Detection Of Methylation Research Articles

Utility of SHOX2 and RASSF1A gene methylation detection on the residual cytology material from endobronchial ultrasound-guided transbronchial needle aspiration.

This study aims to assess the effectiveness of Short Stature Homeobox 2 (SHOX2) and RAS Association Domain Family 1 Isoform A (RASSF1A) gene methylation detection in residual liquid-based cytology (LBC) materials from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) and investigate the diagnostic accuracy of a comprehensive diagnostic approach. Between June 2022 and May 2023, a total of 110 cases that underwent EBUS-TBNA were enrolled in the study. SHOX2 and RASSF1A genes methylation detection using the residual cytological material, LBC, and cell block (CB) were conducted for each EBUS-TBNA case. The sensitivity and specificity of cytology, CB histopathology, SHOX2, and RASSF1A methylation in diagnosing EBUS-TBNA samples were determined based on follow-up data. Among the 72 cases confirmed as pulmonary carcinomas, the methylation test yielded positive results in 24 adenocarcinoma cases, 10 squamous cell carcinoma cases, and 14 small cell carcinoma cases. The sensitivity of the comprehensive diagnosis (combining LBC, CB, and methylation detection) in distinguishing metastatic pulmonary epithelial malignancies in mediastinal and hilar lymph nodes or masses from benign lesions was higher (97.22%, 70/72) than that of morphological diagnosis alone (LBC and CB) (88.89%, 64/72; P < 0.05). SHOX2 and RASSF1A methylation detection demonstrates a high sensitivity and negative predictive value in the identification of pulmonary epithelial malignancies and holds promise as a valuable ancillary approach to enhance morphological diagnosis of EBUS-TBNA.

Ultrasensitive Detection of Tumor Suppressor Gene Methylation by Piezoelectric Sensing Based on Enrichment of Transcription Activator-Like Effectors.

The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.

Recent Advances in Studies of Genomic DNA Methylation and Its Involvement in Regulating Drought Stress Response in Crops.

As global arid conditions worsen and groundwater resources diminish, drought stress has emerged as a critical impediment to plant growth and development globally, notably causing declines in crop yields and even the extinction of certain cultivated species. Numerous studies on drought resistance have demonstrated that DNA methylation dynamically interacts with plant responses to drought stress by modulating gene expression and developmental processes. However, the precise mechanisms underlying these interactions remain elusive. This article consolidates the latest research on the role of DNA methylation in plant responses to drought stress across various species, focusing on methods of methylation detection, mechanisms of methylation pattern alteration (including DNA de novo methylation, DNA maintenance methylation, and DNA demethylation), and overall responses to drought conditions. While many studies have observed significant shifts in genome-wide or gene promoter methylation levels in drought-stressed plants, the identification of specific genes and pathways involved remains limited. This review aims to furnish a reference for detailed research into plant responses to drought stress through epigenetic approaches, striving to identify drought resistance genes regulated by DNA methylation, specific signaling pathways, and their molecular mechanisms of action.

Direct transposition of native DNA for sensitive multimodal single-molecule sequencing

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

DNA methylation markers in esophageal cancer.

Esophageal cancer (EC) is a prevalent malignancy characterized by a low 5-year survival rate, primarily attributed to delayed diagnosis and limited therapeutic options. Currently, early detection of EC heavily relies on endoscopy and pathological examination, which pose challenges due to their invasiveness and high costs, leading to low patient compliance. The detection of DNA methylation offers a non-endoscopic, cost-effective, and secure approach that holds promising prospects for early EC detection. To identify improved methylation markers for early EC detection, we conducted a comprehensive review of relevant literature, summarized the performance of DNA methylation markers based on different input samples and analytical methods in EC early detection and screening. This review reveals that blood cell free DNA methylation-based method is an effective non-invasive method for early detection of EC, although there is still a need to improve its sensitivity and specificity. Another highly sensitive and specific non-endoscopic approach for early detection of EC is the esophageal exfoliated cells based-DNA methylation analysis. However, while there are substantial studies in esophageal adenocarcinoma, further more validation is required in esophageal squamous cell carcinoma. In conclusion, DNA methylation detection holds significant potential as an early detection and screening technology for EC.

