Development And Validation Of A 32-plex InDels Panel For Forensic Purpose And Population Genetic Analysis In Five Chinese Populations

Background Insertion/deletion polymorphisms(InDels)have attracted more and more attention in the field of forensic medicine because of their characteristics of short amplicons and low mutation rate.Traditionally,short tandem repeats(STRs)are the most commonly used genetic markers depending on their high polymorphism and available allele frequency data of different nations.Despite these advantages,STR markers are limited by relatively high mutation rate and poor performance on highly degraded DNA samples.InDel genetic markers could make up for the shortcomings of traditional STR genetic markers and provide new solutions for forensic personal identification and paternity testing in the face of difficult cases.However,the commercial InDel kit which has been developed recent years is mainly designed for European populations and showed relatively lower genetic polymorphism in Chinese populations.Therefore,constructing an InDel multiplex panel with high genetic polymorphism in Chinese Han population is of great value to forensic practice.Objective To construct a novel multiplex panel containing 32 InDels with high genetic polymorphism in Chinese Han population,to evaluate the genetic polymorphism and forensic efficiency values of this multiplex assay in Chinese Han,Xinjiang Kazakh,Xinjiang Uygur,Xinjiang Mongolian and Xinjiang Hui populations,and to analyze the population genetic relationships between the 5 Chinese populations and 26 reference populationsfrom the 1000 Genome Project.Methods⑴32 InDel loci were selected from the 1000 Genome Project(phase 3)or published studies,primers were designed for each locus using the Primer3 and Auto Dimer software,and four fluorescent dyes were labeled at the 5’end of the forward or reverse primer for each locus according to the arranged groups to construct a multiplex panel;⑵Genomic DNA of 804 samples from five different ethnic groups was extracted by the Chelex-100 method,and the extracted DNA was amplified by PCR and was typed by capillary electrophoresis using the 32-plex InDel multiplex panel;⑶Sensitivity,species specificity,and performance on degraded DNA for the 32-plex InDels multiplex panel were evaluated;⑷Forensic parameters of each population were calculated and linkage disequilibrium was analyzed;⑸According to the genotype data of five different ethnic groups in this study and the 26populations from the 1000 Genome Project,genetic distance,phylogenetic tree,principal component analysis(PCA),multidimensional scale analysis(MDS)and STRUCTURE analysis were carried out.Results⑴The 32-plex InDels panel constructed in this study showed satisfying amplification and genotyping results;⑵The multiplex panel was highly sensitive to DNA template amounts as low as 31.25pg;⑶The panel was specific for human species and cannot obtain complete genotypes of the five animal samples in this study;⑷The panel could obtain complete genotypes for degraded DNA template;⑸The MAF of the 32 InDels for Hubei Han population was greater than0.4 except three loci,and forensic parameters showed high genetic polymorphism in Hubei Han population.The CPD and CPE were 0.999999999999966 and 0.9982,respectively;⑹The results of D_A genetic distance,phylogenetic tree,PCA,MDS and STRUCTURE analysis indicated that among the five studied populations,the closest genetic relationship was between Hubei Han and Xinjiang Hui,Xinjiang Kazakh and Xinjiang Uygur populations.Hubei Han and Xinjiang Hui had close genetic relationship with East Asian group,and the Xinjiang Mongolian,Xinjiang Kazakh and Xinjiang Uygur populations showed mixed genetic structure of European and Asia.Conclusion Inthis study,a novel 32-plex InDels multiplex panel was established and was proved to be highly polymorphic in Southern Chinese Han population.The CPD was high in Chinese Han population so it could be a useful tool in forensic personal identification analysis.The 32-plex InDels multiplex panel was highly sensitive and could be used for genotyping of low-copy DNA and it showed great advantage on degraded DNA.The population genetic structure analysis showed that the 32 InDels in this study was able to distinguish intercontinental populations in some degree.The Han population from Hubei and the Hui population from Xinjiang showed close relationship with East Asia,whereas Xinjiang Mongolian,Xinjiang Kazak and Xinjiang Uygur population showed mixed genetic backgrounds of European and Asian.