| Objective:Of the 23 pairs of human chromosomes,22 are autosomes and the other pair is sex chromosomes.In different genders,the male sex chromosome composition is XY and the female is XX.The Y chromosome is only present in male cells and belongs to the proximal centromere chromosome.There are five types of polymorphic genetic markers on the human Y chromosome,including satellite DNA,small satellite DNA,microsatellite DNA,InDel,and SNP.The significance of the analysis of the Y chromosome is that the Y chromosome exists only in male individuals,and the Y chromosome STR is stably inherited by the father to the offspring.The Y chromosome-specific STR locus is characterized by haploid paternal inheritance and is unique in forensic identification compared with the autosomal STR locus.With the development of science and technology,the progress of sequencing technology is also changing with each passing day.The sequencing technologies currently in use include Sanger sequencing,pyrosequencing,and next-generation sequencing technologies that are rapidly evolving.The next-generation sequencing technology can sequence hundreds of thousands to millions of DNA molecules in multiple samples at a time,with sequencing accuracy of over 99%.The high-quality data generated by a single sequencing is sufficient at an average level.Covering the human genome more than 30 times,it provides a new technical means for DNA analysis.The DYF155S1 locus is the small satellite polymorphism site on the first Y chromosome reported,with length polymorphism and sequence polymorphism,and there are differences in repeat sequence between different ethnic groups.Therefore,this study intends to explore the following three aspects: 1.Using the next-generation sequencing technology to analyze the Y chromosome STR and its flanking SNP,explore the accuracy of the next-generation sequencing technology in the Y chromosome STR core sequence determination and explore the next-generation sequencing technology to solve The role of the mixed spot test.The secondary PCR and sanger sequencing technology were used to study the samples of four populations including Chinese Han population,and the genetic polymorphism and difference between groups of DYF155S1 locus were discussed.3.Carry out population genetics research and explore the Y-STR data characteristics of the Korean population in Yanbian,Jilin Province,China,and provide basic data for the application of Y chromosome STR locus in forensic science.Methods:In this study,20 unrelated parental samples were selected,and two unrelated individual samples were selected to form mixed plaque samples with similar DNA content.The two groups determined four samples of father-son relationship.All samples were subjected to Y chromosome STR typing using the ABI Y Filer Plus kit.A sequence-specific primer was designed for each 1.5Kb range upstream and downstream of each STR locus of the Y chromosome,and a DNA fragment containing each Y chromosome STR core sequence was amplified.The amplified DNA fragment was randomly fragmented under the action of a transposase carrying a linker at both ends,and the linker was ligated at the end of the fragment.Repair the nick at the transposition junction,then perform a PCR reaction to enrich the library and add the full length linker required for sequencing.Fragment lengths were screened using AMPure XP Beads to allow target fragments ranging in length from 300-400 bp.The library was mixed,denatured,and added to the Illumina HiSeq sequencing platform for high-throughput parallel sequencing.The two ends of the library were sequenced separately(ie paired-end,PE),so each sample generated reads1(R1)and reads2(R2).)Two data files.Quality control and quality inspection were performed on the sequencing raw data.After confirming the reliability of the original data,the original data was compared with the reference sequence to obtain the STR and SNP data of each sample.Observe whether the core sequence of the Y chromosome STR locus of each sample can be accurately read out,and whether the STR locus of the mixed sample can be accurately classified by data comparison,and whether the haplotype of different samples can be assembled.The DYF155S1 locus of 200 samples from 4 groups in China: Han,Korean,Tibetan and Uygur were investigated by secondary PCR and Sanger sequencing.The genetic analysis of DYF155S1 locus among the four ethnic groups was statistically analyzed.Polymorphism and differences between groups.Statistical analysis was performed on Y-staining STR data of 257 Korean male samples from Yanbian,Jilin Province,China.According to different regions,the corresponding forensic parameters include haplotype number,haplotype diversity,random matching probability,number of haplogroups,and so on.Principal component analysis was performed using R language based on the haplogroup frequencies of 10 populations to determine the genetic distance between different populations.Results:1.This study established a method for sequencing and analyzing the Y chromosome STR locus by combining regional capture technology with next-generation sequencing technology.The feasibility of studying the STR locus repeats and their repetition times by the next-generation sequencing technology was discussed.It is proved that the next-generation sequencing technology of single repetitive sequence STR loci can be better analyzed by sequencing,and the number of repetitions is more.Or the sequencing unit has more STR sites and the sequencing results are not good.The results show that the use of next-generation sequencing technology to study the more complex STR loci such as DYS385,DYS387S1 locus repeats and their number of repetitions has certain limitations,and further research is needed.2.The results of this study indicate that the use of next-generation sequencing technology to study the repeated sequence of STR loci and the number of repetitions of mixed samples has certain limitations and needs further study.3.A total of 5 DYF155S1 locus repeats were found in this study,which were type 1,type 3,type 4,type 6 and type 7.Including the present study,nine types of repeats of the DYF155S1 locus have been found.The study found that the structure of DYF155S1 locus has obvious ethnic differences.The type 6 repeats found in this study were only detected in Uighur and Tibetan individuals,and the type 7 repeats were only detected in Tibetan individuals and Korean individuals.4.In this study,48 Han individual samples were detected in 48 structural compositions,50 Uyghur individual samples were detected in 48 combinations,50 Korean individuals detected 49 structural components,and 50 Tibetan individuals were examined.There are 42 structural combinations.A total of 200 individuals in the DYF155S1 locus detected a total of 187 structural components,suggesting that the polymorphism of this locus is very high,and is an important genetic marker for forensic identification and personal identification.After analyzing the Y chromosome of 257 male individuals in the Yanbian area,the GD value of the DYS385 locus in the 19 STR loci was 0.9666,and the GD value of the DYS391 locus was the lowest,0.2260.Among the 18 STR loci except DYS385,the number of DYS391 alleles was the least,which was 3,and the DYS456 and DYS447 loci had the most alleles,8 of them.4.Genetic distance analysis was carried out among 10 males in the Yanbian area and the YHRD database in Beijing Han,Jiangsu Han,Jilin Han,Yunnan Han,Liaoning Hui,Liaoning Korean,Liaoning Manchu,Guangxi Miao,Japanese,and Korean.The analysis results show that the genetic distance between the Koreans in the Yanbian area and the Koreans and Koreans in Liaoning is similar,but far away from other ethnic groups,especially the genetic distance of Guangxi Miao.Conclusion:1.This study used the method of region capture and sequence analysis to analyze the Y chromosome STR locus,and confirmed that the method can analyze the Y chromosome STR and its flanking sequences more efficiently and accurately.2.The polymorphism distribution of DYF155S1 locus in four ethnic groups in China was investigated.It was confirmed that this locus has certain ethnic differences in the distribution of core sequences and arrangement,which can help individual ethnic division.3.A frequency survey was conducted on 19 Y chromosome STR loci in the Korean ethnic group in Yanbian,China,and corresponding data were obtained.Koreans in Yanbian area have similar genetic distances to Korean Koreans and Koreans,but far away from other ethnic groups. |
