The development of massively parallel sequencing (MPS) technology has enabled the discovery of several new types of forensic markers where microhaplotypes are one of these promising novel genetic markers. Microhaplotypes are, commonly, less than 300 nucleotides in length and consist of two or more closely linked single-nucleotide polymorphisms (SNPs). In this study, we have examined a custom-made QIAseq Microhaplotype panel (Qiagen), including 45 different microhaplotype loci. DNA libraries were prepared according to the GeneRead DNAseq Targeted Panels V2 library preparation workflow (Qiagen) and sequenced on a MiSeq FGx instrument (Verogen). We evaluated the performance of the panel based on 75 samples of Swedish origin and haplotype frequencies were established. We performed sensitivity studies and could detect haplotypes at input amounts down to 0.8 ng. We also studied mixture samples with two contributors for which haplotypes, for the minor contributor, were detectable down to the level of 1:100. Furthermore, we executed kinship simulations to evaluate the usefulness of this panel in kinship analysis. The results showed that both paternity and full sibling cases can clearly be solved. When simulating a half sibling versus unrelated case scenario, there were, however, some overlap of the likelihood ratio distributions potentially resulting in inconclusiveness. To conclude, the results of this initial study are promising for further implementation of this microhaplotype assay into the forensic field, although we noticed some primer design issues that could be optimized, which possibly would increase the power of the assay.

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One of the major challenges in forensic genetics is being able to detect very small amounts of DNA. Massively parallel sequencing (MPS) enables sensitive detection; however, genotype errors may exist and could interfere with the interpretation. Common errors in MPS-based analysis are often induced during PCR or sequencing. Unique molecular indices (UMIs) are short random nucleotide sequences ligated to each template molecule prior to amplification. Applying UMIs can improve the limit of detection by enabling accurate counting of initial template molecules and removal of erroneous data. In this study, we applied the FORCE panel, which includes ~5500 SNPs, with a QIAseq Targeted DNA Custom Panel (Qiagen), including UMIs. Our main objective was to investigate whether UMIs can enhance the sensitivity and accuracy of forensic genotyping and to evaluate the overall assay performance. We analyzed the data both with and without the UMI information, and the results showed that both genotype accuracy and sensitivity were improved when applying UMIs. The results showed very high genotype accuracies (>99%) for both reference DNA and challenging samples, down to 125 pg. To conclude, we show successful assay performance for several forensic applications and improvements in forensic genotyping when applying UMIs.