The investigation of biomarkers is vital for the detection, monitoring and treatment of diseases such as cancer. Many diagnostic and prognostic biomarkers have already been established for use in the clinic – however, a new generation of novel biomarkers is required to detect disease earlier and increase both sensitivity and specificity of detection.

For many cancers, especially hematological (blood) cancers, the detection and measurement of DNA methylation represents a promising biomarker – for example, in regions of the genome known as CpG islands that are frequently methylated. However, many of the current tools used to investigate methylation are unable to capture cell-to-cell distinctiveness, are restricted to small samples or have technical limitations.

Researchers in a recent publication have detailed a new high-confidence method to analyze methylation in B cells. They combined a methylation analytics approach with a high-throughput single-cell multi-omics workflow – Tapestri – from Mission Bio. Technology Networks spoke with Anjali Pradhan, senior vice president of product management at Mission Bio, to discuss this new application for DNA methylation analysis, and its potential in uncovering new biological insights and biomarkers that may guide the future of cancer research.

Sarah Whelan (SW): How important is methylation as a biomarker for hematological malignancy? For example, disease detection, progression monitoring and therapeutics development?

Anjali Pradhan (AP): DNA methylation is a key epigenetic player in the abnormal initiation, development and progression of some of the most aggressive forms of hematopoietic neoplasms – such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL) and diffuse large B cell lymphoma (DLBCL) – often acting in synergy with other epigenetic alterations. It also contributes to the acquisition of drug resistance.

Compared to other epigenetic mechanisms, DNA methylation is stable and less prone to random noise, which makes these methylation-based biomarkers well-suited for analysis in both research settings and clinical applications. Over the last decade, there have been significant advancements in identifying disease‐specific DNA methylation signatures in an attempt to diagnose and monitor patients and formulate a personalized therapeutic course of action. A class of drugs known as DNA hypomethylating agents (HMA), which can reverse DNA methylation, are now being used as standard-of-care therapy for intermediate and higher-risk MDS and AML patients.           

SW: Can you briefly explain what scTAM-seq is and how it works?

AP: scTAM-seq is a powerful method for investigating DNA methylation dynamics at single-cell and single-nucleotide resolution, in a high-throughput manner. The method involves the use of methylation-sensitive restriction enzymes (MSREs) to selectively cut unmethylated recognition sites while leaving methylated sites intact.

Briefly, isolated cells are loaded on the Tapestri® Platform, where they first undergo encapsulation to form isolated droplets that contain protease enzymes to allow access to genomic DNA. The lysate, including the now histone-free DNA, then undergoes a second encapsulation step, where the contents are merged with the MSRE, barcoding beads, PCR primers (that contain a single cut site) and reagents, to exclusively target CpG sites in the genome. Sequencing libraries are prepared and sequenced on any of the Illumina next-generation sequencing (NGS) platforms, and reported out as corresponding NGS reads after sequencing. Tapestri’s multiomic capabilities allow the investigator to combine scTAM-seq with simultaneous analysis of somatic mutations and cell surface marker expression.

Figure 1: Overview of the scTAM-seq workflow. Credit: Bianchi, A. et al., Genome Biology (2022)

SW: What advantages does the scTAM-seq approach have over other methylation profiling techniques?

AP: Traditionally, the most common approach for assessing DNA methylation has been through genome-wide bulk sequencing. However, methylation signals obtained from bulk sequencing are averaged out, thereby concealing the ability to uncover cell-to-cell differences in methylation signatures involved in health and disease.

More recently, several single-cell methylation methods were developed that could reveal these signatures offering insights into cellular heterogeneity.

Genome-wide single-cell approaches, such as bisulfite sequencing, come with unique challenges. Technically, genome-wide methylation data are sparse and genomic coverage is rather limited even for deeply sequenced samples. Additionally, bisulfite conversion can cause the degradation of DNA, leading to excessive sample loss.

Targeted single-cell approaches that use MSREs have been limited to a throughput of less than 100 cells per experiment, with less than 60 investigated CpGs per cell and high false-positive rates due to incomplete digestion of unmethylated CpGs.

To address the existing gaps in current single-cell DNA methylation methods, scTAM-seq leveraging Tapestri® was developed by a group of investigators from the labs of Dr. Renée Beekman and Dr. Lars Velten at Centre for Genomic Regulation (CRG), Barcelona, Spain.

This method has several advantages over other single-cell methylation approaches. First, it’s high-throughput, and can directly profile 650 biologically relevant CpG sites in up to 10,000 cells. It also enables DNA methylation assessment with high accuracy, achieving a median false-positive rate lower than 0.2% combined with a median false-negative rate as low as 7%. And scTAM-seq is multiomics capable, enabling the simultaneous assessment of DNA methylation (epigenetics), somatic mutations (genotype) and surface marker expression (phenotype), letting researchers gain an incredibly rich picture of clonal architecture within a tumor.

SW: How has this technique advanced our understanding of DNA methylation dynamics?

AP: Out of the 28 million CpGs in the human genome, a relatively small subset change their methylation state and are informative on what’s going on inside a cell. scTAM-seq facilitates the assessment of these biologically relevant CpGs at high-resolution, high-confidence and high-throughput, thereby offering an accurate and cost-effective solution to understanding the influence of DNA methylation dynamics in normal biology and disease.

SW: Do you think this technique could have an impact on the future of cancer diagnosis and treatment? If so, how?

AP: Yes, most definitely. Tumors derived from different tissues show unique patterns of DNA methylation changes – in other words, methylation markers differ from one cancer type to another. There have been significant efforts to distill down the large discovery efforts to a manageable number of methylation markers. scTAM-seq approaches can help shape the future of cancer diagnosis by identifying the most relevant tumor-specific diagnostic and prognostic methylation markers, whose methylation is both specific to tumor type and informative of tumor stage. scTAM-seq can also facilitate epigenetic drug discovery by identifying targets for epigenetic drugs, monitoring the therapeutic efficacy and identifying alterations that contribute to drug resistance.

Anjali Pradhan was speaking to Dr. Sarah Whelan, Science Writer for Technology Networks.