted sequencing genotyping technology: Targeted sequencing genotyping (GBTS) technology is a technique that achieves deep resequencing by enriching target loci.

[0006] In the process of evaluating police dog breeding, targeted sequencing genotyping can be used to assess the genetic diversity within and between police dog breeds, providing a reference for police dog breeding, which is conducive to optimizing breed structure and protecting the genetic diversity of police dogs.

[0007] Currently, there are no liquid phase chips specifically designed for police dogs. Summary of the Invention

[0008] The purpose of this invention is to provide a 10K liquid phase chip for police dogs, its design method, application, and reagent kit. This 10K liquid phase chip for police dogs can perform genotyping on police dogs and can be used for breed identification and diversity analysis of police dog populations.

[0009] This invention is achieved through the following technical solution:

[0010] A 10K liquid phase chip for police dogs is disclosed. The genotyping sites of the 10K liquid phase chip for police dogs include 10,463 SNP sites. The physical locations of the 10,463 SNP sites are determined based on whole genome sequence alignment of the canine reference genome CanFam 3.1. The 10,463 SNP sites are shown in Table 1.

[0011] The 10,463 SNP loci of this invention were obtained through sequencing analysis and screening of DNA samples from police dogs. These 10,463 SNP loci can serve as the basis for genotyping of police dogs. The liquid-phase chip designed based on these 10,463 SNP loci can quickly and cost-effectively achieve genotyping of the target SNP loci. The obtained genotyping data can be used for breed identification of police dogs and analysis of police dog population diversity.

[0012] In a preferred embodiment, probes designed based on gene sequences covering 10,463 SNP sites are also included.

[0013] A design method for a 10K liquid phase chip for police dogs includes the following steps:

[0014] S1. Target SNP locus information collection and quality control screening: The raw data obtained from sequencing is screened for quality control to obtain quality-controlled sequencing data;

[0015] S2. Sequence preprocessing and SNP detection: The quality-controlled sequencing data is compared with the canine reference genome CanFam 3.1 to locate the position of the quality-controlled sequencing data on the reference genome. After generating the bam file, it is sorted and deduplicated in sequence, and then the SNPs at the target sites are detected for variation.

[0016] S3. SNP site screening and filling: The SNP sites obtained in step S2 are screened and filled to obtain 10463 SNP sites.

[0017] S4. Probe Design and Preparation: Nucleotide sequences of probes were designed for 10,463 SNP sites, and the sequences were screened.

[0018] Specifically, in step S1, the samples used for sequencing include Kunming dogs, Belgian Malinois, Rottweilers, Beagles, Labradors, German Shepherds, and Springer Spaniels. The quality control screening process is as follows:

[0019] First, remove invalid and low-quality data before performing quality control. The conditions for quality control include:

[0020] 1) Remove adapter sequences; 2) If the number of N bases in the sequencing fragment exceeds 10, remove the paired end fragments; 3) When the proportion of low-quality bases in the sequencing fragment exceeds 40% of the fragment length, remove the paired end fragments.

[0021] Specifically, in step S2, when performing mutation detection on the target site SNP, SNP sites with a coverage depth <5× are removed, and heterozygous mutation sites supported by fewer than 4 segments per allele in the heterozygous genotype are eliminated.

[0022] Specifically, in step S3, functional loci are first identified, and then background loci are added. The process of identifying functional loci involves using genome-wide association analysis (GWAS) to screen for SNP loci associated with important traits in police dogs. Background loci are selected based on the following criteria: uniform distribution of the locus across the genome, MAF greater than 0.1, genotype deletion rate less than 0.1, and heterozygosity less than 0.5.

[0023] Specifically, in step S4, the probe screening requirements are: length of 110bp, GC content controlled between 30% and 70%, number of homologous regions not exceeding 5, and for each single nucleotide site, two nucleotide sequences with 60%-70% overlap covering the site must be designed as probe sequences for that site.

[0024] A reagent kit including a 10K liquid phase chip for police dogs.

[0025] Application of 10K liquid phase chip or reagent kit in genotyping of police dogs.

[0026] Application of 10K liquid phase chip or reagent kit in police dog breed identification.

[0027] Application of 10K liquid phase chip or reagent kit for police dog population diversity analysis.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] 1. The 10463 SNP loci of this invention are obtained through screening DNA samples from police dogs. These 10463 SNP loci can serve as the basis for genotyping police dogs. A liquid-phase chip designed based on these 10463 SNP loci can perform cluster analysis on quasi-police dogs to identify their breed. Cluster analysis can also determine the kinship between individual police dogs. If some police dogs are found to be too closely related, this may lead to inbreeding depression, resulting in decreased fertility and weakened immunity. Based on such results, subsequent breeding plans can be adjusted by introducing police dogs from other regions with more distant bloodlines, thereby effectively improving the genetic diversity of the population and laying the foundation for breeding better working police dogs.

