Performance Validation Of MultipSeq^? AI-Y-STR-Multi Panel Based On Next Generation Sequencing

Objective:To make forensic validation for the Y-STR included in the Multipseq^?AI-Y-STR-Multi Panel and evaluate the forensic efficiency of the kit,providing the basic data and technological means for constructing a more efficient Y-STR testing system.Methods:The validation of the Multipseq^? AI-Y-STR-Multi Panel was performed following the Chinese judicial administration industry standard "Specification for high-throughput sequencing of chromosomal genetic markers and its forensic applications"（SF/T 0070-2020）and Scientific Working Group on DNA Analysis Methods（SWGDAM）guidelines.The concordance,sensitivity,reproducibility,specificity,stability and mixture study were conducted to evaluate the performance of this panel.Simultaneously,parallel detection was performed by capillary electrophoresis（CE）using Goldeneye^? Y-Plus kit.Results:1.Concordance study:In our study,fifty-one male individuals were tested by NGS technology,the results of which were subsequently compared with CE results.A total of 1020 alleles were detected in 51 male samples,and the concordance rate was 100%,suggesting that the sequencing results were reliable.2.Sensitivity study:The Multipseq^? AI-Y-STR-Multi Panel showed high sensitivity,and the limit of detection was as lower as 125 pg.Full profile could be detected when the input amount was higher than 0.125ng.The average loci detection rates deceased to 90%,70%and 60%when the input amounts were 62.5 pg,31.25 pg and 15.625 pg,respectively.3.Reproducibility and repeatability study:The control DNA 9948 were tested in triplicate using different instruments and operators,which obtained fully concordant profiles.4.Specificity study:In the species specificity study,the male monkey could be easily distinguished from the human males.The effective alleles（depth>100×）of monkey were detected only in 6 loci（DYS385B,DYS393,DYS437,DYS481,DYS635 and Y-GATA-H4）,and no amplification were observed on female samples.5.Inhibitor study:Five commonly used PCR inhibitors（humic acid,urea,melanin,indigo and hematin）were applied to pinpoint the window for the inhibitor effects.Urea did not influence the outcome even when the concentration was 500 ng/μL.Melanin had limited influence on the test,and complete loci detection was observed when hematin was higher than 10 μM.Humic acid did not influence the outcome even when the concentration was 10 ng/μL,but no result could be obtained with the concentration of 17 ng/μL and 25 ng/μL.Indigo showed some impact on the sequencing outcome when the concentration was 8 μM.The results proved that the 20 Y-STR testing system was stable enough for forensic genetic application.6.Mixture study:Both the male/male samples and the female/male samples were prepared to evaluate the identification ability for mixtures.In the male/female mixtures,only the male DNA was successfully amplified even in the presence of excess female DNA（400 ng female DNA mixed with 250 pg、125 pg、62.5 pg、31.25 pg and 15.625 pg male DNA 9948）.When the input amount of male DNA was lower to 125 pg,the allele detection rate was 100%.The detection rates decreased to 90%,85%and 70%with the input amount of male DNA decreased.In the male/male mixtures,the control DNA 9948 and 2800 M were mixed with different ratios（49:1,19:1,9:1,3:1 and 1:1）with a final amount of 1 ng.The results showed that only mixture in the ratio of 1:1 could obtain all alleles from two samples.Conclusions:Although insufficient performance were observed in two male mixtures analysis,our validation study demonstrated that the Multipseq^? AI-Y-STR-Multi Panel had high sensitivity,reproducibility and inhibitor resistance,which could be used for preliminary family investigation and detection of male/female mixtures.In conclusion,our results demonstrated that the kit could be applied in forensic practice and population genetics.