Studies On Sequence Feature And Genetic Polymorphism Of RM Y-STRs In Hebei Han Population

Objective:Y-STR has the characteristics of male peculiarity and patrilineal inheritance,so it has special application value in sexual assault investigation,paternal paternity test,male pedigree inference of missing persons and so on.At present,most of the routinely used Y-STR genetic markers show the same haplotype in the same patrilineal family,which is beneficial to the identification of the same paternal family,but it is impossible to distinguish different male individuals in the same patrilineal family.In recent years,it has been found that there is a rapid mutation Y-STR（rapidly mutating Y-STR,RM Y-STR）o⁴n Y chromosome,and its mutation rate is more than 1×10-2,which has higher genetic polymorphism than common Y-STR,so it may have high application value in the identification of male relatives.In order to further study the sequence structure and genetic characteristics of RMY-STR,capillary electrophoresis（capillaryelectrophoresis,CE）typing and next generation sequencing（nextgenerationsequencing,NGS）were used to explore the gene sequence characteristics,genetic polymorphism and mutation rate of RMY-STR locus in Hebei Han population,to evaluate its forensic application value,and to provide basic data for forensic application of RMY-STR locus.To provide a new technical scheme for forensic medicine to solve the individual identification of male relatives.Methods:1.Microreader allele RM-Y ID System(（including 15 RM Y-STR loci）was used to detect CE in 475 unrelated individuals and 500 pairs of father-child samples of Hebei Han nationality.Genetic polymorphism and mutation rate analysis of RM Y-STR loci were carried out in Hebei Han population.2.Using the NGS-RM Y-STR typing system（including 19 RM Y-STR loci）constructed by our laboratory,44 parent-child pairs and 2800m DNA were used for library construction,library quality control and computer sequencing.The fastq files of NGS offline data were analyzed by wintermute software modified version and STRait Razor V3.0 software,the sequence characteristics of RM Y-STR loci were obtained,and the results of this system were compared with those of CE.The typing differences between father and son pairs were compared.Results:1.Analysis of genetic polymorphism of RM Y-STR locusA total of 676 alleles were detected in 15 RM Y-STR loci of 475 unrelated male individuals of Han nationality in Hebei Province,and the number of alleles detected at each locus ranged from 8（DYS630）to 239（DYF399S1）.A total of 474 haplotypes were detected in 475 unrelated males,and the haplotype polymorphism of 15 RM Y-STR loci was as high as 0.999991,and the recognition ability wa⁵s 99.79%.There were also 52 multiple alleles in 33 male individuals,involving 10 loci.2.Analysis of sequence feature of RM Y-STR locus2.1 All libraries involved in sequencing meet the quality control standards.DYS547,DYS526 II,DYS526I and DYS713 loci were removed in the subsequent NGS data analysis because of the existence of non-specific products.The analysis threshold of DYS449 loci was set to 19.0%,and the analysis threshold of other loci was 11.0%.The average Do C of 64 samples and3 2800m is 78643±42879×（average±SD）,the overall average Do C of loci is4797±5215×（average±SD）,and the average scores of Allele%,Stutter%and Other%of all loci are 84%,11%and 4%,respectively.2.2 The NGS-RMY-STR typing system showed good specificity except for non-specific amplification of DYS526I,the results of NGS typing of 13 loci common to Microreader genotyping RM-Y ID System were the same as those of CE typing,and the results of 6 repeated sequencing of positive standard2800m were the same,indicating that the system had good specificity,consistency and repeatability.2.3 A total of 1498 alleles were detected in 64 samples by NGS,74 more alleles and 128alleles based on length polymorphism were detected by CE.20abnormal multi-allele genotypes were detected by NGS typing method,involving 5 loci.Compared with CE,NGS can identify the sequence differences between multi-alleles and infer the source of multi-allele variation by comparing the depth of sequencing.3.Investigation of mutation rate of RM Y-STR locusThe mutation rate of 15 RMY-STR loci of 500 father-son pairs was investigated.138 mutation events were found in 120 father-son pairs,including134 one-step mutation,3 two-step mutation and 1 three-step mutation.24.0%father and son could be distinguished.The mutation rate of each locus was between 2.00×10^-3 and 4.20×10^-2,and the average mutation rate was 1.62×10^-2.Kruskal Wallis test showed that the mutation was related to the father’s childbearing age.NGS method detected 11 mutations in 44 parent-child pairs of 64 samples.NGS⁶ detection method locked the location and specific sequence of mutations,providing more valuable information.Conclusion:In this study,NGS and CE methods were used to analyze the sequence and genetic characteristics of RMY-STR loci in Hebei Han population.It was found that the haplotype diversity of 15 RMY-STRs in Hebei Han population was 0.999991,with a high genetic polymorphism,and the average mutation rate was 1.62x10-2,which could distinguish 24%father-son pairs.The RMY-STR typing system constructed in our laboratory has good stability,consistency and repeatability,obtains the sequence characteristics of RMY-STRs,detects more alleles,defines the mutation location and sequence,and infers the variation source of abnormal alleles according to the sequencing depth,which provides valuable genetic markers and technical methods for individual identification of male relatives.