Detection Of DNA Methylation Research Articles

An intra-stimuli resonance energy transfer-based electrochemiluminescence biosensor for DNA methylation detection

Aberrant DNA methylation, as one of the promising biomarkers, is intimately connected to a variety of genetic and tumor diseases. Herein, we constructed an innovative intra-stimuli electrochemiluminescence resonance energy transfer (ECL-RET) biosensor for methylated DNA detection with high sensitivity and accuracy. Glucose oxidase (GOD) was modified at the terminal of assist DNA as functional conjugation (assist DNA-GOD), which then hybridized with methylated target DNA and hairpin DNA to form Y-shaped DNA ternary conjugate. Ru(bpy)32+ encapsulated silica nanospheres (RuSiO2 NPs) were uniformly coated with silver nanoparticles (Ag NPs) and the formed RuSiO2@Ag nanocomposites were introduced to the terminal of hairpin DNA via amide bond. The combination of the ECL donor (RuSiO2 NPs) and acceptor (Ag NPs) on one nanostructure yielded an intra-molecular ECL-RET. Afterward, the presence of H2O2 as the product of GOD catalyzing glucose induced the etching of Ag NPs and subsequent recovery of ECL signal from RuSiO2 NPs. Thus, due to the dependence of the ECL enhancement on the target concentration, this method achieved the quantitative analysis of methylated target DNA with good selectivity, repeatability, and practical utility.

Short-Term Microplastics Exposure to the Common Mysid Shrimp, Americamysis bahia: Effects on Mortality and DNA Methylation

Primary consumers of microplastics are often zooplankton species such as the mysid shrimp, Americamysis bahia. Ingesting and interacting with these plastics can cause stress and lead to death. In the presence of some environmental stressors, gene expression may be altered without changing DNA sequences via the epigenetic methylation of the DNA. Mysid shrimp were exposed to 5-micrometer fluorescent polystyrene microbeads at different concentrations and different lengths of time. No significant effects were observed on mortality within 72 h, but mortality increased significantly thereafter. Microplastics were consumed by mysids and adhered to the mysid carapace and appendages. An ELISA-like (Enzyme-Linked Imuunosorbent Assay) colorimetric assay was employed to assess mysid DNA for differences in global percent methylation. No significant difference in the average percent methylated DNA nor difference in the number of methylation detections between treatments was found. This is one of few studies that has investigated DNA methylation effects due to microplastics-induced stress and the first study to detect DNA methylation in any member of the order Mysida.

DNA Methylation Biomarkers in Cancer Diagnostics

Abstract Cancer development and progression are characterized by intricate genetic and epigenetic alternations, with DNA methylation playing a crucial role in this transformative process. Recent advancements in DNA methylation mapping technologies have significantly contributed to the identification of novel cancer biomarkers. We provide a short overview of Conformité Européenne-marked in vitro diagnostic tests designed to detect DNA methylation changes in oncology practice.

Differential DNA methylation landscape of miRNAs genes in mice liver fibrosis.

Patients with chronic liver disease were found nearly all to have liver fibrosis, which is characterized by excess accumulation of extracellular matrix (ECM) proteins. While ECM accumulation can prevent liver infection and injury, it can destroy normal liver function and architecture. miRNA's own regulation was involved in DNA methylation change. The purpose of this study is to detect DNA methylation landscape of miRNAs genes in mice liver fibrosis tissues. Male mice (10-12 weeks) were injected CCl4 from abdominal cavity to induced liver fibrosis. 850K BeadChips were used to examine DNA methylation change in whole genome. The methylation change of 16 CpG dinucleotides located in promoter regions of 4 miRNA genes were detected by bisulfite sequencing polymerase chain reaction (BSP) to verify chip data accuracy, and these 4 miRNA genes' expressions were detected by RT-qPCR methods. There are 769 differential methylation sites (DMS) in total between fibrotic liver tissue and normal mice liver tissue, which were related with 148 different miRNA genes. Chips array data were confirmed by bisulfite sequencing polymerase chain reaction (R = 0.953; P < 0.01). GO analysis of the target genes of 2 miRNA revealed that protein binding, cytoplasm and chromatin binding activity were commonly enriched; KEGG pathway enrichment analysis displayed that TGF-beta signaling pathway was commonly enriched. The DNA of 148 miRNA genes was found to have methylation change in liver fibrosis tissue. These discoveries in miRNA genes are beneficial to future miRNA function research in liver fibrosis.

