Method for deducing abundance of each family in population based on mixed pool simplified genome sequencing

Abstract

The invention provides a method for deducing the abundance of each family in a group based on mixed pool simplified genome sequencing, which comprises the following steps of: (1) establishing a parent 2b-rad library, and extracting SNP (Single Nucleotide Polymorphism) genotypes and sites by using a 2b-rad standard process; combining the SNP genotypes of the 2n parents into genotypes of n parent pairs according to a male parent and female parent relationship; comparing the genotypes of the parent pairs, and determining family specific tags; (2) extracting offspring individual tissues, mixing, and carrying out DNA extraction and 2b-rad library building sequencing on the mixed sample tissues; counting the sequencing depth of all the specific tag sites and calculating the secondary allele frequency of the specific tag of the mixed sample filial generation; and (3) calculating the proportion of each family in the offspring according to the sequencing depth of the specific tag site and the secondary allele frequency of the specific tag of the mixed offspring. According to the method, the overall feature distribution of the family can be accurately evaluated without sequencing each individual, so that the cost is greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the field of aquatic biological breeding, and in particular relates to a method for inferring the abundance of each family in a population based on pooled simplified genome sequencing. Background Art

[0002] When multiple families are mixed and farmed, inferring the overall characteristics of a single family requires sequencing each individual individually to determine their genetic relationships. While this strategy accurately infers the overall characteristics of a family, it often comes with extremely high library construction and sequencing costs.

[0003] To save costs during breeding, different families are often mixed in the same pond. Farms often prioritize families based on their overall trait distribution, or by comparing traits across families. Using existing technologies, inferring the overall traits of individual families in mixed-breeding situations is prohibitively expensive. High sequencing costs significantly limit family selection. Being able to infer overall traits for families without knowing the pedigrees of individual individuals in mixed-breeding situations would effectively address this cost issue. Summary of the Invention

[0004] Given the limitations of existing technologies, we have developed a cost-effective analytical process and algorithm to infer the overall characteristics of each family when the progeny of multiple lines are mixed and reared, based on the family as the basic unit of production. Current technology requires large-scale library construction and sequencing, which consumes significant manpower and resources. However, our pooled sequencing and family splitting technology can reduce this cost by 40-50 times. Furthermore, our technology can be combined with currently popular pooled whole-genome selection and GWAS techniques to analyze, evaluate, and predict the overall characteristics of a family.

[0005] The technical solutions of the present invention are as follows: A method for inferring the abundance of each family in a population based on pooled simplified genome sequencing, comprising the following steps: (1) Establish a parental 2b-rad library and extract SNP genotypes and loci using the 2b-rad standard process; merge the SNP genotypes of the 2n parents into the genotypes of n parent pairs based on the paternal-maternal relationship; compare the genotypes of these n parent pairs to determine the family-specific signature; (2) Extract tissues from offspring individuals and mix the tissues of offspring individuals to obtain mixed tissues; extract DNA from the mixed tissues and sequence the 2b-rad library; count the sequencing depth of all specific tag sites and calculate the minor allele frequency of the mixed offspring specific tag; (3) Based on the sequencing depth of the specific tag site and the minor allele frequency of the specific tag in the mixed offspring, the proportion of each family in the offspring is calculated.

[0006] Specific instructions: 2b-RAD is a simplified RAD genotyping method based on type IIB restriction endonucleases. The specific library construction and sequencing process is as follows: 1. Tissue extraction: This technique requires mixing of individual tissues. During the experiment, it is necessary to ensure that the tissue and volume of each individual are similar. This experiment uses shrimp muscle tissue. To prevent DNA degradation during sampling, it is immersed in anhydrous ethanol. After sampling, the tissue is frozen with liquid nitrogen and ground into powder in a mortar using a grinding rod. The biological tissue is thoroughly mixed.

[0007] 2. DNA Extraction: Use a marine animal tissue genomic DNA extraction kit. DNA samples should be analyzed by agarose gel electrophoresis. The main band should be clear and intact, without degradation, and the concentration should be greater than 40 ng / µL.

[0008] 3. DNA quality control: Determine the concentration of purified DNA using a NanoDrop and analyze sample bands using 1% agarose gel electrophoresis. Qualified samples were diluted to 100 ng / uL for library construction.

