Using unique molecular identifiers to improve allele calling in low-template mixtures

Crysup, Benjamin; Mandape, Sammed N.; King, Jonathan L.; Muenzler, Melissa; Kapema, Kapema Bupe; Woerner, August E.

doi:10.1016/j.fsigen.2022.102807

Cited by 12 publications

(3 citation statements)

References 30 publications

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“…The positive effect on the genotype accuracy was also mainly observed for the lower amounts of DNA. Woerner and Crysup et al [ 23 , 25 ] previously applied UMIs in a forensic context, focusing on STRs. Consistent with our findings, their results demonstrated that incorporating UMI reads led to improved genotype calling.…”

Section: Discussionmentioning

confidence: 99%

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

Staadig

Hedman

Tillmar

2023

Genes

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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.

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Section: Discussionmentioning

confidence: 99%

“…A sensitive and accurate detection of low-level DNA variants could potentially be a successful technological improvement for the field of forensic genetics. At present, and as far as we know, only a few studies applying UMI from a forensic perspective have been conducted [ 23 , 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

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

Staadig

Hedman

Tillmar

2023

Genes

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“…This inefficiency becomes particularly pronounced in duplex UMI methods, where both DNA strands require redundant sequencing [51]. Furthermore, inaccuracies in quantifying unique molecules can arise because of errors within the UMI sequences [52].…”

Section: Molecular Barcodingmentioning

confidence: 99%

Clinical Practice Guideline for Blood-based Circulating Tumor DNA Assays

Lee,

Cho,

Kim

et al. 2024

Ann Lab Med

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Circulating tumor DNA (ctDNA) has emerged as a promising tool for various clinical applications, including early diagnosis, therapeutic target identification, treatment response monitoring, prognosis evaluation, and minimal residual disease detection. Consequently, ctDNA assays have been incorporated into clinical practice. In this review, we offer an indepth exploration of the clinical implementation of ctDNA assays. Notably, we examined existing evidence related to pre-analytical procedures, analytical components in current technologies, and result interpretation and reporting processes. The primary objective of this guidelines is to provide recommendations for the clinical utilization of ctDNA assays.

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Feasibility study of a single‐primer extension‐based microhaplotype NGS system

Zhu,

Shu,

Wang

et al. 2024

Electrophoresis

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DNA degradation has been a thorny problem in forensic science. Shortening the amplicon length of the genetic markers improves the analysis of degraded DNA effectively. Microhaplotype (MH) has been proposed as a potential genetic marker that can be used for degraded DNA analysis. In the present study, a 146‐plex MH‐next‐generation sequencing (NGS) system with an average Ae of 6.876 was constructed. Unlike other MH studies, a single‐primer extension (SPE)‐based NGS library preparation method was used to improve the detection of MH markers for degraded DNA. SPE employs a locus‐specific and universal primer to amplify target fragments, reducing the necessity for complete fragment sequences. SPE might effectively mitigate the impact of degradation on amplification. However, SPE produces amplicons of varying lengths, posing challenges in allele calling for SPE‐NGS data. To address this issue, this study proposed a flexible allele‐calling strategy to improve amplicon detection. In addition, this study evaluated the forensic efficacy of the system using 12 low‐template samples (from 1 ng to 7.8 pg), 10 mock‐degraded DNA with various degrees of degradation, and 8 forensic casework samples. When the template is as low as 7.8 pg, our system can accurately detect at least 37 loci and achieves a random match probability (RMP) of 10−30 using the complete allele‐calling strategy. Eighty‐two loci can be detected, and RMP can reach 10−54 using a flexible allele‐calling strategy. After 150 min of 98°C treatment, 36 loci can still be detected, and an RMP of 10−5 can be obtained using the flexible allele‐calling strategy. Furthermore, the number of single nucleotide polymorphism detected at different DNA amounts and degradation levels suggests that the SPE method combined with a flexible allele‐calling strategy is effective.

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Using unique molecular identifiers to improve allele calling in low-template mixtures

Cited by 12 publications

References 30 publications

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

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

Clinical Practice Guideline for Blood-based Circulating Tumor DNA Assays

Feasibility study of a single‐primer extension‐based microhaplotype NGS system

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