Mesenchymal stem cells reverse thymus aging by reprogramming the DNA methylation of thymic epithelial cells

BackgroundA decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration. Mesenchymal stem cells (MSCs) treating thymic ageing is a promising strategy, but the DNA methylation modification mechanism in TECs remains unclear. MethodsAged rhesus monkeys were treated with MSCs to establish a thymic senescence model, and hematoxylin-eosin (HE) staining, immunofluorescence staining, and ELISA were performed to observe the structure and function of the thymus. TEC aging model and MSCs co-culture system were established to detect DNA methylation modification and transcriptomic changes, correlation analysis between transcription factor methylation and mRNA expression, and q-PCR, immunofluorescence staining, and Western blot were used to identified key genes. ResultsMSCs improved the structure and function of thymus in elderly macaque monkeys; reduced the expression levels of β-Gal, P16, and P21; and increased the activity of aging TECs. There were 501 genes with increased methylation in the promoter region in the treated group compared with the untreated group, among which 23 genes were involved in the negative regulation of cell growth, proliferation and apoptosis, while 591 genes had decreased methylation, among which 37 genes were associated with promoting cell growth and proliferation and inhibiting apoptosis. Furthermore, 66 genes showed a negative correlation between promoter methylation levels and gene transcription; specifically, PDE5A, DUOX2, LAMP1 and SVIL were downregulated with increased methylation, inhibiting growth and development, while POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 were upregulated with decreased methylation, promoting cell growth. ConclusionMSCs improve the structure and function of aged thymus, which involves the regulation of DNA methylation profiles and a decrease in the methylation level of the transcription factor NGF to specifically upregulate KRT17 and FOXJ1 to promote the proliferation of TECs.

Fluorimetric detection of DNA methylation by cerium oxide nanoparticles for early cancer diagnosis

In this study, a very sensitive fluorescence nano-biosensor was developed using CeO2 nanoparticles for the rapid detection of DNA methylation. The characteristics of CeO2 nanoparticles were determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) spectroscopy, UV–visible spectroscopy, and fluorescence spectroscopy. The CeO2 nanoparticles were reacted with a single-stranded DNA (ssDNA) probe, and then methylated and unmethylated target DNAs hybridized with an ssDNA probe, and the fluorescence emission was measured.Upon adding the target unmethylated and methylated ssDNA, the fluorescence intensity increased in the linear range of concentration from 2 × 10−13 - 10−18 M. The limit of detection (LOD) was 1.597 × 10−6 M for methylated DNA and 1.043 × 10−6 M for unmethylated DNA. The fluorescence emission intensity of methylated sequences was higher than of that unmethylated sequences. The fabricated DNA nanobiosensor showed a fluorescence emission at 420 nm with an excitation wavelength of 280 nm. The impact of CeO2 binding on methylated and unmethylated DNA was further demonstrated by agarose gel electrophoresis.Finally, the actual sample analysis suggested that the nanobiosensor could have practical applications for detecting methylation in the human plasma samples.

