Validation And Population Data Of The 19 X-STR Loci Multiplex System

backgroundShort tandem repeats(STRs),the second generation of DNA genetic markers,are characterized by their wide distribution in the genome,high genetic polymorphism information content,rapid and efficient detection,simple data analysis and standardization and high degree of automation,Is widely used in practical applications of forensic work,genetic testing and gene diagnosis,organ transplantation,plant and animal breeding,gene mapping,population genetic research,population evolution and other basic applications.Comparing with the extensive application of autosomal STR and Y chromosome STR amplification system in forensic practice,the establishment of the X chromosome STR related multiplex amplification system and the development of commercial kits have been in a slow development stage.However,the X-chromosome STR has an important and even irreplaceable role in the complex parentage relationship and the practice of identifying mixed material samples.Now the most widely used kit is the ARGUS X12 kit from QIANGEN.The AGCU X19 fluorescent detection system selected in this paper has more linkage groups and higher resolution than the group.The present study conducted the validation of the system and investigated for polymorphism of Henan Han,Zhejiang Han,Ningxia Hui,Xinjiang Uygur,Tibet,Guangxi Zhuang,Guangxi Yao and Guangxi Mulao ethnic groups,providing basic data for forensic practice and population genetic.Aims:The multiplex of the 19 X-STR loci(DXS7423,DXS10148,DXS8378,DXS10162,DXS7132,DXS10079,DXS10103,DXS10101,HPTRB,DXS10074,DXS10135,DXS10159,DXS10164,DXS10075,DXS6809,DXS6789,DXS7424,DXS101,DXS10134)amplification system was verified to the effectiveness of applications,and population surveys in China was conducted for providing the basis data.Based on the allele:frequencies of the X-STR loci,genetic distances and population genetic distances were analyzed for different populations,and it also has an effect in population genetics,anthropology,ethnology,archeology,and intangible cultural heritage protection.Method:According to the validation guidelines of Scientific Working Group on DNA Analysis Methods(SWGDAM),the 19 X-STR multiplex systems were validated.The 2157 Samples of Henan Han,Zhejiang Han,Ningxia Hui,Xinjiang Uygur,Tibetan,Guangxi Zhuang,Guangxi Yao and Guangxi mulao nationalities in our country were collected.The AGCU X19 fluorescence detection kit was used to detect the X-chromosome STR typing and the alleles were calculated Distribution,gene locus independence test,statistical genetic polymorphism,individual identification,non-father exclusion and other forensic parameters to verify the composite amplification system of forensic application efficacy,and then analyze the genetic distance between each other,genetic relationship and Population migration evolution.ResultsThe validation shows that the AGCU X19 fluorescence detection system has a sensitivity of 0.125ng.Most of the alleles can be detected in 0.0625ng template samples.There is no specific amplification of non-human DNA.The system can tolerance the common inhibitors of forensic practice work(300 ?mol/L heme concentration,10 mmol/L indigo concentration,50? g/?l humic acid concentration,2.5 mmol/L ion,400 ?mol/L hemoglobin concentration,1200 mM EDTA concentration).The deviation of allele fragments was less than 0.2bp,which was lower than 0.5bp,tolerance of genetic analysis.The detection system inhibits the same typing on different instruments and different laboratories.It is repeatable.The stutter ratio is less than 15%,which does not affect the normal classification,so the result is reliable.Most of the common case samples can be completely typed.The system can effectively distinguish between the advantages and disadvantages of donors by using a mixed sample with a mixing ratio from 1:1 to 1:9.When the PCR reaction components of the system are between 0.75 × and 1.25 ×,the complete typing can still be achieved.When one of the components is reduced to 0.5 ×,allele loss will occur,and the tolerance of the system to changes in component concentations is known,which meets actual needs.The number of PCR cycle gradient experiments showed that the number of cycles was between 28 and 30,and the complete typing could be obtained,and the fluorescence value increased with the number of cycles,and the amplification was well balanced.When the number of cycles is greater than/equal to 31,there will be fluorescence infiltration of individual alleles,but the amplification equilibrium is still good.Therefore,in practical applications,the number of PCR cycles can be adjusted within a certain range based on the amount of sample template.The system can obtain reliable results between the annealing temperatures of 58 ??62 ?.The product fragment fluorescence peak and specificity are best at 60 ?.When the final extension time of the system is 30min,the allele A shoulder can be minimized.In summary,according to the SWGDAM validation guideline,the AGCU X19 fluorescence detection system meets the requirements of forensic practice.In the investigation of population polymorphism,19 X-STR loci were detected in 2,157 unrelated individual species in 8 ethnic groups in Henan Han,Zhejiang Han,Xinjiang Uygur,Tibetan Tibetan,Ningxia Hui,Guangxi Zhuang,Guangxi Yao,and Guangxi Yi.353 alleles were detected.the allele frequency ranged from 0.0013 to 0.7094,the highest was the 10 allele of Guangxi Yao DXS10164;19 X-STR loci had polymorphism information content is between 0.4106 to 0.9277.The paired loci,that are in linkage disequilibrium in different populations,may be located in the same linkage cluster or in different linkage clusters,and not all the paired loci in the same linkage cluster are in linkage disequilibrium.Therefore,the linkage disequilibrium state of the paired loci is related not only to the physical distance and the genetic distance,but also to the genetic structure of the population.When 19 X-chromosomal STR loci were used as a combination of seven linkage clusters,the cumulative individual recognition power of the CDPf reached 1.000 million and the CDPm was Between 0.999 999 999 402 265 to 0.999 999 999 996 670,CMECt ranged from 0.999 999 999 188 144 to 0.999 999 999 995 949,and CMECd ranged from 0.999 999 942 863 498 to 0.999 999 999 638 495.Having high individual identification and non-parental exclusion the multiplex can provide valuable information for forensic practice.In the analysis of the genetic relationship among the selected eight ethnic groups,Tibetans and Uygurs were divided' into one branch,and their genetic relationship with the other six people was far.The Henan Han,Zhejiang Hans,Ningxia Huis,Guangxi Zhuangs,and Guangxi Mulaos have closer genetic relationships.The three ethnic groups in Guangxi and Ningxia Hui are divided into one branch,but Guangxi Yao ethnic group has a long genetic relationship with the other three ethnic groups,which may be related to the remote sampling site.At the same time,in the analysis of genetic relationships with Japanese,black Africans,and European Caucasians,differences in genetic relationships between ethnic can be derived.Conclusion:The 19 X-STR fluorescence detection systems selected in this study have good sensitivity,species-specificity,and mixed sample testing capabilities,with good repeatability,consistency,and accurate typing.The CPD and CMET of the system in the eight ethnic groups meet the actual application needs,and can provide effective technical means for the identification of complex parental rights and the testing of mixed samples.The polymorphism survey was conducted on eight ethnic groups to provide basic data for forensic applications and genetic studies.