High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations

Abstract

SummaryIncreasing evidence of functional and transcriptional heterogeneity in phenotypically similar cells examined individually has prompted interest in obtaining parallel methylome data. We describe the development and application of such a protocol to index-sorted murine and human hematopoietic cells that are highly enriched in their content of functionally defined stem cells. Utilizing an optimized single-cell bisulfite sequencing protocol, we obtained quantitative DNA methylation measurements of up to 5.7 million CpGs in single hematopoietic cells. In parallel, we developed an analytical strategy (PDclust) to define single-cell DNA methylation states through pairwise comparisons of single-CpG methylation measurements. PDclust revealed that a single-cell epigenetic state can be described by a small (<1%) stochastically sampled fraction of CpGs and that these states are reflective of cell identity and state. Using relationships revealed by PDclust, we derive near complete methylomes for epigenetically distinct subpopulations of hematopoietic cells enriched for functional stem cell content.

Polycomb contraction differentially regulates terminal human hematopoietic differentiation programs

“…Whole genome bisulfite libraries were constructed as previously described 61 minutes. The Bisulfite converted single stranded DNA was converted to double stranded DNA through 1 cycle of PCR with random hexamer as previously described 61 followed by standard illumine library construction. Libraries were aligned using Novoalign V3.02.10 (www.novocraft.com) to human genome assembly GRCh37 (hg19).…”

Mathematical modeling with single-cell sequencing data

“…Future work and applications 526 Future applications of this approach is to explore hypothesis in the resolution of 527 single-cell genomics and study altered and novel cell states with genetic and epigenetic 528 alterations in various biological systems and pathogenesis [46]. Incorporating effects of 529 external perturbations such as therapy is our interest as well.…”

Epiclomal: probabilistic clustering of sparse single-cell DNA methylation data

“…Advances in technology 7 and laboratory protocols have allowed the generation of high-throughput sequencing 8 data of individual cells [3][4][5][6]. In particular, single-cell whole-genome bisulfite sequencing 9 (sc-WGBS) techniques have been developed to assess the epigenetic diversity of a 10 population of cells [7,8]. Because of the limited amount of DNA material, the generated 11 sc-WGBS data are usually sparse, that is, data from a large number of CpG sites are 12 missing and/or are subject to measurement error.…”

“…An increasing amount of sc-WGBS data has been generated from various cell types 16 including: mouse embryonic stem cells [9,10], human hematopoietic stem cells [7,11], 17 human hepatocellular carcinoma [12], mouse hepatocytes and fibroblasts [13], human 18 and mouse brain cells [14] and human cell lines [15]. In order to assess the epigenetic 19 diversity in these different cell populations, a variety of non-probabilistic methods have 20 been considered.…”

“…Introduction 1 DNA methylation of the fifth cytosine position (5mC) is a well studied epigenetic mark clustering methods over previously published and synthetic data sets, plus a novel large 57 scale sc-WGBS data set from breast cancer xenografts [22,23] generated using state of 58 the art methodology [7]. 59…”