Abstract 2417: Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic

Abstract Omega Therapeutics has developed a novel platform of programmable epigenomic mRNA medicines capable of modifying chromatin state to specifically tune gene expression at the pre-transcriptional level. Epigenomic controllers (ECs) unlock targets that have been historically considered “undruggable,” with one of the most elusive being the MYC oncogene. A direct MYC-targeting anti-cancer agent has previously remained intangible, largely due to the absence of a drug binding pocket and tight autoregulation. A clinical trial is underway (MYCHELANGELO, NCT05497453) to investigate pre-transcriptional inhibition of MYC with OTX-2002 in patients with hepatocellular carcinoma (HCC). OTX-2002, a first-in-class mRNA therapeutic delivered via lipid nanoparticles (LNP), encodes two proteins that durably modify chromatin, in part, through CpG DNA methylation at the MYC locus. Using both liquid and solid biopsy sampling from in vivo studies, we investigated whether target engagement for OTX-2002 could be assessed by MYC methylation. Detection of DNA methylation has long held promise as an oncology biomarker given its functional roles in various cancer types and the potential signal afforded by methylated CpGs. Indeed, DNA methylation has been shown to serve as a robust analyte in liquid biopsy-derived multi-cancer early detection (MCED) and minimal residual disease (MRD) tests that have recently been utilized in the clinic. Many of these platforms are founded on complex models that leverage methylation signals across &amp;gt;1 million CpGs that can span tens of megabases of genomic space. We faced a distinct challenge when compared to these MCED/MRD tests in developing a pharmacodynamic methylation assay for OTX-2002 as the target region consists of just a few kilobases. With a tissue specific LNP delivery system, ultra-high sensitivity was required to identify rare ctDNA events from the larger cfDNA population. To this end, we designed a minimal hybridization/capture panel that targeted ~50 kb of genomic space, effectively allowing for ultra-deep methylation sequencing of MYC. When this technique was paired with enzymatic (EM) conversion for methylation detection and supported by an analysis pipeline focused on epiallele identification, this assay was able to detect methylation down to the theoretical limit in a dilution series of control genomic DNA, 1 in 104 copies of MYC. This degree of sensitivity translated to successful preclinical detection of on-target methylation by OTX-2002 from DNA extracted from plasma samples collected from mice-bearing human HCC xenografts. Overall, we present a non-invasive and exquisitely sensitive method of assessing target engagement and site-specific pharmacodynamic activity of a novel epigenomic medicine that can be directly translated to the clinical setting. Citation Format: Justin Chen, William Senapedis, Stephen Siecinski, Elmer Figueroa, Adam Katz, Samuel Mildrum, Yaoyu E. Wang, Houda Belaghzal, Kayleigh Gallagher, Graeme Hodgson, Charles W. O'Donnell, Thomas G. McCauley. Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2417.

Abstract 2291: Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation

Abstract Background: Current genomic and epigenomic profiling of cancer tissue DNA or cfDNA (cell-free DNA) in liquid biopsy relies upon separate, time- and sample-consuming technologies for somatic variant detection or methylation analysis. Here we describe workflow and performance of the Agilent Bravo automated liquid handling platform with the Agilent Avida targeted enrichment solution for next generation sequencing of somatic variants and methylation profiling. This solution can effectively analyze low-input tumor DNA or cfDNA samples. The Avida Duo workflow enables highly sensitive detection of single nucleotide variant (SNV), insertions and deletions (INDEL), copy number variation (CNV) and DNA methylation profiles from a single sample, without any sample splitting. Methods and Results: Panels, reagents, and automated workflows for Avida DNA, Avida Methyl, and Avida Duo Methyl (combined DNA &amp; methylation) kits were developed to accommodate up to 96 samples on the Bravo NGS workstation. The automated solution supports independent single-day workflows for somatic variants or methylation sample preparation. The Avida Duo analysis combines both workflows without sample splitting, streamlining the process and reducing sample consumption. Leveraging a focused cancer hotspot panel, we demonstrate excellent reproducibility and low allele frequency (SNV at sub 1%) variant detection in cfDNA samples and reference standards with as little as 10ng DNA input. We exhibit similar performance with a larger ~300 kb target region tumor profiling panel. Finally, we demonstrate DNA methylation detection in the standalone or combined (“Duo”) workflows across differentially methylated regions (DMRs) with a panel of ~3400 targets. Conclusions: Automation of the Avida targeted enrichment solution with the Bravo NGS workstation enables a sensitive, high-throughput, end-to-end analysis for detection of genomic alterations and DNA methylation changes from a single low-input cfDNA or tumor DNA sample. Citation Format: Ashraf Wahba, Tony Ho, Sarah Johns, Aswati Aravind, Heng Wang, Neelima V. Mehendale, Gilbert P. Amparo, Khine Win, Manuel Gomez, Grace Zhao, Douglas N. Roberts. Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2291.