[0030] 2. This invention achieves deep resequencing of target sites by reducing the abundance of DNA high-throughput sequencing libraries, thereby significantly reducing the cost of genotyping. Attached Figure Description

[0031] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0032] Figure 1 This is a statistical distribution diagram of 10,463 SNP loci on the chromosome of a police dog in Embodiment 1 of the present invention;

[0033] Figure 2 This is a graph showing the results of gap analysis on the genome for 10,463 SNP sites in Example 1 of the present invention;

[0034] Figure 3 This is a diagram showing the distribution of 10,463 SNP sites in the gene structure in Example 1 of the present invention;

[0035] Figure 4 This is a statistical chart of the MAF distribution of 10463 SNP sites in Example 1 of the present invention;

[0036] Figure 5 The PCA1-PC2 distribution map of the population PCA analysis of 10463 SNP sites in Example 4 of this invention;

[0037] Figure 6 The PCA1-PC3 distribution map of the population PCA analysis of 10463 SNP sites in Example 4 of this invention;

[0038] Figure 7 The PCA2-PC3 distribution map of the population PCA analysis of 10463 SNP sites in Example 4 of this invention;

[0039] Figure 8A three-dimensional PCA diagram of the population of 10,463 SNP sites in Example 4 of this invention;

[0040] Figure 9 This is the phylogenetic tree constructed based on 10463 SNP sites in Embodiment 4 of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The illustrative embodiments and descriptions of this invention are for illustrative purposes only and are not intended to limit the invention. The embodiments described below are some, but not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known structures, materials, or methods are not specifically described to avoid obscuring the invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are commercially available. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art.

[0043] Example 1:

[0044] Design and fabrication of a 10K liquid phase chip for police dogs

[0045] 1) Target SNP locus information collection and quality control screening:

[0046] Using whole-genome resequencing data from 231 individuals across 7 police dog breeds, SNP loci obtained through sequencing analysis were used for subsequent screening.

[0047] The target SNP locus information was obtained from resequencing data of 30 Kunming dogs (KMQ), 32 Malinois (MLNAQ), 35 Rottweilers (LWN), 29 Beagles (BGQ), 35 Labradors (LBLD), 35 German Shepherds (DM), and 35 Springer Spaniels (SBG). The raw sequencing data were preprocessed to remove invalid and low-quality data, resulting in cleanreads. Quality control steps included: ① Adapter trimming removal; ② Removal of paired reads if the N base content in a read exceeded 10; ③ Removal of paired reads if the proportion of low-quality (Q≤20) bases in a read exceeded 40% of the read length.

[0048] 2) Sequence preprocessing and SNP detection:

[0049] The clean reads after quality control were aligned with the canine reference genome using BWA software (CanFam 3.1) to locate their positions on the reference genome, generating a BAM file. The BAM file was then sorted using samtools, and all PCR repeat reads were deduplicated using Picard software. Finally, GATK software was used to detect variants at the target SNP sites, removing SNPs with a coverage depth <5× to ensure the accuracy of the genotyping results. Heterozygous mutation sites were retained only if each allele in the heterozygous genotype had at least four supporting reads; otherwise, they were discarded. This resulted in 17,357,384 SNP sites for subsequent analysis.

[0050] 3) SNP site screening and filling:

[0051] ① Functional locus screening: Functional loci are used directly as the framework for design loci, and the regions outside the functional loci are filled in. In this embodiment, a genome-wide association analysis strategy is used to screen for SNP loci associated with important traits in police dogs.

[0052] Using whole-genome resequencing data from seven police dog breeds, and based on the phenotypic data and statistical results of each breed, after quality control, genome-wide association analysis was performed using the GLM model in PLINK software. A total of 5482 SNPs that were significantly associated with phenotypic indicators were obtained and incorporated into the chip design as functional sites.

[0053] ② Background site screening: Background sites were selected based on resequencing data from 7 police dog breeds: Based on the principle of uniform genome distribution, sites with MAF greater than 0.1, genotype deletion rate less than 0.1, and heterozygosity less than 0.5 were selected, resulting in 183,090 SNP sites.

