Validation And Application Of A New X-STR Multiplex System

Background:In the past decades,short tandem repeats(STRs)have become the cornerstone for forensic laboratories to carry out human identification studies and paternity testing.X chromosome STRs(X-STRs)can be a powerful complement to the results of autosomal STR analysis in forensic experiments,especially for the analysis of some complex genetic relationships.At present,the most widely used X-STR multiplex detection kits in domestic and overseas markets are the AGCU-X19 STR kit,Typer X19 kit containing 19 loci and the Investigator Argus X-12 Kit containing 12 loci,respectively.However,there are too many overlapped X-STRs loci between these kits,and the detection efficiency is also wasted.In practical applications,the number of available X chromosome loci cannot meet the demand.Besides,the X-STR loci contained in those multiplex system clustered into several linkage groups.In forensic kinship tests and individual identifications,the likelihood should be calculated based on haplotype frequencies.However,haplotypes may recombine during meiosis,and haplotypes in female samples are difficult to determine,and the probability of pairwise linkage disequilibrium within a linkage group is higher,which makes the practical application of X-STRs subject to many restrictions.In this paper,we intended to verify the reliability and efficiency of a newly developed X-STR multiplex systems,named the MicroreaderTM 19X Direct ID System,which contained 19 X-STR loci(DXS6795,DXS6803,DXS6807,DXS9907,DXS7423,DXS6810,GATA172D05,DXS101,DXS9902,DXS7133,GATA31E08,DXS6800,DXS981,DXS10162,DXS6809,DXS10135,HPRTB,GATA165B12 and DXS10079)and the sex determination locus of AMEL.Method:According to the SWGDAM developmental validation guidelines and specification of X-STR testing for forensic purpose(SF/Z JD0105006-2018),we did a series of validation experiments,which included PCR conditions,sensitivity,species specificity,stability,DNA mixtures,concordance,stutter,sizing precision and population studies.The genetic polymorphism of this new X-STR multiplex system was investigated using 200 unrelated individuals from Guangdong Han population,and the allele frequencies and forensic parameters were calculated and compared to the AGCU-X19 STR kit.Result:For the new X-STR multiplex system,the optimal cycling number is 29 as recommended.The recommended optimal annealing temperatures was 60℃.We recommended that the final extension time should be 15 min or more.In order to obtain the complete profiles as much as possible,we determined that the amount of template DNA should be 250 pg or more.Only chimpanzee and macaque showed weak cross-reactivity,and no peaks were observed for the other animals.Conclusion:These studies showed that the MicroreaderTM 19X Direct ID System is highly sensitive,stable,and able to accurately identify mixed samples.This new X-STR multiplex system have high cumulative PDF and MEC in Guangdong Han population,meeting the requirements of complex kinship analysis and individual identification.In conclusion,we developed and evaluated the forensic efficiency of a new X-STSs multiplex system,which can provide a new method for DNA analysis in forensic genetics and enlarge the X-STR reference database.