Exploring the ageing methylome in the model insect, Nasonia vitripennis

BackgroundThe ageing process is a multifaceted phenomenon marked by the gradual deterioration of cellular and organismal functions, accompanied by an elevated susceptibility to diseases. The intricate interplay between genetic and environmental factors complicates research, particularly in complex mammalian models. In this context, simple invertebrate organisms have been pivotal, but the current models lack detectable DNA methylation limiting the exploration of this critical epigenetic ageing mechanism.This study introduces Nasonia vitripennis, the jewel wasp, as an innovative invertebrate model for investigating the epigenetics of ageing. Leveraging its advantages as a model organism and possessing a functional DNA methylation system, Nasonia emerges as a valuable addition to ageing research.ResultsWhole-genome bisulfite sequencing unveiled dynamic alterations in DNA methylation, with differentially methylated CpGs between distinct time points in both male and female wasps. These changes were associated with numerous genes, enriching for functions related to telomere maintenance, histone methylation, and mRNA catabolic processes. Additionally, other CpGs were found to be variably methylated at each timepoint. Sex-specific effects on epigenetic entropy were observed, indicating differential patterns in the loss of epigenetic stability over time. Constructing an epigenetic clock containing 19 CpGs revealed a robust correlation between epigenetic age and chronological age.ConclusionsNasonia vitripennis emerges as a promising model for investigating the epigenetics of ageing, shedding light on the intricate dynamics of DNA methylation and their implications for age-related processes. This research not only expands the repertoire of ageing models but also opens avenues for deeper exploration of epigenetic mechanisms in the context of ageing.

Abstract 3493: Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay

Abstract The abnormal changes in DNA methylation are linked to the early stages of carcinogenesis. Identifying these epigenetic changes in circulating tumor DNA (ctDNA) can reveal potential biomarkers for the early diagnosis of various cancers. However, analyzing such data poses bioinformatics challenges due to the lack of sensitivity in detecting the low abundance ctDNA signals in biopsy samples, which are often overwhelmed by the complexity of libraries containing hundreds of targeted regions. Read-level methylation analysis holds the promise of more in-depth DNA methylation detection due to the wide coverage and high sensitivity of rare signals. However, this approach is hindered by the absence of a standardized workflow capable of generating interpretable reports suitable for both bench scientists and professional bioinformaticians. Here, we present a bioinformatics workflow that examines next-generation sequencing (NGS) data and characterizes the read-level methylation patterns of amplicons. Compared to other currently available tools, our method is designed to work with high-multiplex, large-scale targeted assays. It effectively eliminates the undesired noise derived from sequencing byproducts such as false CpG calls, dimers, and off-target alignments. Additionally, to accommodate the substantial volume of data generated by state-of-the-art NGS platforms, the workflow enables parallel processing of samples compatible with both cloud-based and on-premises computing resources. This workflow provides a comprehensive per-sample visualization of DNA methylation patterns and reports read-level methylation results in a “pattern-as-a-feature” table. In this table, the occurrence of an amplicon epiallelic haplotype (pattern) for every sample is represented as a “feature column” and is aggregated with all patterns discovered in the experiment. These read-level patterns, along with other information, can be used to develop machine learning algorithms to reiteratively harvest true predictive features and penalize confounding signals in predicting cancer diagnosis. Citation Format: Mingda Jin, Masatomo Kaneko, Steven Cen, Hongtao Li, Wei Guo, Xinyi Zhou, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, Andre L. Abreu, Jeffrey Bhasin, Xiaojing Yang, Xi-Yu Jia. Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3493.

Periodic magnetic modulation enhanced electrochemical analysis for highly sensitive determination of genomic DNA methylation

DNA methylation aberrations have a strong correlation with cancer in early detection, diagnosis, and prognosis, which make them possible candidate biomarkers. Electrochemical biosensors offer rapid protocols for detecting DNA methylation status with minimal pretreatment of samples. However, the inevitable presence of background current in the time domain, including electrochemical noise and variations, limits the detection performance of these biosensors, especially for low concentration analytes. Here, we propose an ultrasensitive frequency-domain electrochemical analysis strategy to effectively separate the weak signals from background current. To achieve this, we employed periodic magnetic field modulation of magnetic beads (MBs) on and off the electrode surface to generate a periodic electrochemical signal for subsequent frequency-domain analysis. By capturing labeled MBs with as low as 0.5 pg of DNA, we successfully demonstrated a highly sensitive electrochemical method for determination of genome-wide DNA methylation levels. We also validated the effectiveness of this methodology using DNA samples extracted from three types of hepatocellular carcinoma (HCC) cell lines. The results revealed varying genomic methylation levels among different HCC cell lines, indicating the potential application of this approach for early-stage cancer detection in terms of DNA methylation status.