[0009] 4. Library Construction: A simplified genomic library of Litopenaeus vannamei was constructed using the 5-tag concatenated simplified genome MisoRAD technology developed independently by the laboratory. The specific experimental steps are as follows: (1) DNA digestion: Use BsaXI enzyme to digest the purified DNA to obtain DNA fragments with a length of 27 bp.

[0010] (2) Ligation: The five samples were ligated together using the adaptorA and adaptorB linkers at 16°C for 1 h. The adaptorA is ACACTCTTTCCCTACACGACGCTGTTCCGATCTNNN (SEQ ID NO. 1) and the adaptorB is GTGACTGGAGTTCAGACGTGTGCTCTTCACGACAGTCTNNN (SEQ ID NO. 2).

[0011] (3) Amplification and enrichment: PCR amplification and enrichment were performed, and the products were recovered by gel electrophoresis. The recovered products were then amplified again and purified using a PCR product purification kit.

[0012] (4) Short tag concatenation: Use Lgu enzyme to remove the linker fragment and use T4 ligase to concatenate the five tags.

[0013] (5) Library detection and sequencing: Use electrophoresis to detect library fragments and use Qubit to quantify. Then use the second-generation sequencing platform to sequence the mixed library.

[0014] Furthermore, in step (3), the proportion of each family was calculated using the least squares method and the Lagrange multiplier method.

[0015] Furthermore, in step (2), the tissues of the offspring individuals are mixed in equal amounts.

[0016] Furthermore, the specific tag means that less than half of the parent pairs have this base pair at this site.

[0017] Furthermore, the group is an aquatic organism group.

[0018] Furthermore, the aquatic organisms include fish and crustaceans.

[0019] Furthermore, the crustaceans include shrimps.

[0020] Furthermore, the shrimp includes Penaeus vannamei.

[0021] Compared with the prior art, the present invention has the following advantages: This technology can sequence a pool of offspring with unknown pedigrees and determine the proportion of each family. When different families are mixed in a single pool, this technology can assess the overall distribution of family characteristics without sequencing every individual, significantly reducing costs.

[0022] To evaluate the accuracy of this technology in estimating pedigree proportions after pooling progeny, we conducted an experimental validation experiment. Progeny with known pedigrees were mixed and sequenced in proportion. The technology was then used to perform pedigree splitting, and the results were compared with the actual proportions. The results demonstrated that this technology accurately generated pedigree splitting. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Operation flow chart of the present invention.

[0024] Figure 2 : Exploration of the accuracy of family splitting. Analysis results of 50 offspring.

[0025] Figure 3 : Exploration of the accuracy of family splitting. Analysis results of 115 offspring. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0027] In a breeding production environment where the family is the basic breeding unit, seedlings from multiple families are mixed and cultured in one breeding environment. The following operations are performed: Experimental steps: (1) Take the parental tissue sample. Use 2b-rad to construct a library for sequencing of the parental tissue alone.

[0028] (2) Extract tissues from offspring individuals and mix them at equal mass to obtain mixed tissues. Use a kit to extract DNA from the mixed tissues and sequence the 2B-rad library.

[0029] Analysis steps: (1) The 2n parental sequencing results were used to extract SNP genotypes and sites using the 2b-rad standard process.

[0030] (2) Combine the SNP genotypes of the 2n parents into the genotypes of n parent pairs based on the paternal and maternal relationships.

[0031] (3) Compare the genotypes of these n parent pairs and determine the family-specific signature (a specific signature means that less than half of the parent pairs have this base pair at this site).

[0032] (4) Calculate the minor allele frequency of the hybrid generation-specific tag site.

[0033] (5) The proportion of each family was calculated using the least squares method and the Lagrange multiplier method.

[0034] The specific algorithm is as follows:

[0035] Example: 1. Establish 2b-rad libraries of seven family parents: Muscles from each parent were cut and frozen in liquid nitrogen for storage. 50 mg of muscle samples were taken from each parent. DNA from each individual sample was extracted using the phenol-chloroform extraction method. 2b-rad libraries of different samples of whiteleg shrimp were constructed using the 2b-rad library construction kit, and then sequenced and analyzed.

