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Updated: Jun 20, 2025
Rare Event Detection Using Error-corrected DNA and RNA Sequencing
Published on: August 3, 2018
Unique molecular identifier-based amplicon sequencing of microhaplotypes for background noise mitigation
Ye-Lim Kwon1, Kyoung-Jin Shin1
1Department of Forensic Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea; Department of Forensic Medicine, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.
This study introduces MH-UMIseq, a novel system using unique molecular identifiers (UMIs) to significantly reduce background noise in microhaplotype (MH) massively parallel sequencing (MPS). MH-UMIseq enhances forensic marker analysis by improving the detection limits of MHs, especially with higher DNA input amounts.
Area of Science:
- Forensic Genetics
- Molecular Biology
- Next-Generation Sequencing Technologies
Background:
- Microhaplotypes (MHs) are valuable forensic markers due to their high polymorphism.
- Massively parallel sequencing (MPS) is widely used for MH analysis, but background noise limits detection.
- Unique molecular identifiers (UMIs) are effective in mitigating sequencing and PCR errors.
Purpose of the Study:
- To design and evaluate a UMI-based amplicon sequencing system (MH-UMIseq) for simultaneous amplification of 46 MHs.
- To assess the performance of MH-UMIseq in reducing background noise across various input DNA amounts.
- To determine the impact of UMI technology on the resolution of MH analysis in forensic applications.
Main Methods:
- Development of a four-step library preparation protocol for MH-UMIseq: barcoding PCR, nuclease reaction, boosting PCR, and indexing PCR.
- Performance evaluation using Illumina NextSeq 550 and MiniSeq with 31 sets and varying DNA inputs (5 ng, 1 ng, 200 pg).
- Analysis of UMI data using fgbio, STRait Razor 3.0, and Visual Microhap.
Main Results:
- MH-UMIseq substantially suppressed background noise, achieving average not suppressed noise proportions of 0.1% (5 ng), 0.3% (1 ng), and 0.7% (200 pg).
- Background noise reduction was more effective with higher DNA input amounts (≥1 ng).
- The number of UMI families correlated with DNA copy number and system resolution, indicating improved performance with increased DNA input.
Conclusions:
- MH-UMIseq effectively mitigates background noise in microhaplotype massively parallel sequencing.
- The system demonstrates superior resolution compared to conventional MPS for deconvoluting complex mixtures with ≥1 ng of DNA.
- MH-UMIseq offers a promising advancement for forensic genetic analysis, enhancing the reliability and sensitivity of microhaplotype profiling.
