Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Next-generation sequencing (NGS) workflows can introduce artifacts, complicating the detection of low-frequency somatic variants.
• Unique molecular identifiers (UMI) are used to mitigate NGS artifacts, with Duplex-seq being a highly effective method leveraging DNA strand complementarity.
• Existing Duplex-seq protocols often require pair-end sequencing and lengthy target enrichment steps.

Purpose of the Study:

• To develop a simplified protocol for duplex unique molecular identifier (UMI) retrieval in multiplex PCR-based enrichment and sequencing.
• To demonstrate the accurate detection of single nucleotide variants (SNVs) at very low allele fractions using the new method.
• To evaluate the utility of in vitro DNA reference materials for benchmarking low-level variant detection.

Main Methods:

• Development of a novel protocol for duplex UMI capture during multiplex PCR enrichment and sequencing.
• Application of the protocol to reference materials for assessing SNV detection accuracy.
• Analysis of potential artifacts introduced during the preparation of in vitro DNA reference materials.

Main Results:

• Accurate detection of known SNVs at 0.1-0.2% allele fractions was achieved using duplex UMI.
• The developed method simplifies the workflow compared to existing targeted duplex sequencing approaches.
• Low-level base substitution artifacts were identified in in vitro DNA reference materials, potentially limiting their use for benchmarking.

Conclusions:

• The new targeted sequencing method effectively uses duplex UMI to remove NGS artifacts within a simplified workflow.
• This approach enhances the accuracy of detecting low-frequency somatic variants.
• Caution is advised regarding the use of in vitro DNA reference materials for validating very low-level variant detection due to potential introduced artifacts.