Determining method for sequencing enzyme digestion combination in sequencing genotyping technology

Abstract

The invention provides a determining method for sequencing enzyme digestion combination in a sequencing genotyping technology. The determining method comprises the following steps that 1, restriction enzyme digestion site predicting is performed on a target genome, and the number of enzyme digestion segments obtained through different enzyme digestion modes is counted; 2, a joint sequence and a PCR amplification primer sequence at the two ends of each enzyme digestion segment are designed according to the predicted enzyme digestion segments in the various enzyme digestion modes in the step 1; 3, sequencing libraries are constructed through a GBS technology for the different enzyme digestion modes; 4, sequencing is performed through the sequencing libraries constructed in the step 3; 5, SNP marker sites are obtained according to sequencing results; 6, the specific enzyme digestion combination for the target genome is determined according to the number of the SNP marker sites and the enzyme digestion segment sizes which are obtained through different enzyme digestion combinations.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for determining a combination of sequencing restriction enzymes in sequencing genotyping technology. Background technique [0002] Genetic molecular markers (measurable and inheritable polymorphisms among different individuals within one or more populations) firmly occupy the core position of modern genetics and are also important for disciplines such as population genetics, ecology and developmental biology. research direction. The current mainstream genetic markers have been developed to the third generation, that is, single nucleotide polymorphisms (Single Nucleotide Polymorphisms, SNP) molecular markers. This kind of genetic marker is characterized by the substitution of a single base, and generally consists of only two bases. It is a dimorphic marker, and the difference in length between the first-generation RFLP and the second-generation STR is used as a genetic marke...

AI Technical Summary

Problems solved by technology

However, different simplified genome sequencing methods are quite different in terms of library construction strategy, single-enzyme digestion / double-enzyme digestion combination selection, and sequencing platform selection, which will significantly affect the efficiency and cost of subsequent typing

For example, in the RAD sequencing method, the library construction strategy is complicated, and too many steps will interfere with the results of subsequent experiments; the frequency and distribution of different restriction endonucleases on the genomes of different species are quite different. Species, which enzyme to use for experiments becomes the decisive factor in determining the number and cost of SNPs obtained in experiments; 2b-RAD technology uses type IIB restriction endonucleases, although 2b-RAD technology can obtain enzyme-digested fragments at the whole genome level, but The size of this enzyme-digested fragment is only 25-35bp. According to the average frequency of genome-wide variation, too short enzyme-digested fragments are difficult to enrich SNP sites, which will cause a large loss of sequencing data; When comparing the repetitive regions of the genome, a large number of alignment errors will be brought about, and it will also strongly interfere with the typing accuracy of SNPs, thereby seriously interfering with downstream applications

When analyzing SNP marker sites, single enzyme digestion is not conducive to the screening of enzyme fragments in subsequent experiments, while some double enzyme digestion combinations have the disadvantage of too many or too few enzyme fragments, and too many enzyme fragments will increase the efficiency of the experiment. Cost, too few enzyme fragments will reduce the density of SNP mining, which will affect the subsequent biological analysis, and some enzyme digestion combinations will affect the efficiency of enzyme digestion due to genome methylation

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