Development Of Linked Autosomal STRs Typing Panel Based On Massively Parallel Sequencing And Preliminary Study On Discerning The Same Class Kinship

Objective:Currently,the short tandem repeat(STR)remains the mainstream genetic marker for personal identification and paternity in forensic DNA testing due to its high polymorphism.The likelihood ratios were calculated to be the same using the independent STR loci included in most commercial STR kits to identify the three second-degree kinships: grandparent-grandson,uncle-nephew,and half siblings,and therefore the independent STR loci commonly used in forensic DNA testing could not be used to complex kinship identification of the same degree kinship identification.Massively parallel sequencing(MPS)technology can detect a large number of STR loci and analyze multiple DNA samples.In this study,an autosomal-linked STR loci typing system was constructed based on MPS,and a set of MPS sequencing data analysis processes and methods were established.Through the forensic assessment of the system and the analysis of population genetics and related forensic parameters,and through Merlin software simulated and generated 10,000 pairs of grandparents,uncles and nephews,half-siblings and full-siblings to get their classification scores,provided a new detection method and preliminary research data for the same degree kinship in the field of complex kinship identification.Methods:(1)Development MPS panel: According to certain criteria,173 autosomal linked STR loci were selected from the Rutgers Map v3 genetic map.The selected loci were searched for the Hg38 reference sequence on the UCSC website and the location of the target region of each STR loci was determined Next,upload the bed file to the AIdesign probe / primer design platform for primer design of the STR target fragment and the fragment length of the STR locus ranges from 150 bp to 270 bp and develop a multiplex PCR targeting capture amplification typing panel.(2)Construction and sequencing of MPS libraries: Use the QIAamp? DNA Investigator kit to extract DNA from 110 northern Han ethnic unrelated individuals as blood card samples,QIAGEN DNeasy Blood & Tissue kit to extract blood DNA from family samples,and use DNA samples construction MPS libraries with MultipSeq? Custom Panel kit,using Qubit 4.0 and QuantStudio 5 Real-time fluorescence quantitative PCR instrument to quantify these libraries,and using Qseq100 for libraries quality control,and finally qualified libraries were sequenced on the Illumina MiSeq FGx sequencing platform.(3)MPS sequencing data analysis: The sequencing quality of the sample fastq files was evaluated using FASTQC software,then the sample fastq files were analyzed using STRaitRazor 3.0 software under the corresponding STR typing profile of this panel,then the relevant data were extracted using the MPS data analysis process established in our laboratory,and finally the data Kwere visualized.(4)Forensic validation studies of the panel: Random selection of 2 samples for the same batch of 3 library constructions and 3 different batches of library construction repeatability studies and the random selection system of 20 STR loci for single locus amplification and accuracy of Sanger sequencing experiment;Use 9948 control DNA to prepare libraries with DNA input amounts of 10 ng,5ng,2.5ng,1ng,0.5ng,0.25 ng,and 0.125 ng for sensitivity experiments.Mix with 9947 A and 9948 control DNA at different ratios of 1: 1,1: 2,1: 4,1: 9,2: 1,4: 1,and 9: 1 and the mixed samples were studied.Species specific study of blood DNA from 7 animal samples of pigs,dogs,cattle,sheep chicken,rabbit and mouse.Population genetics analysis was performed on 110 unrelated individuals of the northern Han nationality.(5)Pedigree sample analysis: Analyze 4 family samples including five samples of grandparents,parents and child to verify whether the STR of each combination is closely linked.Use Merlin software to simulate the generation of grandparent-grandchild,uncle and nephew,half-sibs and full-sibs of 10,000 pairs each,and calculate the lnLR value,and finally the classification score value of each kinship pair is obtained.Results:(1)Library quality control: Libraries fragment length in the range of 310 ~ 400 bp,neither primer dimers of small fragments nor tail peaks of large fragments;Relative fluorescence unit value(RFU)increase accordingly with library concentration;the sequencing data in the sample fastq file can reach more than 98% of the bases above Q30.(2)MPS data analysis: The average total coverage depth of the sample is 302393 ×,and the proportion of the main peak is 0.8255;the average coverage depth of 173 STR loci is 2617 ×;a total of 14759 heterozygotes were detected in 173 loci of 110 unrelated individuals and the average allele heterozygosity balance ratio was 0.881;the average composition of Allele%,Stutter% and Noise% in the sequence composition ratio of 173 STR loci were 83.0%,4.4% and 12.6%,respectively.(3)Forensic validation studies of the panel: The same batch of sequencing and different batches of sequencing parameters have similar results and the STR loci have the same typing.Randomly select 20 STR loci for single locus amplification MPS sequencing results and the results of each STR locus verified by Sanger are consistent with the typing results of panel;when the DNA input is less than 2.5ng,Allele% gradually decreases,and the STR loci appears loss when less than 0.25ng;when the mixing ratio is 1:4,1:9 or 4:1,9:1,the STR typing of the sample with the secondary proportion in the mixed sample will be difficult to distinguish between stutter and other peaks or its true typing;the p-value obtained by Hardy-Weinberg equilibrium test after Boniferroni correction has 8 allele frequencies of STR loci that do not meet Hardy-Weinberg equilibrium,namely D1S1660,D5S1473,D5S1459,D6S1284,D15S644,D11S1983,D13S1492 and D20S1146;the genetic polymorphism of each STR locus,polymorphism information content,personal recognition ability and heterozygosity observations all performed well.(4)Pedigree sample analysis: The analysis of the results of the typing and genetic relationships of the 4 family samples including grandparents,parents,and child showed that a total of 66 clusters of 173 STR loci were closely linked;Using classification rates to evaluate the ability of panel to distinguish between grandparent-grandson,uncle-nephew,half-siblings,and fullsiblings kinship pairs,the results showed that the classification rates of grandparent-grandson pairs,uncle-nephew pairs,half-siblings pairs and the full-siblings pairs the relationship pair relationship pair are 0.7247,0.5276,0.2154,and 0.9954,respectively.In other words,the correct rate of distinguishing from other three kinships is 72.47% for grandparent-grandson,52.76% for unclenephew,21.54% for half-siblings,and 99.54% for full-siblings.Conclusion:In this study,a complex PCR targeted capture amplification typing panel and analysis method consisting with 173 autosomal linked STRs was established based on MPS,which has well forensic parameter results with good species specificity,sensitivity of 1 ng,better accuracy and repeatability,and better mixture detection ability and the ability to distinguish the grandparentgrandson,uncle-nephew,half-siblings,and full-siblings.The 173 linked STR loci in this study can initially provide a new detection method and analysis idea for the identification of same degree kinship in complex kinship identification.However,due to the insufficient number of genetic markers and the imperfect calculation method for the panel,the accuracy of classification cannot be achieved to a more ideal value,and further improvement and optimization are needed.