Development Of A High Resolution Y-SNP Haplogroup Multiplex System For Forensic Purpose

Objective: The non-recombining region(NRY)genetic marker of Y chromosome has the characteristics of patrilineal inheritance,haplotype inheritance and small effective population,which has become an ideal marker for studying human evolution and population genetics.Y-short tandem repeat(Y-STR)and Y-single nucleotide polymorphism(Y-SNP)are two of the most important genetic markers of NRY region.The phylogenetic tree constructed by Y-SNP for haplogroup division is one of the main methods for inferring geographic ancestry and population.The distribution of Y-SNP or haplogroups in different regions and populations shows certain characteristics.Therefore,the Y-SNP information of male samples from unknown sources can be used to classify the haplogroups,so as to infer their geographical origin or the population to which they belonged.In addition,Y-SNP haplogroup combined with Y-STR data can also be used for family traceability,so as to assist in case detection,which has important forensic significance.Therefore,in this study,a group of valuable Y-SNP markers would be seleted according to various regions of Y chromosome genetic structure characteristics,building a high resolution integrated Y haplogroup phylogenetic tree and being suitable for Chinese population,which would be aimed to provide a basis for inferring the geographical,ethnic and pedigree traceability of male samples from unknown sources in China,and providing a new technical method for forensic practice.Methods: A total of 183 Y-SNP loci were selected according to literature survey and the 2017 ISOGG website(Version: 12.334),and a Y-SNP multiplex system was developed based on Mass ARRAY system and MALDI-TOF MS technology.Based on the results of previous studies and relevant literature,381 Y-SNP were screened with reference to the latest version of ISOGG website(Version: 15.58)in2019 Based on next-generation sequencing technology,a complete and highresolution Y-SNP multiplex system suitable for Chinese population was developed.According to the "Guidelines for Validation of DNA Analysis Methods(2016)" proposed by the Scientific Working Group on DNA Analysis Methods(SWGDAM),the forensic performance of the two systems were evaluated in terms of repeatability,accuracy and consistency,sensitivity,specificity,mixtures,suitability of forensic case samples and population studies.Results: Based on Mass ARRAY system and MALDI-TOF MS,a multiplex system containing 183 Y-SNP loci was successfully developed(named 183-plex Panel),and the amplified fragment size was about 80～120 bp.The panel were divided into five pools,named Pool 1,Pool 2,Pool 3,Pool 4,and Pool 5,including 36,42,40,35 and 32 Y-SNP makers,respectively.The call rate of each Pool was 99.72%,99.05%,96.25%,98% and 96.25%,respectively,with an average call rate of97.89%.Based on the next-generation sequencing technology,a genotyping system containing 381 Y-SNPs was successfully developed(named NGS Panel).The amplified fragment size ranged from 160 to 280 bp,and the average call rate of over 20 X was 95.81%.The haplogroup results of the two systems were consistent and the accuracy was up to 100%.The sensitivity of 183-plex Panel was low,and complete profile could be obtained at 10 ng of DNA input.NGS Panel was highly sensitive,and complete 381 Y-SNP makers could be genotyped when the DNA input was as low as 1.25 ng.However,for both systems,the correct direction of haplogroup branching was obtained even at a low concentration of DNA input(78.125 pg).In terms of mixture detection by NGS Panel,the haplogroup showed a common main haplogroup when the male-male mixture ratio was 1:1,1:3 and 3:1.When the mixing ratio was 1:9,1:19,9:1 and 19:1,haplogroup presented as a result of the dominant component of the male haplogroup.Both systems were suitable for different types of case samples;And for biometric materials from different tissues of the same person,haplogroup result was consistent with organizational identity.In the population study,the results of 183-plex Panel of a total of 1419 unrelated male individuals from 9 different populations in China showed that haplogroup distribution was different among different Chinese populations.For example,O haplogroup was most distributed in the Han population,which was accounted for70%～86%;C haplogroup was more distributed in the Xinjiang Mongolian and was accounted for 67%.While D haplogroup was more distributed in the Qinghai Tibetan,and R haplogroup was more distributed in the Xinjiang Uygur population.Which were accounted for 62% and 34%,respectively.A total of 134 terminal haplogroups were obtained from 9 populations,and the haplogroup diversity value(HD)was 0.95531,among which the Han populations had the highest HD value,and HD was 0.96572 from Shandong Han and 0.94596 from Jiangsu Han,respectively.A total of 93 terminal haplogroups were detected by NGS Panel in200 Han population,and the HD was as high as 0.9883,which was higher in resolution and system efficiency than that of 183-Plex Panel.NGS Panel could distinguish two unrelated individuals sharing Y-STR haplotypes,and could achieve the purpose of distinguishing different pedigrees at haplogroup level.Conclusion: In this study,183 Y-SNP multiplex system based on Mass ARRAY system and MALDI-TOF MS technology and the 381 Y-SNP multiplex system based on next-generation sequencing technology were developed.The performance of the two systems was evaluated in terms of repeatability,accuracy and consistency,sensitivity,specificity and applicability of forensic materials.The results indicated that the two systems had good overall performance.The 183-plex Y-SNP Panel has a certain ability of distinguishing in Chinese population,and the haplogroup distribution of different populations of Chinese shows specificity,and the haplogroup resolution is higher especially in the Han population;While NGS Panel has the advantages of high sensitivity,high resolution and high system efficiency,which can achieve the purpose of distinguishing different families at haplogroup level.The combined application of high-resolution Y-SNP haplogroup constructed in this study and Y-STR data plays a very helpful role in the application of forensic genealogy tracing or family investigation.In the case of sufficient amount of DNA template and equipped with experimental equipment,combined with the advantages of low cost saving,time saving and simple data processing,183-plex Y-SNP Panel can be considered for judicial application,especially for the construction of Y-SNP database.If laboratory conditions permit,NGS panel is recommended to obtain high resolution haplogroup results,with more detailed haplogroup division and more accurate inference.The population haplogroup data obtained in this study provide a basis for the population structure and anthropology of forensic genetics in China.