Independent Development Of Multi-allelic SNP System Through MPS,Construction Of Six-color Fluorescence Multiplex Panel Of Diallelic DIP And Mini STR By CE And Insights Into The Genetic Structure Of Chinese Tibetan Group Based On DIP Data

Background:In forensic practice,current protocols of forensic investigation are based on the analysis of PCR product size or DNA sequence.Routine forensic analysis using short tandem repeat(STR)often result in fail DNA analysis if the biological samples encountered are degraded or low-template DNA.Mini STR is a common supplementary genetic marker for degradation DNA detection.However,the number of mini STR loci in the same reaction system is limited,which leads to the low cumulative identification efficiency of all loci in the system.Single nucleotide polymorphism(SNP)and deletion or insertion polymorphism(DIP)demonstrate the main characteristics of low mutation rates and small amplified fragments which are appropriate for detecting highly degraded samples.Multiple allelic SNPs(multi-allelic SNPs)have more than two alleles at a nucleotide position.Using SNPs with more than two alleles provides greater discrimination capacity than diallelic SNPs,allowing fewer markers to be typed in order to achieve suitable results;multi-allelic SNPs also give a clearer signal whether to the presence of mixtures by detecting the presence of the third or fourth allele.DIPs manifest as length polymorphisms,supporting the multiple DIP genotyping in capillary electrophoresis instrument and owning simple operation method and low cost.In order to improve the genotyping success rate and increase the cumulative identification efficiency of forensic identification for highly degraded samples,this study aims to construct two forensic identification systems based on two different sequencing methods,and to provide effective new tools for the genotyping of the degradation samples.Studying the genetic structure of each ethnic group is helpful to dissect the genetic background and trace back to the ethnic origin.Tibetan people have lived in the Qinghai-Tibet Plateau(mean elevation over 4500 m)for generations,and can adapt well to the high-altitude environment.Due to the relatively closed geographical environment,Tibetans have preserved their representative physical characteristics and genetic information,thereby become an important research group in human genetics.In this study,genetic background of Chinese Tibetan groups were revealed by 91 DIP loci.Methods and contents:(1)Based on the public SNP database and some customized screening criteria,27 multi-allelic SNPs were selected out and genotyped on the massively parallel sequencing(MPS)platform.Sequencing performance evaluation of this MPS panel,as well as the genetic polymorphism investigations and forensic application efficiency assessment of these loci were conducted in Chinese Mongolian and Chinese Kazak groups.(2)On the basis of capillary electrophoresis platform,a six-dye fluorescent labeling system including 59 diallelic DIP loci,two mini STR loci,2 Y-DIP loci and one Amelogenin gene was developed.According to the validation guidelines for DNA analysis methods formulated by the Scientific Working Group on DNA Analysis Methods,the validation studies were carried out.Moreover,the genetic distribution characteristics and forensic application efficiency of this new system were investigated in Han Chinese in Hunan(CHH),Chinese Tibetan in Qinghai and Tebit(CTQ and CTT)groups.(3)The forensic population structure dissection and genetic relationship analysis of two Chinese Tibetan groups and other 26 reference populations were performed based on 91 diallelic DIP loci data and multitudinous bioinformatics analysis software,such as STRAF version 1.0.5,Arlequin version 3.5,DISPAN program,Genepop version 4.7,TBtools,Phylip version 3.695,MEGA version 7.0,Origin version 2021,STRUCTURE version 2.3.4,R version 3.5.3,CLUMPP version 1.1.2,Distract version 1.1,Infocalc version 1.1,Snipper version 2.5,and so on.Results and conclusions:(1)The sequencing performance of the MPS multi-allelic SNP system was suitable for forensic identifications,and all multi-allelic SNP loci involved herein all identified three and four variational nucleotides in the CMX and CKX groups.Various forensic statistical parameters demonstrated that the MPS panel was an effective system for individual identification and paternity testing.(2)The new system based on capillary electrophoresis platform was strong stability,high sensitivity,good specificity and suitable for the forensic degraded sample detection.The mini STR loci in the system can easily prompt the mixed samples.The Y-DIP loci can identify gender more accurately.These 59 autosomal DIP loci had high polymorphisms and strong discrimination power in CHH,CTQ and CTT groups.This new system will be an effective new tool for the forensic personal identification and kinship identification in East Asian population,and is also easy to be popularized and applied in primary forensic DNA laboratories.(3)The genetic structure between the two Tibetan groups and the 5 populations from East Asia was most similar.At the optimal K value of 3 in STRUCTURE analysis,the proportions of East Asian ancestral composition in the CTQ and CTT groups were 0.8932 and 0.9107,respectively.(4)Forensic ancestral informativeness assessments suggested that several multi-allelic SNP and diallelic DIP loci could be regarded as ancestry informative markers inferring individual biogeographic origins as well as contributing to the forensic anthropology and population genetic researches.