Set of 15 SNP-SNP Markers for Detection of Unbalanced Degraded DNA Mixtures and Noninvasive Prenatal Paternity Testing

Abstract

Unbalanced and degraded mixtures (UDM) are very common in forensic DNA analysis. For example, DNA signals from criminal suspects are masked by a large amount of DNA from victims, or cell-free fetal DNA (cffDNA) in maternal plasma is masked by a high background of maternal DNA. Currently, detecting minor DNA in these mixtures is complex and challenging. We developed a new set of SNP-SNP microhaplotypes with short amplicons, and we successfully genotyped them using the new method of amplification-refractory mutation system PCR (ARMS-PCR) combined with SNaPshot technology based on a capillary electrophoresis (CE) platform. This panel reflects a high polymorphism in the Southwest Chinese Han population and thus has excellent potential for mixture studies. We evaluated the feasibility of this panel for UDM detection and noninvasive prenatal paternity testing (NIPPT). Fifteen SNP-SNPs detected minor DNA of homemade DNA mixtures, with a sensitivity of 0.025–0.05 ng and a specificity of 1:1,000. In addition, the panel successfully genotyped degraded DNA from single and mixed samples. Finally, 15 SNP-SNPs were applied to 26 trios. All samples displayed positive results with at least one marker to detect cffDNA. Besides, all fetal alleles in maternal plasma were confirmed by genotyping fetal genomic DNA from amniocentesis and paternal genomic DNA from peripheral blood. The results indicated that the SNP-SNP strategy based on the CE platform was useful for UDM detection and NIPPT.

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis

“…The sensitivity, accuracy, polymorphism, forensic parameters, degraded samples, mixtures, and animal samples of the panel performed adequately, thereby indicating that our panel was a powerful forensic tool and could provide a good supplement and enhancement to existing detection methods. Based on our previous studies of 15 SNP-SNP MHs [ 16 , 17 ], we comprehensively optimized the MH screening, sequencing, and analysis protocols in this study.…”

“…At present, most MHs of reported panels are selected from published articles [ 8 ].Therefore, the current screening method is not systematic, and its genome coverage is not extensive. The number of MHs in some panels is small, and the detection platform still uses first-generation sequencing [ 16 , 17 , 32 , 40 ]. Moreover, the analysis methods of some MPS panels, such as Flfinder [ 11 ] and MHtyper [ 41 ], are more suitable for their own research analyses.…”

“…However, our MPS-based panel was able to observe all complete MH genotypes ( Table 2 and Table S7 ). For the degraded mixtures, a ratio of 1:10 was selected for analysis because it was the lowest limit at which STR could detect the mixture, and matched the actual proportion of cell-free fetal DNA (cffDNA) in maternal plasma (range 5–20%, average 9–10%) [ 17 , 54 ]. We set 1, 3, and 5 µL of DNA input to explore the effects of sequencing genotypes corresponding to different sequencing inputs.…”

“…However, when two or more loci are heterozygous, Sanger sequencing cannot determine the cis-trans relationship between alleles of a single SNP in genomic DNA [ 6 , 14 ], i.e., the haplotype phase [ 15 ]. Our previous research showed that although the capillary electrophoresis (CE) platform can phase MHs, it only resolved those composed of two SNPs, with a low detection throughput at one time [ 16 , 17 ]. However, MPS can compensate for the deficiencies in Sanger sequencing and CE platforms.…”

State of the Art for Microhaplotypes

“…Non-invasive prenatal paternity testing is possible using fetal DNA in maternal circulation [ 33 ]. The large number of alleles of the more polymorphic loci provides a high likelihood that the fetus’s genotype differs from the maternal genotype.…”