Construction And Validation Of A 74AIM-SNPs Multiplex System Based On Capillary Electrophoresis

BackgroundIn the long process of human migration and evolution,there are SNP loci with large frequency differences among people in different geographic regions.Such SNP loci are called ancestry informative SNPs(AISNPs).In previous study,we developed a 74 ancestry informative single nucleotide polymorphisms(AISNPs)panel that could distinguish 10 biogeographic groups of global populations(Sub-Saharan Africa,North Africa,Europe,Southwest Asia,South Asia,North Asia,East Asia,Southeast Asia,Pacific and Americas).The 74 AISNPs panel can further divide East Asian into North Asia,East Asia,and Southeast Asia while maintaining the performance at global differentiation.However,the system has not yet been implemented in capillary electrophoresis platforms which is commonly used in forensic DNA laboratories.And this deficiency can raise difficulties to popularize this system.In order to overcome the shortcomings,a multiplex system based on SNaPshot technique was developed to make it applicable to detect the genotypes of 74 AISNPs by capillary electrophoresis platforms which is widely used in forensic DNA laboratories.Furthermore,the present study conducted validations of the multiplex system to provide a research basis for the popularization and application of the 74SNPs ancestry inference panel in the forensic DNA laboratory.MethodThe sequence information of 74SNPs were downloaded from the Ensembl Genome Database website.We used Primer Premier 5 software to design PCR primers,and used Primer-BLAST tool provided by NCBI website to verify primer specificity.Based on SNaPshot technique,the 74 AISNPs multiplex system were developed,and referred to the forensic science SWGDAM verification guide,validation studies,such as PCR conditions,species specificity,sensitivity studies and stability studies,accuracy studies,case-type samples simulations,were tested on the multiplex system.A total of 537 unrelated individuals were collected from Han Chinese in Qinghai,Yao in Guangdong,Cantonese in Guangdong,Teochew in Guangdong and Hakka in Guangdong.The 74SNPs typing data of individuals were obtained through the constructed multiplex system,and the genetic structure and ancestral component ratio of the tested population were further analyzed by using allele frequency calculation,principal component analysis,phylogenetic tree analysis,STRUCTURE analysis,and LR value calculation.ResultThe constructed 74AIM-SNPs composite detection system performed PCR amplification and single base extension reaction in three reaction tubes,and the three reaction tubes contain 28,25,and 21 locus,respectively.The detection system can detect and obtain 74SNPs typing data under the condition of 36～44 PCR cycle number without the generation of non-specific peaks.The system can be tested at PCR annealing temperature of 51～59℃ to obtain reliable results.Chimpanzees and rhesus monkeys generated 64 and 32 locus,respectively,and cattle,dogs,pigs,sheep,mice,chickens showed no amplification except for the "T" typing peak detected at rs10496971.The detection system was of great sensitivity.The developed multiplex system showed a certain tolerance to common inhibitors such as hemoglobin,EDTA,calcium ion and indigo.The detection results of 10 different genotyping samples were consistent with the results of Sanger sequencing.The developed 74 multiplex SNPs system was applicable to routine forensic casework samples including blood gauzes,blood cards,buccal swabs,seminal stains,hair folliciles and swabs of bottleneck.The 74SNPs genotyping data of the test population were used for principal component analysis,phylogenetic tree analysis,STRUCTURE analysis,and calculation of LR values.The analysis results were consistent with the actual geographic location of the test population.Conclusion:The developed system performed well in genotyping accuracy,peak height balance and sensitivity and it is applicable to routine forensic casework samples.Our study make the 74SNPs ancestry inference panel applicable to capillary electrophoresis platforms and provide a research basis for the popularization of the 74SNPs ancestry inference panel in the forensic DNA laboratory.