Circulating Tumor DNA (ctDNA) and Its Role in Gynecologic Malignancies.

Circulating tumor DNA (ctDNA) refers to small fragments of DNA released into the bloodstream by cancer cells. It is obtained through "liquid biopsy;" which most commonly refers to plasma or blood samples, but can be obtained from a number of bodily fluids including ascitic fluid, saliva, and even urine and stool. ctDNA is detected via polymerase chain reaction (PCR) or next-generation sequencing (NGS). The DNA from these samples is analyzed for the detection of point mutations, copy-number alterations, gene fusion, and DNA methylation. These results have the potential for use in cancer diagnosis, determining prognosis, targeting gene-specific therapies, and monitoring for/predicting disease recurrence and response to treatment. ctDNA offers an alternative to tissue biopsy; it is less invasive and can be monitored serially over time without multiple procedures. Moreover it may have the ability to detect disease recurrence or predict behavior in a way that solid tissue biopsies, tumor marker surveillance, and imaging cannot. Recent explosion in interest in ctDNA shows promising developments for widespread adoption of these techniques in cancer care. However, the use of ctDNA in diagnosis and treatment of gynecologic malignancies is currently limited, compared to adoption in other solid-organ tumors such as breast and colorectal cancers. Compared to other cancer types, there appear to be fewer comprehensive studies and clinical validations specifically focusing on the use of ctDNA in gynecologic cancers. More research is needed in this area to advance the potential for use of ctDNA in ovarian, endometrial, and cervical cancers before this can be routinely adopted to improve care for patients with gynecologic malignancies.

A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes

BackgroundLong-read sequencing can enable the detection of base modifications, such as CpG methylation, in single molecules of DNA. The most commonly used methods for long-read sequencing are nanopore developed by Oxford Nanopore Technologies (ONT) and single molecule real-time (SMRT) sequencing developed by Pacific Bioscience (PacBio). In this study, we systematically compare the performance of CpG methylation detection from long-read sequencing.ResultsWe demonstrate that CpG methylation detection from 7179 nanopore-sequenced DNA samples is highly accurate and consistent with 132 oxidative bisulfite-sequenced (oxBS) samples, isolated from the same blood draws. We introduce quality filters for CpGs that further enhance the accuracy of CpG methylation detection from nanopore-sequenced DNA, while removing at most 30% of CpGs. We evaluate the per-site performance of CpG methylation detection across different genomic features and CpG methylation rates and demonstrate how the latest R10.4 flowcell chemistry and base-calling algorithms improve methylation detection from nanopore sequencing. Additionally, we show how the methylation detection of 50 SMRT-sequenced genomes compares to nanopore sequencing and oxBS.ConclusionsThis study provides the first systematic comparison of CpG methylation detection tools for long-read sequencing methods. We compare two commonly used computational methods for the detection of CpG methylation in a large number of nanopore genomes, including samples sequenced using the latest R10.4 nanopore flowcell chemistry and 50 SMRT sequenced samples. We provide insights into the strengths and limitations of each sequencing method as well as recommendations for standardization and evaluation of tools designed for genome-scale modified base detection using long-read sequencing.

Photoelectrochemical sensor based on AuNPs@WO3@TpPa-1-COF for quantification of DNA methylation levels.

A"signal-off" photoelectrochemical (PEC) sensing platform has been designedfor the ultrasensitive detection of DNA methylation levels and multiple methylated sites. The platform employs tungsten trioxide and TpPa-1-COF loaded by gold nanoparticle (AuNPs@WO3@TpPa-1-COF) composite material as the photoactive component and p-type reduced graphene (rGO) as an efficient quencher. The PEC signal of AuNPs@WO3@TpPa-1-COF composite is effectively quenched in the presence of p-type rGO, because p-type rGO can compete with AuNPs@WO3@TpPa-1-COF to deplete light energy and electron donors. In addition, a hybrid strand reaction (HCR) amplification strategy fixes more target DNA and then combines with rGO-modified anti-5-methylcytosine antibody to facilitate ultrasensitive DNA methylation detection. Under optimal conditions, DNA methylation can be measured within a linear concentration range of 10-14 to 10-8 M, with an exceptionally low detection limit of 0.19 fM (S/N = 3). At the same time, the platform can conduct quantitative determination of multi-site methylation, with the linear equation △I = 44.19LogA + 61.43, and the maximum number of methylation sites is 5. The sensor demonstrates high sensitivity, excellent selectivity, and satisfactory stability. Furthermore, theproposed signal-off PEC strategy was successfully employed to detect DNA methylation in spiked human serum samples, with recoveries ranging from 93.17 to 107.28% and relative standard deviation (RSD) ranging from 1.15 to 5.49%.

