FORK-seq

FORK-seq maps genome replication at single-molecule resolution in Saccharomyces cerevisiae using nanopore sequencing and BrdU (Bromodeoxyuridine) pulse-chase labelling to profile replication tracks at approximately 200-nucleotide resolution.

Key Features:

• Single-molecule mapping: Profiles individual DNA molecules with nanopore sequencing to map replication tracks at ~200-nucleotide resolution.
• BrdU quantification: Quantifies incorporation of BrdU (Bromodeoxyuridine) along pulse-chased replication intermediates.
• Single-cell dynamics reconstruction: Reconstructs DNA replication dynamics at the single-cell level from replication tracks.
• Initiation and termination detection: Identifies individual initiation and termination events on single molecules.
• Population-level validation: Reproduces population-based replication directionality profiles from single-molecule data.
• Heterogeneity detection: Reveals cell-to-cell heterogeneity in replication initiation and termination locations.
• Novel event discovery: Detects initiations at previously unidentified locations (9%) and terminations at previously unidentified locations (18%).
• Large-scale analysis: Applied to a dataset of 58,651 replication tracks.

Scientific Applications:

• High-resolution replication mapping: Study genome replication architecture in Saccharomyces cerevisiae at ~200-nt resolution.
• Initiation/termination variability analysis: Analyze per-cell variability of replication initiation and termination events.
• Genomic stability and replication stress research: Investigate genomic stability and replication stress responses that depend on accurate DNA replication.
• Discovery of novel replication loci: Identify previously unannotated initiation and termination sites.

Methodology:

Quantification of BrdU incorporation along pulse-chased replication intermediates from nanopore-sequenced single molecules, mapping of replication tracks, reconstruction of DNA replication dynamics at single-cell level, reproduction of population replication directionality profiles, and identification of individual initiation and termination events.