Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing

Area of Science:

• Genomics and Molecular Biology
• Epigenetics and Chromosome Biology

Background:

• Replication timing (RT) domains are fundamental units of chromosome organization, implicated in cellular development and disease.
• Existing genome-wide RT mapping methods are limited by the need for large cell numbers, hindering studies on rare cell types or single cells.

Purpose of the Study:

• To introduce a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq).
• To enable high-resolution analysis of replication timing at the single-cell level.

Main Methods:

• scRepli-seq utilizes whole-genome amplification (WGA) of genomic DNA (gDNA) from individual S-phase cells.
• Next-generation sequencing (NGS) determines copy-number differences between replicated and unreplicated DNA.
• Haplotype-resolved scRepli-seq is achieved using single-nucleotide polymorphism (SNP)/indel data to distinguish homologous chromosomes.

Main Results:

• The protocol is experimentally feasible within 3 days before sequencing.
• Computational pipelines for data quality control, normalization, and binarization are provided.
• scRepli-seq successfully maps replication timing domains in single cells.

Conclusions:

• scRepli-seq overcomes limitations of conventional methods, enabling single-cell resolution of replication timing.
• This technique provides a powerful tool for investigating the role of replication timing in biological processes and diseases at an unprecedented resolution.