scTAM-seq enables targeted high-confidence analysis of DNA methylation in single cells

Abstract

Single-cell DNA methylation profiling currently suffers from excessive noise and/or limited cellular throughput. We developed scTAM-seq, a targeted bisulfite-free method for profiling up to 650 CpGs in up to 10,000 cells per experiment, with a dropout rate of less than seven percent. scTAM-seq focuses sequencing coverage on informative, variably methylated CpGs that in many tissues and cell types make up a minor fraction of all CpGs. By applying scTAM-seq to B cells from blood and bone marrow, we demonstrate that it can resolve DNA methylation dynamics across B-cell differentiation at unprecedented resolution, identifying intermediate differentiation states that were previously masked. scTAM-seq additionally queries surface protein expression and somatic mutations, thus enabling integration of single-cell DNA methylation information with cell atlas data, and opening applications in tumor profiling. In summary, scTAM- seq is the first high-throughput, high-confidence method for analyzing DNA methylation at single-CpG resolution across thousands of single cells.

Single cell cancer epigenetics

“…Some years later, genomewide MSRE-based single cell technologies were developed, such as single-cell CpG-island sequencing (scCGI-seq) and epigenomics and genomics of single cells analyzed by restriction (epi-gSCAR), achieving up to 18.8% genome coverage [49,50]. Additionally, a novel targeted bisulfite-free method, named single cell-targeted analysis of the methylome (scTAM-seq), directly profiles 650 specific CpG sites in up to 10 000 cells using a commercial microfluidic platform [51]. By avoiding the use of bisulfite, we can ensure better DNA integrity, although nonbisulfite-based technologies still exhibit similar coverage to those that are bisulfite based.…”

“…Single cell-sequencing and spatial technologies to study the different epigenetic mechanisms in cancer. Abbreviations: ACT-seq, antibody-guided chromatin tagmentation sequencing [170]; CLEVER-seq, chemicallabeling-enabled C-to-T conversion sequencing [46]; CoBATCH, combinatorial barcoding and targeted chromatin release [171]; epigenomic MERFISH, epigenomic multiplexed error robust fluorescence in situ hybridization [108], scABA-seq, single cell restriction endonuclease AbaSI sequencing [45]; scATAC-seq, single cell sequencing assay for transposase-accessible chromatin [59]; scBS-seq, single cell bisulfite sequencing [41]; scChIC-seq, single cell chromatin immunocleavage sequencing [76]; scChIL-seq, single cell chromatin integration labeling [169]; scChIP-seq, single cell chromatin immunoprecipitation followed by sequencing [75]; scCUT&Tag, single cell cleavage under targets and tagmentation [174]; scDamID, single cell DNA adenine methyltransferase identification [84]; scHi-C, single cell Hi-C [82,83]; sciMAP-ATAC, single cell combinatorial indexing on microbiopsies assigned to positions for the assay for transposase accessible chromatin [109]; sci-MET, single cell combinatorial indexing for methylation analysis [152]; scMAB-seq, single cell methylase-assisted bisulfite sequencing [47]; scMNase-seq, single cell micrococcal nuclease sequencing [74]; scRRBS-seq, single cell reduced representation bisulfite sequencing [39,40]; scTAM-seq, single cell targeted analysis of the methylome [51]; scTEM-seq, single cell transposable element methylation sequencing [155]; spatial ATAC-seq, spatially resolved chromatin accessibility profiling [110]; spatial CUT&Tag, spatial histone modification profiling with cleavage under targets and tagmentation [107]; uliCUT&RUN, ultra-low-input cleavage under targets and release using nuclease [172].…”