Applying Unique Molecular Indices with an Extensive All-in-One Forensic SNP Panel for Improved Genotype Accuracy and Sensitivity

Abstract

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.

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“…The FORCE panel was first tested using an in-solution hybridization capture approach to accommodate severely degraded DNA (<75 bp fragments) [1]. In 2023, Staadig et al evaluated the FORCE panel utilizing the QIAseq Targeted DNA chemistry (QIAGEN) and MiSeq sequencing as the basis for the laboratory workflow [27]. The QIAseq enrichment technology employs a unique primer design, which makes the method more amenable to degraded DNA than traditional PCR enrichment.…”

“…Due to limitations of the enrichment approach (i.e., single primer extension), 10 SNPs were excluded from the initial panel design, resulting in a total of 5,497 SNPs in the QIAseq FORCE panel (4,073 total iiSNP and kiSNPs, all categorized herein as iiSNPs). The QIAseq FORCE method was shown to produce accurate genotype calls down to 125 pg DNA input [27]. In addition to the MiSeq (or MiSeq FGx in RUO mode), the QIAseq FORCE panel can also be sequenced on higher throughput instruments (e.g., NextSeq) [1].…”