LUMI-PCR

LUMI-PCR enhances ligation-mediated PCR (LM-PCR) workflows for cloning and quantitative mapping of genomic integration sites on the Illumina sequencing platform.

Key Features:

• Novel Adapter Design: Employs an Illumina-compatible adapter with inverted forked adapter strand orientation to prevent amplification of non-target DNA while preserving sequence diversity for cluster generation.
• Unique Molecular Identifiers (UMIs): Integrates UMIs to reduce PCR amplification bias and improve quantitation of relative integration abundance at saturating sequencing coverage.
• Reduced PCR Cycles: Merges ligation and library generation steps to reduce total PCR cycles without sacrificing coverage or fidelity.
• High Reproducibility and Coverage: Produces reproducible quantitation of clonal integrations and deep coverage of subclonal integrations, demonstrated with replicate libraries from murine leukemia virus-infected spleen samples.
• Scalability and Automation: Compatible with 96-well formats and automated liquid handling systems (e.g., Beckman Biomek) for high-throughput processing.
• Informatics Pipeline: Includes a pipeline that maps reads, constructs integration contigs, and quantitates integration abundance using both fragment lengths and UMIs.
• Optimization Suggestions: Provides recommendations for adapting the protocol to target other DNA sequences.

Scientific Applications:

• Integration Site Identification: Identifying integration sites of insertional mutagens, integrating vectors, and naturally occurring mobile genetic elements using Illumina sequencing.
• Quantitative Analysis in Complex Mixtures: Quantitating relative abundance of integrations within complex mixtures, including clonal and subclonal populations.
• Insertional Mutagenesis Screens: Analyzing cell populations undergoing selection in insertional mutagenesis screens to assess clonal dynamics.

Methodology:

The informatics pipeline maps reads, constructs integration contigs, and quantitates integration abundance using fragment lengths and UMIs.