Method and device for detecting SNP marker sites based on low-depth sequencing

A technology of deep sequencing and marking loci, applied in the field of genetics, can solve problems such as wrong typing results, filling that cannot guarantee high accuracy, poor timeliness, etc., and achieve high accuracy results

Pending Publication Date: 2021-06-01
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most studies on livestock and poultry species rely on high-depth sequencing data of small samples to construct reference haplotype databases. There have been a large number of research reports. When the number of markers is at the million level, there will be tens of thousands or even hundreds of thousands of wrong typing results, and this strategy has high computational complexity and poor timeliness, which is still not conducive to breeding practice

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  • Method and device for detecting SNP marker sites based on low-depth sequencing
  • Method and device for detecting SNP marker sites based on low-depth sequencing
  • Method and device for detecting SNP marker sites based on low-depth sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Experimental materials

[0060] Using 3000 individual ear tissue samples from the core breeding herd of Duroc, the genome was extracted and diluted to 40ng / μL.

[0061] 2. Experimental method

[0062] 2.1 Low-depth DNA library construction and sequencing

[0063] This example uses Tn5 digestion to carry out DNA library construction instructions, specifically:

[0064] (1) Embed Tn5 proenzyme with specific Tn5ME-A / Tn5Merev and Tn5ME-B / Tn5MErev joints at 72°C for 2 hours to obtain Tn5 working enzyme with cut-paste activity, dilute the working enzyme to 16.5ng / μL, In 4 μL 5x TAPS-MgCl 2 , Digest 50ng of genome in the reaction system of 2μL dimethylformamide (DMF) and Nuclease-free water, the condition is 55℃ for 10min.

[0065] (2) Add 3.5 μL of 0.2% SDS to each reaction, and incubate again at 55° C. for 10 min. Subsequently, a PCR reaction was carried out, and 96 different indexes were included in the primers to distinguish individuals.

[0066] The PCR program i...

Embodiment 2

[0076] This example is used to illustrate the accuracy and timeliness of the method for detecting SNP marker sites provided by the present invention.

[0077] 1. Experimental materials

[0078] Genomes were extracted from blood samples of 3000 individuals in Huiyang bearded chicken and Lingnan yellow chicken distant-origin deep hybrid family and diluted to 40 ng / μL.

[0079] 2. Experimental method

[0080]The basic methods of low-depth DNA library construction and sequencing steps, polymorphic site identification and screening, reference haplotype database construction, candidate sample mutation typing and accuracy assessment are the same as in Example 1. The differences include: the average sequencing depth of each individual is about 0.8×; the reference genome uses the chicken GRCg6a (INSDC Assembly GCA_000002315.5, Mar 2018) version; since the genome heterozygosity and complexity of the hybrid population is much higher than that of the pure line population, Therefore, the...

Embodiment 3

[0084] This example is used to illustrate the influence of the sequencing depth and sample size of each sample on the accuracy of genotyping during the construction of the reference haplotype database.

[0085] The experimental materials and experimental methods used in this embodiment are the same as those in Example 2. In the construction of the reference haplotype database, different reference sample sizes (200, 500, 1000, 1500, 2000, 3000, 4000) and the sequencing depth of each sample (0.05×, 0.1×, 0.2×, 0.3×, 0.5 ×), the accuracy was assessed by comparing the final obtained genotyping results with the high-depth data.

[0086] The result is as image 3 shown. It can be seen from the figure that the average sequencing depth of each sample reaches more than 0.2×, and when the sample size exceeds 1500, the accuracy of genotyping is basically stable (maintained above 98.78%), and no longer increases with the sequencing depth and number of samples. Significant changes occur...

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Abstract

The invention relates to the field of genetics, in particular to a method and a device for detecting SNP marker sites based on low-depth sequencing. The method comprises the following steps: acquiring genome DNA of an individual to be detected; performing individual low-depth whole genome sequencing on the genome DNA, and comparing a sequencing result to a reference genome to obtain polymorphic site information; based on a hidden Markov model, performing genetic typing on the polymorphic site information by utilizing a reference haplotype database, wherein the reference haplotype database comprises mutation site information of a breeding population to which the to-be-detected individual belongs. According to the invention, the low-depth sequencing data of a single sample is utilized to carry out high-accuracy and standardized genotyping of the SNP sites of tens of millions of orders of magnitude of the whole genome in an extremely short time.

Description

technical field [0001] The invention relates to the field of genetics, in particular to a method and device for detecting SNP marker sites based on low-depth sequencing. Background technique [0002] Single nucleotide polymorphisms (Single nucleotide polymorphisms, SNP) are currently the most mainstream genetic markers, which are numerous in the genome, widely distributed, and have good genetic stability. SNP is widely used in the analysis of genetic mechanisms of various traits, selection evolution research and genome prediction in human, animal and plant research. [0003] Different research contents have different requirements for the number of genetic markers. Among them, the research content that needs to use genome-wide high-density markers mainly includes genome-wide association analysis and animal and plant genome selection analysis. In genome-wide association analysis, the use of higher-density genome-wide genetic markers can more accurately identify the true causa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B20/20G16B20/50G16B50/30G16B30/10
CPCG16B20/20G16B20/50G16B30/10G16B50/30
Inventor 胡晓湘王宇哲朱迪任江丽李宁
Owner CHINA AGRI UNIV
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