Mapping Replication Timing in Single Mammalian Cells

Abstract

Replication timing (RT) is the temporal order in which genomic DNA is replicated during S phase. Early and late replication correlate with transcriptionally active and inactive chromatin compartments, but mechanistic links between large‐scale chromosome structure, transcription, and replication are still enigmatic. A proper RT program is necessary to maintain the global epigenome that defines cell identity, suggesting that RT is critical for epigenome integrity by facilitating the assembly of different types of chromatin at different times during S phase. RT is regulated during development and has been found to be altered in disease. Thus, RT can identify stable epigenetic differences distinguishing cell types, and can be used to help stratify patient outcomes and identify markers of disease. Most methods to profile RT require thousands of S‐phase cells. In cases where cells are rare (e.g., early‐stage embryos or rare primary cell types) or consist of a heterogeneous mixture of cell states (e.g., differentiation intermediates), or when the interest is in determining the degree of stable epigenetic heterogeneity within a population of cells, single‐cell measurements of RT are necessary. We have previously developed single cell Repli‐seq, a method to measure replication timing in single cells using DNA copy number quantification. To date, however, single‐cell Repli‐seq suffers from relatively low throughput and high costs. Here, we describe an improved single‐cell Repli‐seq protocol that uses degenerate oligonucleotide–primed PCR (DOP‐PCR) for uniform whole‐genome amplification and uniquely barcoded primers that permit early pooling of single‐cell samples into a single library preparation. We also provide a bioinformatics platform for analysis of the data. The improved throughput and decreased costs of this method relative to previously published single‐cell Repli‐seq protocols should make it considerably more accessible to a broad range of investigators. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Whole Genome Amplification (WGA) of single cells and sequence library construction. Basic Protocol 2: Deriving and displaying single‐cell replication timing data from whole genome sequencing.

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer

“…DNA from microdissected KPC tumour tissue was subject to whole-genome amplification as described before 55 . TruSeq indexed Illumina sequencing libraries where then constructed from WGA DNA and subjected to sparse whole-genome sequencing at a depth of roughly 3 million sequencing reads, enough to enable copy number ascertainment at a bin resolution of ~100 kb.…”

Emergence of replication timing during early mammalian development

“…To understand when and how RT emerges during development, we used single-cell Repli-seq 5 , 19 in preimplantation mouse embryos (Fig. 1a,b ).…”

Optimized Repli-seq: Improved DNA Replication Timing Analysis by Next-Generation Sequencing

“…Repli-seq. Single-cell Repli-seq (scRepli-seq) has also been achieved (Dileep and Gilbert 2018;Takahashi et al 2019;Miura et al 2020;Bartlett et al 2022), and it allows estimation of cell-to-cell variability in RT. However, scRepli-seq is low throughput, very laborious and expensive, requires the generation of multiple single-cell libraries per sample, and has low resolution.…”

“…This alternative allows measuring RT without the need of cell purification by flow cytometry; however, it requires costly deep sequencing (≥30X coverage) and the dynamic range of the data obtained is even lower than that of the S/G1 Repli-seq. Single-cell Repli-seq (scRepli-seq) has also been achieved (Dileep and Gilbert 2018; Takahashi et al 2019; Miura et al 2020; Bartlett et al 2022), and it allows estimation of cell-to-cell variability in RT. However, scRepli-seq is low throughput, very laborious and expensive, requires the generation of multiple single-cell libraries per sample, and has low resolution.…”