DIP-STR (Insertion/Deletion Polymorphism–Short Tandem Repeat) markers are based on polymorphic primers, they enable the deconvolution of highly unbalanced two-person DNA mixtures by selectively amplifying alleles from the minor contributor. These markers are particularly effective for forensic applications, where the DNA of the person of interest is present as a minor contributor. Among the promising applications of DIP-STR markers, are also cases of microchimerism, such as the analysis of cell-free fetal DNA (cffDNA) circulating in the blood of pregnant women, with the aim of enabling non-invasive prenatal paternity testing (NIPPT). DIP-STR markers have already demonstrated their potential for detecting cffDNA as early as seven weeks of gestation, and this ability is particularly relevant in cases involving pregnancies resulting from rape.

To date, DIP-STR analysis has primarily relied on capillary electrophoresis (CE), a cost-effective method, which is, however, limited to the detection of a single informative marker per reaction, thereby requiring relatively large amounts of DNA. 

This project aims to expand the potential of DIP-STR markers for forensic applications by implementing a novel analytical protocol. Here, we report the developmental validation of two multiplex panels comprising 24 and 20 DIP-STR markers, optimized for massive parallel sequencing (MPS) on an Illumina MiSeq platform. Sequencing results obtained from single-source DNA samples showed good intra- and inter-run reproducibility and repeatability, with sensitivity down to 62 pg of DNA input and full concordance with CE data. Species-specificity testing confirmed that the markers are specific to human DNA. The panels were further evaluated on plasma samples collected from ten pregnant women to explore their performance in NIPPT and revealed detection of the fetal alleles starting from the first trimester of pregnancy. In addition, with an appropriate sequencing depth, the multiplex panels applied to simulated DNA mixtures are expected to allow specific amplification of the minor contributor in highly unbalanced mixtures (with ratios up to 1:100), while accounting for potential allelic drop-outs. 

Overall, our results demonstrate that MPS sequencing enhances the multiplexing capacity of DIP-STR markers. This approach enables the simultaneous analysis of multiple informative loci in a single experiment, it increases the amount of genetic information recovered per sample and improves the ability to resolve complex mixtures. By expanding the number of loci that can be interrogated, MPS facilitates the analysis of DIP-STRs in forensic applications and opens the way for the development of a new method to perform NIPPT.

Authors

• Laura Carrara (School of Criminal Justice, Faculty of Law, Criminal Justice and Public Administration, University of Lausanne, Switzerland)
• Franco Taroni (School of Criminal Justice, Faculty of Law, Criminal Justice and Public Administration, University of Lausanne, Switzerland)
• Diana Hall (Forensic Genetics Unit, University Center of Legal Medicine, Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland)