| ObjectiveTo estabilish a high-throughput Y-STR multiplex system based on MiSeq next generation sequencing platform,then perform genetic polymorphism study in Han ethnic minority from Jiangsu Province so that population genetic data and forensic parameters could be obtain,which were fundmental data for forensic application.Methods:.1.74 candidate Y chromosome Short Tandem Repeat loci and One sex-identification marker Amelogenin locus were selected via publish paper and Commercial kits,specific primer pairs were designed by using primer premier 5.0 sofeware,After the synthesis of the primers,primers for NGS were verified by general PCR followed with agarose gel electrophoresis.the 2800M DNA standard(Promega)was used as the template.2.After multiplex PCR,a TruSeq(?)DNA PCR-Free HT Kit was used to constructe the NGS library,and then the library was constructed for real-time fluorescence quantification and quality tested.The primer pairs and multiplex amplification system were optimized.Finally a 67 Y-STR loci and One sex-identification marker Amelogenin locus multiplex system was established.3.A total of 149 FTA Oral swabs sample were collected.DNA were extracted using PrepFiler Automated Forensic DNA Extraction Kit.and DNA were quantified by QubitTM dsDNA HS Assay Kit.4.The configuration files of 68 loci were recorded in Notepad software and The FastQ files generated by MiSeq FGx platform were analyzed by STRait Razor 3.0 software to obtain allele typing and sequence information of each loci.5.Population genetic analysis and parameter evaluation of this multiplex system were analyzed using Arlequin Version 3.5 sofeware.The genetic diversity and haplotype diversity of population samples were calculated by direct method.When analyzing the sequences of each locus in the sample data,Cluster X software was used to carry out multi-sequence alignment.Origin Pro 9.0 drawing software was used to map and analyze the data.Result:1.In this study,a multiplex system consisting of 67 Y-STR loci and One sex-identification marker Amelogenin locus was estabilished.The length range of the amplified fragment was below 350bp.This system was sensitive which could obtain satisfied genotype using lng genomic DNA.2.A total of 149 haplotypes were observed in 149 unrelated males from Jiangshu Han-ethnic population.The overall haplotypediversity(HD)and discrimination capacity(DC)were 0.999999......and 0.999999.....,respectively.Their value of gene diversity(GD)ranginged from 0.1275 to 0.9969.The GD values of 53 out of the 67 loci were over 0.6.A total of 814 alleles were detected from 67 Y-STR loci,including sequence polymorphism in the repeat region and sequence polymorphism in the flanking region.There were 349 more sequence polymorphisms than length polymorphisms.3.DYF387a/b,DYS404Sla/b and DYF399Sla/b/c loci showed abnormal genotyping in 149 unrelated male individuals of Han nationality in Jiangsu province.There were 5 samples with allelic abnormalities of DYF387a/b locus which showed as tri-allelic patterns.1 samples showed tri-allelic pattens in DYS404Sla/b locus.6 samples showed qua-allelic patterns in DYF399Sla/b/c locus,and one sample showed pen-allelic patterns in DYF399Sla/b/c locus.4.This research studied the sequence polymorphisms of 67 Y-STR loci,and the 45 Y-STR loci had different core sequences of the same length Different bases were observed in the flanking sequence of 13 Y-STR loci(DYS385a/b、DYS388、DYS389 I、DYS390、DYS439、DYS459a/b、DYS481、DYS522、DYS531、DYS576、DYS626).Compared with previous studies,380 new repeat region variation were found in 50 Y-STR loci(DYS19、DYS385a/b、DYF387Sla/b、DYS389Ⅱ、DYS391、DYF399Sla/b/c、DYS404Sla/b、DYS439、DYS443、DYS444、DYS446、DYS447、DYS448、DYS449、DYS458、DYS459a/b、DYS460、DYS485、DYS505、DYS508、DYS510、DYS518、DYS520、DYS527a/b、DYS531、DYS552、DYS557、DYS587、DYS593、DYS596、DYS612、DYS617、DYS622、DYS626、DYS627、DYS630、DYS641、DYS644、DYS645、DYS710、DYS720、Y-GATA-A10、Y-GATA-H4),and 13 new flank variants were found in 11 Y-STR(DYS385a/b、DYS388、DYS389 I、DYS390、DYS439、DYS459a/b、DYS481、DYS522、DYS531.DYS626)loci.5.In this study,stutter sequences were analyzed on 27 Y-STR loci whose repeat structure was compound repeat structure.The stutter sequence of 10 Y-STR loci(DYS390、DYS437、DYS447、DYS448、DYS520、DYS552、DYS587、DYS593、DYS596、DYS622)accounted for less than 10%of the target genes.The stutter sequence of 15 Y-STR loci(DYS19、DYF387a/b、DYS389 Ⅱ、DYS449、DYS510、DYS518、DYS527a/b、DYS557、DYS626、DYS627、DYS630、DYS635、DYS720)accounted for more than 10%and less than 20%of the target genes.The stutter sequence of DYS710 and DYS612 loci was higher,accounting for 0.3372±0.087 and 0.4188±0.0688 of the target gene,respectively.Nonspecific amplification of 2bp replication slippage occurred in DYS19 and DYS710 1oci.Conclusion:1.A multiplex system consisting of 67 Y-STR loci and One sex-identification marker Amelogenin locus was established,which was high-throughput,sensitive,accurate and stable.The system can be applied to the practice of forensic science.2.Population genetics study indicated that most of the studied loci were high polymorphism markers in Han,and the obtained forensic parameters and population genetic data can provide basic data for forensic medicine practice and human genetics research.3.The results showed that 45 Y-STR loci had the same length but different sequences,among which 13 Y-STR genes had different bases in flanking sequences.369 new repeat region variation were found in 50 Y-STR loci,and 13 new flank variants were found in 11 Y-STR loci.The different bases of the flanking sequence can improve the ability of individual recognition and the efficiency of forensic identification,and play an auxiliary role in the individual recognition of Y-STR loci.The new sequence variation provides more genetic polymorphisms for Y-STR loci and provides basic data for population genetics studies.4.It was found that DYF387a/b,DYS404Sla/b and DYF399Sla/b/c loci showed abnormal genotyping in Jiangsu Han population.The abnormal typing of Y-STR loci can improve individual recognition ability,but special attention should be paid to the occurrence of this abnormal typing in the identification of mixed spots. |