[0054] Through the above steps, a total of 188,572 functional and background SNPs were selected. Sites that could not be uniquely aligned on the genome, sites avoiding linkage (by calculating linkage blocks and selecting only the first few longest linkage blocks within a given interval, with only one site per block to avoid probe waste due to linkage), and sites avoiding complex genomic regions (filtering out sites in simple repetitive sequence regions, sites with flanking homologous regions greater than 5, and sites with GC content <30% or >70% to avoid probe capture failure in complex regions) were finally obtained. A total of 10,463 SNPs were obtained, evenly distributed across the police dog genome. The specific genomic coordinates of these 10,463 SNPs are shown in Table 1, and their distribution on the police dog chromosomes is statistically shown in Table 2. Figure 1 As shown, Figure 1 The horizontal axis represents chromosome number, the left vertical axis represents the number of loci and segments, and the right vertical axis represents chromosome length; the gap analysis results of 10463 SNP loci on the genome are as follows: Figure 2 As shown (Note: All loci were analyzed. The analysis principle was: the distance between two loci was calculated by subtracting the position of the previous locus from the position of the next locus. If the distance between two loci was greater than the genome size / number of loci * 10, it was considered a gap); the distribution of the 10463 SNP loci in the gene structure is shown below. Figure 3 As shown in Table 1 (Note: Statistical distribution of all loci in gene structure. The horizontal axis represents gene structure type, and the vertical axis represents the number of loci under that type), the 10463 SNP loci include 3138 functional loci selected from 5482 SNP loci and 7352 functional loci selected from 183090 SNP loci. When using them, the 10463 SNP loci are used as a whole, that is, the 10463 SNP loci are used together for genotyping of police dogs and identification of police dog breeds; the 10463 SNP loci are shown in Table 1.

[0055] Table 1

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] .

[0117] 4) Design and synthesis of liquid phase probes:

[0118] Nucleotide sequences of probes were designed for 10,463 target SNP sites, and the sequences were screened. Deep resequencing of target sites was achieved by reducing the abundance of high-throughput DNA sequencing libraries, thereby significantly reducing the cost of genotyping. The probe design process must clearly meet the following criteria:

[0119] ① The probe length is 110 bp;

[0120] ② The GC content is controlled between 30% and 70% to ensure stability and hybridization efficiency;

[0121] ③ The number of homologous regions should not exceed 5. The selected regions should avoid simple sequence repetitions and blank regions as much as possible to reduce non-specific binding;

[0122] ④ Design two nucleotide sequences with 60%-70% overlap covering the selected SNP site to ensure site capture;

[0123] ⑤ Based on the nucleotide sequences designed above, two 110bp DNA nucleotide sequences with biotinylate groups modified at the 5' end were synthesized, which are the 10K liquid-phase chip probes for police dogs. The synthesized canine chip probes were mixed in equimolar mass and diluted with EDTA and Tris-HCl buffer to a final concentration of 3pmol / mL to prepare a chip probe mixture for subsequent experiments.

[0124] Ultimately, the police dog 10K liquid phase chip prepared in this embodiment includes a set of nucleotide probes for detecting 10,463 SNP sites.

[0125] The police dog 10K liquid phase chip prepared in this embodiment can be used to prepare a kit, which includes the police dog 10K liquid phase chip and other reagents.

[0126] Example 2:

[0127] The method for obtaining police dog genotype data using the 10K liquid phase chip for police dogs prepared in Example 1 includes the following steps:

[0128] 1. DNA extraction

[0129] Blood samples from police dogs were selected, and DNA was extracted from the samples using a magnetic bead DNA extraction kit (GenoPrep® Genomic DNA Extraction Kit).

[0130] 2. Quality Inspection

[0131] Qubit enables precise quantification of DNA concentration: The dsDNA HS Assay Kit for Qubit is used to precisely quantify samples.

[0132] Agarose gel electrophoresis to detect genome integrity: Take the DNA sample to be tested, spot it on a 1.5% agarose gel, and electrophoresis at 150V for 25 min.

[0133] Qubit ≥ 50 ng / µL, 1% agarose gel integrity > 20 kb.

[0134] 3. Library Construction

[0135] A quantitative amount of DNA was collected and fragmented using the GenoBaits® End Repair Enzyme. The fragmented DNA was then end-repaired and ligated with A-tails. The A-tailed DNA fragments were ligated to sequencing adapters using GenoBaits® Ultra DNA Ligase, and the ligation product was purified using carboxyl-modified magnetic beads (GenoPrep® DNA CleanBeads). The ligation product was then added to barcode-labeled sequencing primers and a high-fidelity PCR reaction system for PCR amplification. Different barcodes were used to distinguish different samples. The amplified product, purified by the carboxyl-labeled magnetic beads, was ready for probe hybridization experiments.

