Construction Of A Multiplex SNP Detection System Based On Second-generation Sequencing Technology And Its Application In Non-invasive Prenatal Paternity Testing

ObjectiveTo construct a single nucleotide polymorphisms(SNP)multiplex amplification system based on the next-generation sequencing(NGS)technology and evaluate its practical application value in forensic identification,especially in a non-invasive prenatal paternity test(NIPPT).This system could provide new ideas for forensic personal identification and paternity testing,and broaden the application prospects.Methods(1)Based on the genetic data from the NCBI database and SNP loci from published literature,SNPs with high forensic application value were screened from the whole human genome.Primers were designed to construct a multiplex SNP amplification system based on the NGS platform and performed forensic validation of accuracy,consistency and reproducibility.(2)A population genetic survey was conducted on 100 unrelated Chinese Han individuals using the system.The forensic parameters of each SNP locus were statistically analyzed to assess the effectiveness of the system and its value in forensic applications.(3)The system was applied to 15 groups of triplicate samples containing maternal,paternal and maternal plasma(including fetal components)for sequencing analysis and paternity determination,and the results were compared with those based on capillary electrophoresis(CE)technology for consistency.At the same time,the suspected fathers of 15 cases were combined with 70 unrelated male individuals to explore the feasibility of this system in NIPPT.(4)Based on the maternal information of each group and the sequencing results of different proportions of simulated pregnant plasma samples to analyze the factors that may affect the success rate of the system.Results(1)In this study,a multiplex detection system containing 627 SNP loci covering 22 pairs of autosomes and two sex chromosomes was constructed based on NGS.All loci were verified to be accurately typed by Sanger sequencing.Valid data and consistent typing results with good reproducibility could be obtained for different specimens such as peripheral blood,hair,nail and saliva swabs from the same individual.The system was applied to 100 Chinese Han unrelated individuals and 15 groups of triplicate samples,including pregnant women’s plasma.All samples were obtained with high-quality sequencing data,and the average sequencing depth of each site was above 30x.(2)The population genetic survey of 100 Chinese Han unrelated individuals showed that the combined probability of discrimination(CPD)for 589 A-SNPs was 1-1.93×10-207,the combined probability of exclusion in duo(CPEduo)and trio(CPEtrio)was 1-1.34×10-24 and 1-3.13×10-52.The cumulative probability of discrimination in female(CPDf)and male(CPDm)individuals for 12 X-SNPs was 0.999985827539 and 0.997674945,the cumulative mean probability of exclusion in duo(CMECduo)and trio(CMECtrio)was 0.783011137340 and 0.944829927254;the haplotype match probability(HMP)for the 26 Y-SNPs was 0.195102041,the haplotype diversity(HD)was 0.793399417,and the discrimination capacity(DC)was 0.142857143.(3)All 15 groups of triplicate samples which including maternal,paternal peripheral blood and maternal plasma,were tested by second-generation sequencing for paternity determination,and the results were consistent with which obtained by the STR typing method based on the CE platform.Comparing 15 groups of suspected fathers with 70 unrelated male individuals,the cumulative paternity indices of biological fathers in 13 identifiable paternity samples were significantly different from those of unrelated male individuals.(4)The sequencing results of different proportions of simulated maternal plasma samples showed that the system was more capable of detecting mixtures.More than 99%of valid SNP locus information could still be obtained when the proportion of cell-free fetal DNA(cffDNA)in maternal plasma was as low as 4%.(5)The fetal fraction in maternal plasma increased with gestational weeks from 6 to 13 weeks,but the relationship between fetal fraction and the age of pregnant women was not significant.ConclusionIn this study,a multiplex detection system containing 627 SNP loci based on NGS was constructed,and it was verified that the accuracy,reproducibility and consistency of the system were good,and the sequencing data of each sample obtained from the assay were of high quality.The system can be used for personal identification and paternity testing and has good prospects for application in NIPPT,which can provide a new detection method and identification ideas for forensic research and practice.