Validation And Forensic Application Of A 6-dye Multiplex System With 45 Y-STR Loci

Background and aim:In recent years,more and more attention has been paid to the construction of Y-STR database.Up to 2018,eight million Y-STR data had been included in the Y-STR database,and it is still increasing at the rate of one million per year.As the capacity of Y-STR database increases rapidly,the number of Y-STR haplotypes also increases,which generates too much information in the comparison of Y-STR haplotypes.Furthermore,it is difficult to distinguish authenticity in paternal family tracing.When application into forensic cases,Y-STR typing can only exclude individuals of the same paternal line,but cannot determine kinship between individuals.If there is a common ancestor,it will increase the difficulty of identification.At present,the most commonly used commercial kits are the AmpFISTRTM Yfiler PlusTM,Powerplex? Y23 and DNA TyperY26,etc.Those kits contain a small number of Y-STR loci and some loci were low-polymorphic in Chinese Han population,which leads low haplotype diversity.Besides,some rapidly-mutating Y-STR loci were included in these kits,which improved the polymorphism efficiency of the system,to a certain extent,but it was also easy to mistakenly exclude the male individuals from the same paternal lineage.Therefore,we are supposed to select Y-STR loci and develop a novel system to meet the needs of Y-STR database construction and paternal family screening.In this study,we aim to evaluate the forensic applicability of AGCU Y LM system and obtain the mutation rates and polymorphism parameters of Guangdong Han population.Methods:A series of validation tests were performed,including PCR conditions,sensitivity,anti-interference ability,mixture,species specificity,reproducibility,sizing precision,stutter analysis,case samples and concordance study,according to the guidance of SWGDAM validation guidelines.A total of 345 DNA-confirmed father-son pairs from Guangdong Han were collected to genotype using AGCU Y LM kit to obtain the mutation information and polymorphism parameters.Results:The results showed that the optimal cycle number was 30,the most suitable annealing temperature was 60℃,and the most optimal final extension time was 20 minutes.To obtain complete profiles,62.5pg was the minimum concentration.The system was of great tolerance to common inhibitors(30 mmol/L indigo,20 ng/μl humic acid,0.2 mmol/L EDTA,200 μmol/L hemoglobin).When the ratio of male and female mixed samples was 1:1-1:100,complete male profiles could be obtained.No specific amplification was detected in non-human DNA samples with the exception of chimpanzees.Reliable and full profiles were obtained for most daily casework samples.Consistent typing was observed in different laboratories and different instruments,which showing great reproducibility.The deviation of the average allele fragments was less than O.lbp,which was within tolerance range of genetic analysis of 0.5bp.Except for DYD481,DYS456,DYS389Ⅱ and DYS393,the stutter ratio of most loci was less than 15%,which did not affect normal genotyping.In 100 population samples,the results of 19 loci overlapped with AmpFISTRTM Yfiler Plus TM were identical.The average mutation rate of 45 Y-STR in Guangdong Han population was 2.38×10-3(95%CI,1.7×10-3-3.3×10-3),the GD value of 41 loci was over 0.5,the HD value was 0.999861,and the DC value was 0.973529.Conclusion:The AGCU Y LM system has high sensitivity,great specificity,high precision,strong anti-interference ability,reproducibility,and is compatible with other commercial Y-STR kits.In general,this amplification system possesses lower mutation rate and high polymorphism in Guangdong Han population,which can meet the needs of Y-STR database construction and forensic DNA testing.