[0136] 4. Liquid phase probe capture

[0137] Take 500 ng of the constructed DNA library, add a mixture of GenoBaits® Panel, GenoBaits® Block I, and GenoBaits® Block II, and concentrate to dryness. Add GenoBaits® 2X Hyb Buffer and GenoBaits® Hyb Buffer Enhancer, and incubate at 65°C for 2 hours to complete the hybridization reaction. Add GenoBaits® DNA Probe Beads to elute and remove unbound DNA, retaining only the target DNA fragment that hybridizes with the GenoBaits® Panel. Enrich and purify the target fragment by PCR amplification to complete the hybridization capture library.

[0138] Build.

[0139] 5. Sequencing

[0140] After library construction, preliminary quantification was performed using Qubit 2.0, followed by accurate quantification of the effective library concentration using qPCR to ensure library quality. Once the library passed the initial testing, it proceeded to the sequencing stage. A DNBSEQ-T7 sequencer was used, with the PE150 sequencing algorithm selected for sequencing.

[0141] 6. Genotype data analysis

[0142] The raw data were quality controlled using FastQC. Then, the sequencing data were aligned to the police dog reference genome CanFam 3.1 using the default parameters of BWA software. SNPs were detected and genotyping was performed using the standard procedure of GATK software.

[0143] Example 3:

[0144] Detection rate test of the 10K liquid phase chip for police dogs prepared in Example 1:

[0145] The 10K liquid crystal chip for police dogs was used to detect 12 samples, and the results are shown in Table 2:

[0146] Table 2

[0147]

[0148] As shown in Table 2:

[0149] The average site detection rate of the 10K liquid phase chip for police dogs prepared in the example was 99.867%, and the test results met the requirements for the development of a liquid phase chip detection kit for police dogs.

[0150] This demonstrates that the 10K liquid phase chip for police dogs of the present invention has high SNP detection accuracy, and can directly perform genotyping on the population and construct a genetic map.

[0151] In addition, the MAF distribution statistics of the 10463 SNP sites in the above 12 samples are as follows: Figure 4 (Note: This is a statistical representation of the MAF values ​​at loci. A higher MAF value indicates better polymorphism at the locus. The horizontal axis represents different ranges of MAF values, and the vertical axis represents the number of loci within each MAF range.)

[0152] Example 4:

[0153] Application of the 10K liquid phase chip for police dogs prepared in Example 1 in the analysis of diversity and breed identification of police dog populations

[0154] Principal component analysis and neighbor-joining (NJ) phylogenetic trees of police dogs were constructed using the 10K liquid phase chip prepared in Example 1. Based on resequencing samples (231 samples from Example 1 (30 Kunming dogs (KMQ), 32 Malinois (MLNAQ), 35 Rottweilers (LWN), 29 Beagles (BGQ), 35 Labradors (LBLD), 35 German Shepherds (DM), and 35 Springer Spaniels (SBG)) and GBTS test samples (12 test samples from Example 2), a population PCA distribution map was drawn using 10463 SNP loci, as shown below. Figure 5-8 As shown; the phylogenetic tree constructed using 10463 SNP loci is as follows Figure 9 As shown.

[0155] Depend on Figures 5-9 It can be known that:

[0156] The police dog 10K liquid phase chip prepared in Example 1 can effectively classify 7 breeds. The 12 test samples in Example 2 can be identified as Kunming police dogs (KMQ) through phylogenetic analysis. That is, the police dog 10K liquid phase chip prepared in Example 1 can be used for police dog breed identification and can be used to assist in kinship identification and police dog genetic structure analysis.

[0157] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A 10K liquid phase chip for police dogs, characterized in that, The genotyping loci of the police dog 10K liquid-phase chip include 10463 SNP loci. The physical locations of these 10463 SNP loci were determined based on whole-genome sequence alignment using the canine reference genome CanFam 3.

1. These 10463 SNP loci were obtained through sequencing analysis of DNA samples from Kunming dogs, Belgian Malinois, Rottweilers, Beagles, Labradors, German Shepherds, and Springer Spaniels. The 10463 SNP loci are shown in the table below. The police dog 10K liquid phase chip also includes probes designed based on gene sequences covering 10,463 SNP sites.

2. A kit comprising the police dog 10K liquid phase chip as described in claim 1.

3. The application of the 10K liquid phase chip for police dogs as described in claim 1 in the genotyping of police dogs.

4. The application of the kit as described in claim 2 in the genotyping of police dogs.

5. The application of the 10K liquid phase chip for police dogs as described in claim 1 in the identification of police dog breeds.

6. The application of the reagent kit as described in claim 2 in the identification of police dog breeds.

7. The application of the 10K liquid phase chip for police dogs as described in claim 1 in the diversity analysis of police dog groups.

8. The application of the kit as described in claim 2 in the diversity analysis of police dog groups.

Images