Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI

Quan Peng,Marcus Lewis,John Dicarlo,Yexun Wang,Chang Xu,Daniel Kim

doi:10.1038/s41598-019-41215-z

Abstract

For specific detection of somatic variants at very low levels, artifacts from the NGS workflow have to be eliminated. Various approaches using unique molecular identifiers (UMI) to analytically remove NGS artifacts have been described. Among them, Duplex-seq was shown to be highly effective, by leveraging the sequence complementarity of two DNA strands. However, all of the published Duplex-seq implementations so far required pair-end sequencing and in the case of combining duplex sequencing with target enrichment, lengthy hybridization enrichment was required. We developed a simple protocol, which enabled the retrieval of duplex UMI in multiplex PCR based enrichment and sequencing. Using this protocol and reference materials, we demonstrated the accurate detection of known SNVs at 0.1–0.2% allele fractions, aided by duplex UMI. We also observed that low level base substitution artifacts could be introduced when preparing in vitro DNA reference materials, which could limit their utility as a benchmarking tool for variant detection at very low levels. Our new targeted sequencing method offers the benefit of using duplex UMI to remove NGS artifacts in a much more simplified workflow than existing targeted duplex sequencing methods.

Highlights

Recent advancement in generation sequencing technology allows people to profile large genomic regions and look for somatic variations efficiently
Both original top and bottom strands can be enriched by single primer extension and their duplex relationship is traceable by the combination of unique molecular identifiers (UMI) and strand barcodes
Using the in-house DNA references prepared from mild sonication, we evaluated the performance of our duplex UMI enabled single primer enrichment workflow, by performing targeted sequencing with the 192 plex panel

Summary

Introduction

Recent advancement in generation sequencing technology allows people to profile large genomic regions and look for somatic variations efficiently. Given enough DNA input, an efficient enrichment process and enough sequencing depth, detecting mutations at very low fractions is still challenged by artifacts accumulated in many steps of the NGS workflow[6,7,8] Those artifacts can arise from DNA base damage during the sample preparation, erroneous base incorporation by DNA polymerase during enrichment and library amplification, and errors from final sequencing readout. In order to better differentiate those NGS artifacts from real mutations at very low allele fractions, people have developed an error-correction mechanism through the incorporation of unique molecular identifiers (UMIs) in the NGS workflow[9,10,11,12] In these protocols, UMIs (carried by short oligos) are attached to endogenous DNA fragments, carried along through enrichment and amplification, and sequenced together with genomic sequences. Changing from single-plex to duplex UMI would be desirable to combine the benefit of a simple enrichment workflow and increased variant calling accuracy

Methods

Results

Conclusion