Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI.
Quan PengChang XuDaniel KimMarcus LewisJohn DiCarloYexun WangPublished in: Scientific reports (2019)
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.
Keyphrases
- single cell
- real time pcr
- rna seq
- randomized controlled trial
- single molecule
- label free
- loop mediated isothermal amplification
- high throughput
- cancer therapy
- circulating tumor
- genome wide
- copy number
- systematic review
- high resolution
- image quality
- magnetic resonance
- magnetic resonance imaging
- cell free
- dna methylation
- electronic health record
- mass spectrometry
- nucleic acid
- quantum dots
- contrast enhanced