The Construction Of 1,245 SNPs Targeted Capture And High-throughput Sequencing System And Its Application In Forensic Medicine

Background:In recent years,with the continuous innovation of modern technology,the forensic evidence test has also developed in a more advanced direction.Among them,the most widely used in modern forensic application is short tandem repeat(STR)as a genetic marker,combined with high-resolution capillary electrophoresis(CE)for individual identification and paternity testing.STR has a high individual recognition efficiency and can solve general forensic problems.Compared with the traditional STR locus,Single nucleotide polymorphisms(SNPs)have shorter amplified fragments,wider distribution among human genome,and lower mutation rate,which can be used to solve forensic complex materials which are still staying challenge using STRs,such as identification of mixed samples(mixing ratio>10:1)or highly degraded samples(<100 bp).Currently,SNP genotyping assays of the commercial SNPs kits are mainly based on single-base primer extension and Sanger sequencing,which can only analyze a few SNPs simultaneously due to multiplexing limitations,so that greatly limited the application of SNP marker.Compared with Sanger sequencing,Next Generation sequencing(NGS)has higher sequencing depth,multi-parallel,and higher accuracy,which offers a revolutionary method of massive parallel sequencing for detecting large number of SNPs as well as analyzing multiple samples in a single reaction,for complementing the shortcomings of the STR-based approach.In order to efficiently genotype the selected SNPs,optimizing the DNA library construction is critical.In this study,we developed a panel including 1,245 SNPs based on Molecular Inversion Probe(MIP)and NGS for forensic applications.Objective:The combination of Molecular Inversion Probe(MIP)technology and Next-Generation Sequencing technology(NGS)was used to establish a SNPs panel to solve the complex issues encountered in forensic practices.Method:(1)SNPs selection criteria:the minor allele frequency(MAF)of the candidate SNPs must be more than 0.3 both in worldwide and East Asia(EAS)populations;the SNPs must be benign regarding human health;no linkage disequilibrium(r~2<0.1).(2)MIP sequences were designed for the targeted sites using MIPGEN software,and the poor quality sites would be removed,such as high GC content or located in the repeat region.(3)Optimizing the MIP-SNPs system in a pilot study to make the panel more balanced.(4)We applied the method to genotype a population of 210 unrelated individuals from the Hubei province of China and assessed the allele frequencies,Hardy-Weinberg equilibrium and linkage disequilibrium.(5)Two groups of artificial mock degraded samples were tested using the MIP-SNP system.(6)Seven trio families were tested to further determine the efficacy of the system.Result:(1)According to the 1000 Genome Project data,all SNPs we selected showed high heterozygosity(MAF>0.3).We applied the MIP-NGS method to genotype 210 unrelated individuals from Hubei Province,China,and assessed allele frequencies,Hardy-Weinberg equilibrium and linkage disequilibrium.More than 95%of the SNPs had MAF?0.2,and no significant deviation or strong linkage was observed for 98%of the SNPs.(2)In addition,we evaluated the performance of the MIP-NGS method using simulated degradation DNA samples.Even if the genomic DNA is sonicated to the~100bp range,we can genotype most SNPs.(3)Finally,we used a low-quality DNA sample to perform paternity testing on seven trio families.The conclusion is completely consistent with the analysis of STR and has a higher confidence index.Meanwhile,the random matching probability is calculated for these 21individuals,and the results have reached the requirements of individual identification.Conclusion:In summary,we have established a high-accuracy and high-performance MIP-SNPs system and genotyping method containing 1,245SNPs,which may be used to solve the complex problems encountered in forensic practices.