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.

Analysis of the diagnostic performance of PAX1/SOX1 gene methylation in cervical precancerous lesions and its role in triage diagnosis.

Methylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex-determining region Y-box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under-researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV-DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV-DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid-based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43-0.62), 0.88 (95% CI: 0.80-0.97), and 0.88 (95% CI: 0.75-1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40-0.58), 0.80 (95% CI: 0.68-0.93), and 0.92 (95% CI: 0.811-1.00), respectively. In HPV16/18-negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.

The endometrial cancer detection using non-invasive hypermethylation of CDO1 and CELF4 genes in women with postmenopausal bleeding in Northwest China.

The objective of this study was to verify the clinical predictive performance of methylated cysteine dioxygenase type 1 (CDO1m) and CUGBP Elav-like family member 4 (CELF4m) in endometrial cancer (EC) women with postmenopausal bleeding (PMB). A single-center, prospective, and case-control study was conducted in the Gansu Provincial Maternity and Child-care Hospital with 138 female postmenopausal patients enrolled in 2022. All patients underwent body mass index (BMI) detection, transvaginal ultrasonography (TVUS) detection, carbohydrate antigen 125 detection, and the cervical exfoliated cell CDO1/CELF4 gene methylation detection to analyze the sensitivity, specificity, and accuracy of different screening tests statistically with the biopsy and/or dilation and curettage (D&C) pathological diagnosis under hysteroscopy as the gold standard. There was no significant difference in age between the EC group and the non-EC group, P = 0.492. Using quantitative polymerase chain reaction (qPCR) technology, we validated the CDO1 and CELF4 methylation detection with 87.5% sensitivity and 95.9% specificity as a useful strategy for the triage of women with PMB for the detection of EC. In addition, 100% of type II EC (n = 6) were positively detected by the CDO1 or CELF4 methylation test. The CDO1 and CELF4 methylation test with high specificity as an auxiliary diagnostic tool or alternative method provides physicians with a reference to distinguish between benign and malignant tumors in women with postmenopausal bleeding, to justify the necessity of using invasive methods to confirm diagnosis.

Profiling age and body fluid DNA methylation markers using nanopore adaptive sampling

DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research.

Profiling genome-wide methylation in two maples: Fine-scale approaches to detection with nanopore technology.

DNA methylation is critical to the regulation of transposable elements and gene expression and can play an important role in the adaptation of stress response mechanisms in plants. Traditional methods of methylation quantification rely on bisulfite conversion that can compromise accuracy. Recent advances in long-read sequencing technologies allow for methylation detection in real time. The associated algorithms that interpret these modifications have evolved from strictly statistical approaches to Hidden Markov Models and, recently, deep learning approaches. Much of the existing software focuses on methylation in the CG context, but methylation in other contexts is important to quantify, as it is extensively leveraged in plants. Here, we present methylation profiles for two maple species across the full range of 5mC sequence contexts using Oxford Nanopore Technologies (ONT) long-reads. Hybrid and reference-guided assemblies were generated for two new Acer accessions: Acer negundo (box elder; 65x ONT and 111X Illumina) and Acer saccharum (sugar maple; 93x ONT and 148X Illumina). The ONT reads generated for these assemblies were re-basecalled, and methylation detection was conducted in a custom pipeline with the published Acer references (PacBio assemblies) and hybrid assemblies reported herein to generate four epigenomes. Examination of the transposable element landscape revealed the dominance of LTR Copia elements and patterns of methylation associated with different classes of TEs. Methylation distributions were examined at high resolution across gene and repeat density and described within the broader angiosperm context, and more narrowly in the context of gene family dynamics and candidate nutrient stress genes.

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.

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.

