DNA mixture analyses of autosomal single nucleotide polymorphisms for individual identification using droplet digital polymerase-chain reaction and massively parallel sequencing in combination with EuroFormix

ABSTRACTDroplet digital polymerase-chain reaction (ddPCR) and massively parallel sequencing (MPS) can be used to detect an extremely low proportion of non-overlapping single nucleotide polymorphisms (SNPs) of a minor contributor in a DNA mixture. Both semi-continuous and continuous probabilistic likelihood ratio (LR) models can be used to interpret the DNA profiles of mixtures. We analysed 28 identity-informative SNPs in 20 DNA mixtures with various minor to major ratios (1:29, to 1:99) by using our customized ddPCR panel and two MPS panels. The minor contributors of the DNA mixtures were correctly inferred using both semi-continuous and continuous LR models of EuroForMix based on the data of our customized ddPCR and one MPS panel. The accuracy rate of minor contributor assignment was 95% and 90% using semi-continuous and continuous LR models, respectively, using the data of the other MPS panel with lower coverage reads. In conclusion, the quantitative genotype data of SNPs generated using ddPCR can be used for the minor contributor inference for DNA mixtures. The performance of a continuous LR model in minor contributor identification in DNA mixtures may not be superior to that of a semi-continuous LR model when the coverage reads of the MPS panel is insufficient.KEYWORDS: Droplet digital PCRmassively parallel sequencingcontinuous likelihood ratiosemi-continuous likelihood ratiosingle nucleotide polymorphism AcknowledgmentsThe authors acknowledge the technical support of Professor Tsang-Ming Ko and Genefile Bioscience Laboratory for ddPCR experiments. We thank Ai-Jiun Jung for data analysis and manuscript typewriting.Disclosure statementNo potential conflict of interest was reported by the authors.Supplemental dataSupplemental data for this article can be accessed online at https://doi.org/10.1080/00450618.2023.2261511Additional informationFundingThis work was supported by the National Taiwan University Hospital Taiwan, R.O.C. [Grant Number NTUH 106-003500].