Influential factors on urine EV DNA methylation detection and its diagnostic potential in prostate cancer.

The value of Extracellular vesicles (EVs) diagnostic markers is widely recognized. However, current research on EV DNA remains limited. This study investigates the biological properties, preprocessing factors, and diagnostic potential of EV DNA. We found that DNA positive vesicles account for 23.3% ± 6.7% of the urine total EV, with a large amount of DNA attached to the outside. EV DNA fragments are large, there is no significant effect on uEV DNA when store urine less than 6h at 4°C. In addition, the influence of different EV extraction methods on methylation detection is also minor. More importantly, RASSF1A methylation in urine total EV DNA can distinguish between PCa and BPH, with an AUC of 0.874. Our results suggest the potential of urine EV DNA as a novel marker for PCa diagnosis. This provides a new idea for the study of urinary tumor markers.

A signal processing and deep learning framework for methylation detection using Oxford Nanopore sequencing

Oxford Nanopore sequencing can detect DNA methylations from ionic current signal of single molecules, offering a unique advantage over conventional methods. Additionally, adaptive sampling, a software-controlled enrichment method for targeted sequencing, allows reduced representation methylation sequencing that can be applied to CpG islands or imprinted regions. Here we present DeepMod2, a comprehensive deep-learning framework for methylation detection using ionic current signal from Nanopore sequencing. DeepMod2 implements both a bidirectional long short-term memory (BiLSTM) model and a Transformer model and can analyze POD5 and FAST5 signal files generated on R9 and R10 flowcells. Additionally, DeepMod2 can run efficiently on central processing unit (CPU) through model pruning and can infer epihaplotypes or haplotype-specific methylation calls from phased reads. We use multiple publicly available and newly generated datasets to evaluate the performance of DeepMod2 under varying scenarios. DeepMod2 has comparable performance to Guppy and Dorado, which are the current state-of-the-art methods from Oxford Nanopore Technologies that remain closed-source. Moreover, we show a high correlation (r = 0.96) between reduced representation and whole-genome Nanopore sequencing. In summary, DeepMod2 is an open-source tool that enables fast and accurate DNA methylation detection from whole-genome or adaptive sequencing data on a diverse range of flowcell types.

Mini-review of DNA Methylation Detection Techniques and Their Potential Applications in Disease Diagnosis, Prognosis, and Treatment.

DNA methylation is the dominant epigenetic mechanism for regulating gene expression in mammals, playing crucial roles in development, differentiation, and tissue homeostasis. Aberrations in DNA methylation are closely associated with the potential onset of various diseases. Consequently, numerous DNA methylation detection techniques have been successively developed. These methods not only facilitate the exploration of disease mechanisms but also hold significant promise for the development of diagnostic and prognostic strategies. In this Perspective, we present a comprehensive overview of commonly employed DNA methylation detection techniques as well as biosensing based on their underlying analytical techniques. For its medical applications, we begin by examining the pathogenesis of different diseases and then proceed to discuss how relevant technologies are applied in the context of these specific medical conditions. Additionally, we briefly discuss the current limitations of these techniques and highlight future challenges in advancing methylation detection and analysis methodologies.