[0036] 2. Establish a progeny mixed pool 2b-rad library: Muscles from each offspring of the pool were cut and frozen in liquid nitrogen for storage. 20 mg of samples were taken from each individual and mixed together. DNA was extracted using the phenol-chloroform method. 2b-rad libraries were constructed for different samples of whiteleg shrimp using the 2b-rad library construction kit, followed by sequencing analysis. We conducted two sets of experiments, each with three replicates. One set of experiments used a total of 50 juvenile shrimp individuals, and the other set used a total of 115 shrimp. The number of shrimp in each family is shown in the following table: Table 1 Number of shrimps from each family used for mixed sampling

[0037] Analysis steps: (1) The 2*7 parental sequencing results were used to extract SNP genotypes and sites using the 2b-rad standard process.

[0038] (2) Combine the SNP genotypes of 2*7 parents into genotypes of 7 parent pairs based on the paternal-maternal relationship.

[0039] (3) Compare the genotypes of these 7 parent pairs and determine the family-specific tag sites (a specific tag means that less than half of the parent pairs have this base pair at this site). The sites where all family-specific tags are located need to be counted: the proportion of parent-specific bases in each family (for example, if the parent genotype is AATT and the specific base is A, its proportion is 2 / 4) (4) Count the sequencing depth of all specific tag sites and calculate the minor allele frequency of the hybrid generation specific tag sites.

[0040] (5) The proportion of each family was calculated using the least squares method and the Lagrange multiplier method.

[0041] We use a bar chart to show the final results of the two sets of experiments and the actual mixed sample input ratio. Figure 2 and Figure 3 As shown: Figure 2 For an experiment with 50 offspring, Figure 3 This experiment involved 115 offspring. We can see that for both sets of experiments, the correlation coefficients between all three replicates and the true feed ratio were above 0.97. This fully demonstrates the reliability of our established method.

[0042] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for inferring the abundance of each family in a population based on pooled simplified genome sequencing, characterized in that: The following steps are involved: (1) Establish a parental 2b-rad library and extract SNP genotypes and loci using the 2b-rad standard process; merge the parental SNP genotypes into n parent-pair genotypes based on the paternal-maternal relationship; compare the genotypes of these n parent-pairs to determine the family-specific signature; (2) Extract tissues from offspring individuals and mix the tissues of offspring individuals to obtain mixed tissues; perform DNA extraction and 2b-rad library sequencing on the mixed tissues; Count the sequencing depth of all specific tag sites and calculate the minor allele frequency of the hybrid generation specific tag; (3) Based on the sequencing depth of the specific tag site and the minor allele frequency of the specific tag in the mixed offspring, the proportion of each family in the offspring is calculated.

2. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 1, characterized in that: In step (3), the proportion of each family was calculated using the least squares method and the Lagrange multiplier method.

3. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 1, characterized in that: In step (2), the tissues of the offspring individuals are mixed in equal amounts.

4. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 1, characterized in that: The 2b-RAD library construction and sequencing process is as follows: (1) Collect individual tissues and mix them, extract genomic DNA from the tissues, perform DNA quality inspection, and use qualified samples for library construction; (2) Library construction: A simplified genome library of Penaeus vannamei was constructed using the 5-tag concatenated simplified genome MisoRAD technology.

5. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 4, characterized in that: The construction method of the library is: 1) DNA digestion: Use BsaXI enzyme to digest the purified DNA to obtain DNA fragments with a length of 27 bp; 2) Connection: The five samples were connected together through the adaptorA and adaptorB linkers; where adaptorA is ACACTCTTTCCCTACACGACGCTGTTCCGATCTNNN, and adaptorB is GTGACTGGAGTTCAGACGTGTGCTCTTCACGACAGTCTNNN; 3) Amplification and enrichment: PCR amplification and enrichment are performed, followed by gel electrophoresis and recovery. The recovered products are then amplified again and purified. 4) Short tag concatenation: Use Lgu I endonuclease to remove the adapter fragment and use T4 ligase to concatenate the five tags; 5) Library detection and sequencing.

6. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 1, characterized in that: The population is an aquatic organism population.

7. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 6, characterized in that: The aquatic organisms include fish and crustaceans.

8. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 7, characterized in that: The crustaceans include shrimps.

9. The method for inferring the abundance of each family in a population based on pooled simplified genome sequencing according to claim 8, characterized in that: The shrimp includes whiteleg shrimp.

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