Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type–specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

Original languageEnglish
Pages (from-to)401-414
Number of pages14
JournalChromosome Research
Issue number4
Early online dateJul 4 2022
StatePublished - Dec 2022

Bibliographical note

Funding Information:
The research reported in this study was supported by NIH R35GM137950 and U54AG076041 to JCRM.

Funding Information:
We thank Laura Niedernhofer and Hai Dang Nguyen for providing the MEFs and U2OS cell lines and Silvia Meyer-Nava and Anala Shetty for critically reading the manuscript.

Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature B.V.


  • DNA replication
  • replication timing
  • genomics
  • library preparation
  • next-generation sequencing

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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