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

Bianchi, Agostina; Scherer, Michael; Zaurín, Roser; Quililan, Kimberly; Velten, Lars; Beekman, Renée

doi:10.1186/s13059-022-02796-7

Cited by 16 publications

(17 citation statements)

References 68 publications

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“…Moreover, according to the available data epigenetic changes can also contribute to the transformation of JMML clones and determine the outcome of the disease 22 . Although the Tapestri technology can be used to assess DNA methylation 23 , here, we did not analyze the methylation profile of WT clones. In our opinion, epigenetic changes are secondary event and occur after mutations in the RAS signaling pathway.…”

Section: Discussionmentioning

confidence: 99%

Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases

Volchkov,

Khozyainova,

Gurzhikhanova

et al. 2023

npj Syst Biol Appl

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Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of early childhood that develops due to mutations in the genes of the RAS-signaling pathway. Next-generation high throughput sequencing (NGS) enables identification of various secondary molecular genetic events that can facilitate JMML progression and transformation into secondary acute myeloid leukemia (sAML). The methods of single-cell DNA sequencing (scDNA-seq) enable overcoming limitations of bulk NGS and exploring genetic heterogeneity at the level of individual cells, which can help in a better understanding of the mechanisms leading to JMML progression and provide an opportunity to evaluate the response of leukemia to therapy. In the present work, we applied a two-step droplet microfluidics approach to detect DNA alterations among thousands of single cells and to analyze clonal dynamics in two JMML patients with sAML transformation before and after hematopoietic stem cell transplantation (HSCT). At the time of diagnosis both of our patients harbored only “canonical” mutations in the RAS signaling pathway genes detected by targeted DNA sequencing. Analysis of samples from the time of transformation JMML to sAML revealed additional genetic events that are potential drivers for disease progression in both patients. ScDNA-seq was able to measure of chimerism level and detect a residual tumor clone in the second patient after HSCT (sensitivity of less than 0.1% tumor cells). The data obtained demonstrate the value of scDNA-seq to assess the clonal evolution of JMML to sAML, response to therapy and engraftment monitoring.

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Section: Discussionmentioning

confidence: 99%

Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases

Volchkov,

Khozyainova,

Gurzhikhanova

et al. 2023

npj Syst Biol Appl

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“…For profiling DNA methylation at single-cell resolution, we used scTAM-seq 30 , which leverages the Mission Bio Tapestri technology to investigate up to 1,000 CpGs in up to 10,000 cells per experiment. Briefly, we loaded 120,000-140,000 cells into the Tapestri machine and followed the default Mission Bio DNA+Protein protocol for V2 chemistry (https://missionbio.com/wp-content/uploads/2021/02/Tapestri-Single-Cell-DNA-Protein-Sequencing-V2-User-Guide-PN_3360A.pdf), with modifications: (i) we added a DNA methylation sensitive restriction enzyme (HhaI) to remove non-methylated targets prior to amplification.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, barcodes were extracted from the raw sequencing files before alignment to the reference genome subset to the CpG panel. Reads mapping to each of the amplicons were quantified to generate a count matrix and DNA methylation states were determined using a cutoff of one sequencing read as in the original scTAM-seq publication 30 . We used those cellular barcodes that had more than 10 sequencing reads in at least 70% of the control (non-HhaI) amplicons.…”

Section: Methodsmentioning

confidence: 99%

“…We present a novel method, EPI-Clone, that extracts both clonal and cell state information from targeted single-cell DNA methylation data. EPI-Clone builds upon scTAM-seq, which uses the commercially available Mission Bio Tapestri platform, to read out methylation of several hundred CpGs in up to tens of thousands of single cells at a time, with a dropout rate of ∼7% 30 . We applied EPI-clone to barcoded hematopoietic progenitor and mature myeloid cells, as well as in native human and mouse hematopoiesis and were able to accurately identify clone of origin for most cells.…”

Section: Introductionmentioning

confidence: 99%

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Somatic epimutations enable single-cell lineage tracing in native hematopoiesis across the murine and human lifespan

Scherer,

Singh,

Braun

et al. 2024

Preprint

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SummaryCurrent approaches to lineage tracing of stem cell clones require genetic engineering or rely on sparse somatic DNA variants, which are difficult to capture at single-cell resolution. Here, we show that targeted single-cell measurements of DNA methylation at single-CpG resolution deliver joint information about cellular differentiation state and clonal identities. We develop EPI-clone, a droplet-based method for transgene-free lineage tracing, and apply it to study hematopoiesis, capturing hundreds of clonal trajectories across almost 100,000 single-cells. Using ground-truth genetic barcodes, we demonstrate that EPI-clone accurately identifies clonal lineages throughout hematopoietic differentiation. Applied to unperturbed hematopoiesis, we describe an overall decline of clonal complexity during murine ageing and the expansion of rare low-output stem cell clones. In aged human donors, we identified expanded hematopoietic clones with and without genetic lesions, and various degrees of clonal complexity. Taken together, EPI-clone enables accurate and transgene-free single-cell lineage tracing at scale.

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“…Next we utilized single-cell Targeted Analysis of the Methylome (scTAM-seq) 41 data from mouse hematopoiesis 42 . In line with our previous observations at the level of chromatin accessibility (Figure 2A), we found that DNA methylation-based heterogeneity was increased in more primitive hematopoietic progenitor cells such as HSCs and early MPPs compared to more differentiated progenitors such as GMPs 11 and pre-B cells (Figure 5C).…”

Section: Epichaos Is Applicable To Single-cell Epigenomics Data From ...mentioning

confidence: 99%

EpiCHAOS: a metric to quantify epigenomic heterogeneity in single-cell data

Kelly,

Scherer,

Braun

et al. 2024

Preprint

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Epigenetic heterogeneity is a fundamental property of biological systems, and is recognized as a potential driver of tumor plasticity and therapy resistance. Single-cell epigenomics technologies have been widely employed to study epigenetic variation between - but not within - cellular clusters. We introduce epiCHAOS: a quantitative metric of cell-to-cell epigenetic heterogeneity, applicable to any single-cell epigenomics data type. After validation in synthetic datasets, we applied epiCHAOS to investigate global and region-specific patterns of epigenetic heterogeneity across diverse biological systems. EpiCHAOS provides an excellent approximation of stemness and plasticity in development and malignancy, making it a valuable addition to single-cell cancer epigenomics analyses.

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

Cited by 16 publications

References 68 publications

Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases

Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases

Somatic epimutations enable single-cell lineage tracing in native hematopoiesis across the murine and human lifespan

EpiCHAOS: a metric to quantify epigenomic heterogeneity in single-cell data

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