Evaluation of DNA methylation levels of SEPT9 and SHOX2 in plasma of patients with head and neck squamous cell carcinoma using droplet digital PCR.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most commonly diagnosed cancer globally. HNSCC develops from the mucosa of the oral cavity, pharynx and larynx. Methylation levels of septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) genes in circulating cell‑free DNA (ccfDNA) are considered epigenetic biomarkers and have shown predictive value in preliminary reports in HNSCC. Liquid biopsy is a non‑invasive procedure that collects tumor‑derived molecules, including ccfDNA. In the present study, a droplet digital PCR (ddPCR)‑based assay was developed to detect DNA methylation levels of circulating SEPT9 and SHOX2 in the plasma of patients with HNSCC. The assay was first set up using commercial methylated and unmethylated DNA. The dynamic changes in the methylation levels of SEPT9 and SHOX2 were then quantified in 20patients with HNSCC during follow‑up. The results highlighted: i)The ability of the ddPCR‑based assay to detect very low copies of methylated molecules; ii)the significant decrease in SEPT9 and SHOX2 methylation levels in the plasma of patients with HNSCC at the first time points of follow‑up with respect to T0; iii)a different trend of longitudinally DNA methylation variations in small groups of stratified patients. The absolute and precise quantification of SEPT9 and SHOX2 methylation levels in HNSCC may be useful for studies with translational potential.

Integrated DNA methylation analysis reveals a potential role for PTPRN2 in Marfan syndrome scoliosis.

Marfan syndrome (MFS) is a rare genetic disorder caused by mutations in the Fibrillin-1 gene (FBN1) with significant clinical features in the skeletal, cardiopulmonary, and ocular systems. To gain deeper insights into the contribution of epigenetics in the variability of phenotypes observed in MFS, we undertook the first analysis of integrating DNA methylation and gene expression profiles in whole blood from MFS and healthy controls (HCs). The Illumina 850K (EPIC) DNA methylation array was used to detect DNA methylation changes on peripheral blood samples of seven patients with MFS and five HCs. Associations between methylation levels and clinical features of MFS were analyzed. Subsequently, we conducted an integrated analysis of the outcomes of the transcriptome data to analyze the correlation between differentially methylated positions (DMPs) and differentially expressed genes (DEGs) and explore the potential role of methylation-regulated DEGs (MeDEGs) in MFS scoliosis. The weighted gene co-expression network analysis was used to find gene modules with the highest correlation coefficient with target MeDEGs to annotate their functions in MFS. Our study identified 1253 DMPs annotated to 236 genes that were primarily associated with scoliosis, cardiomyopathy, and vital capacity. These conditions are typically associated with reduced lifespan in untreated MFS. We calculated correlations between DMPs and clinical features, such as cobb angle to evaluate scoliosis and FEV1% to assess pulmonary function. Notably, cg20223687 (PTPRN2) exhibited a positive correlation with cobb angle of scoliosis, potentially playing a role in ERKs inactivation. Taken together, our systems-level approach sheds light on the contribution of epigenetics to MFS and offers a plausible explanation for the complex phenotypes that are linked to reduced lifespan in untreated MFS patients.

A ratiometric fluorescence assay for the detection of DNA methylation based on an alkaline phosphatase triggered in situ fluorogenic reaction.

The accurate and sensitive quantification of DNA methylation is significant for the early diagnosis of cancer. In this work, an alkaline phosphatase (ALP) triggered in situ fluorogenic reaction between ascorbic acid (AA) and 2,3-DAN was employed as a ratiometric fluorescent probe for the accurate and sensitive detection of DNA methylation with the assistance of ALP encapsulated liposomes. The quinoxaline derivative with a yellow fluorescence emission (I525) was generated from the reaction between AA and 2,3-DAN. Meanwhile, the consumption of 2,3-DAN declined its fluorescence intensity (I386). A ratiometric fluorescent probe (I525/I386) constructed by the above in situ fluorogenic reaction was applied for the accurate detection of DNA methylation. The methylated DNA was first captured by its complementary DNA in 96-well plates. Then, 5mC antibody (Ab) linked liposomes that were encapsulated with ALP recognized and combined with the methylation sites of the target DNA. After the liposomes were lysed by Triton X-100, the released ALP triggered the hydrolysis of ascorbic acid diphosphate (AAP) to form AA, participating in the fluorogenic reaction with 2,3-DAN to produce a quinoxaline derivative. Thus, the ratiometric fluorescence detection of DNA methylation was achieved using I525/I386 values. Using the ALP-enzyme catalyzed reaction and liposomes as signal amplifiers, a low detection limit of 82 fM was obtained for DNA methylation detection. Moreover, the accuracy of the assay could be improved using ratiometric fluorescent probes. We hope that the proposed assay will pave a new way for the accurate determination of low-abundance biomarkers.