RETrace: simultaneous retrospective lineage tracing and methylation profiling of single cells

Abstract

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100× higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.

Differential DNA methylation and changing cell-type proportions as fibrotic stage progresses in NAFLD

“…It may be possible to infer cell type to a high accuracy from single-cell DNAm data alone considering that DNAm profiles can be specific to cell lineage [37]. A method called RETrace estimates cell-type proportions from DNAm measured using single cell reduced representation bisulfite sequencing [38].…”

The Skull’s Girder: A Brief Review of the Cranial Base

“…Such techniques will provide the ability to revisit and refine long-standing models, while asking novel biological questions and uncover new facets of the cranial base. As an example, the advent of single cell genomics provides a platform to lineage trace cell populations—using de novo spontaneous mutations—in post-mortem tissues [ 79 ]. Such methods could help clarify the neural crest-mesoderm interface of the cranial base in primates, including humans, and in comparison to previous models (chick, mouse) assess if and how this boundary has shifted during evolution.…”

Applications of Single-Cell DNA Sequencing

“…The number of microsatellite loci profiled per cell has subsequently increased to achieve greater lineage resolution, with ∼1,000-2,000 loci per cell in two different studies (14,170) and ∼12,000 loci in a third study (157). One of these studies was able to simultaneously profile methylation in the same cells and recovered a known cultured lineage tree with 88% accuracy (170). However, there remain significant challenges associated with artifactual microsatellite mutations that occur during single-cell genome amplification and library preparation (14).…”