Abstract 7025: Advancing methylation analysis: TAPS v1.0: A fast, scalable, and non-destructive sequencing method

Abstract In the pursuit of advancing multi-cancer early detection, the exploration of cell-free DNA (cfDNA) through methylation profiling has emerged as a promising avenue. However, the realization of its full potential is impeded by limitations inherent in conventional methodologies, notably the 'gold standard' bisulfite conversion methods. These approaches pose challenges such as DNA destruction and the generation of low-complexity sequences resulting from the conversion of unmethylated cytosines to thymine. TET Assisted Pyridine-Borane Sequencing (TAPS) holds the potential as a non-destructive, rapid, and scalable alternative for base-level methylation analysis. In this context, we introduce TET Assisted Pyridine-Borane Sequencing version 1.0 (TAPS v1.0), showcasing advancements to the technology originally introduced by Schuster-Böckler, Song, and their collaborators. TAPS v1.0 transforms 5mC into dihydrouracil (DHU) through a series of oxidation and reduction reactions. Ultimately, DHU is recognized as dU during PCR resulting in C-to-T conversions at 5mC sites, leaving unmethylated cytosines, the majority of cytosines, unchanged. The nondestructive nature of 5mC conversion yields significantly higher library yield and complexity compared to BS-seq. By leveraging Watchmaker's proprietary polymerase evolution platform, a DNA polymerase tolerant to DHU was selected for library amplification post DHU conversion, enabling unimpeded detection of 5mC, particularly in densely packed CpG repeats. Coupled with an optimized End Repair and A-tailing chemistry preceding TAPS v1.0, clinically relevant cfDNA samples of low quality and/or quantity can be analyzed in methylation detection pipelines. To evaluate the performance of TAPS v1.0, human genomic DNA with control spike-ins featuring varying methylation statuses were employed. TAPS v1.0 exhibited significantly higher final library yield and greater complexity compared to BS-seq. Similar outcomes were observed in clinically relevant cfDNA samples. Citation Format: Josh Haimes, Craig Marshall, Travis Sanders, Sam Vogel, Martin Ranik, Eduard Casas, Doug Wendel, Leo Karamanof, Bjarne Faurholme, Abre de Beer, Lee French, Ross Wadsworth, Kristina Giordia, Eric van der Walt, Brian Kudlow. Advancing methylation analysis: TAPS v1.0: A fast, scalable, and non-destructive sequencing method [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 7025.

DNA methylation of IFI44L as a potential blood biomarker for childhood-onset systemic lupus erythematosus

BackgroundIFN-induced protein 44-like (IFI44L) promoter methylation has been demonstrated to serve as an effective blood diagnostic biomarker for adult-onset SLE. However, its utility as a diagnostic marker for childhood-onset SLE (cSLE) remains to be verified.MethodsInitially, we conducted a differential analysis of gene methylation and mRNA expression patterns in cSLE whole blood samples obtained from the public GEO database to determine IFI44L gene expression and assess the methylation status at its CpG sites. Subsequently, we collected clinical whole blood samples from 49 cSLE patients and 12 healthy children, employing an HRM-qPCR-based IFI44L methylation detection technique to evaluate its diagnostic efficacy in pediatric clinical practice.ResultsA total of 26 hypomethylated, highly expressed genes in cSLE were identified by intersecting differentially expressed genes (DEGs) and differentially methylation genes (DMGs). GO enrichment analysis for these 26 genes indicated a robust association with type I IFN. Among the overlapping genes, IFI44L exhibited the most pronounced differential expression and methylation. In subsequent clinical validation experiments, IFI44L methylation was confirmed as an effective blood-based diagnostic biomarker for cSLE, achieving an AUC of 0.867, a sensitivity of 0.753, and a specificity of 1.000.ConclusionsIFI44L methylation is a promising blood biomarker for cSLE.ImpactIFI44L promoter methylation was reported to serve as a highly sensitive and specific diagnostic marker for adult-onset SLE. However, the diagnostic efficacy of IFI44L in childhood-onset SLE (cSLE) still remains to be confirmed. In this study, we utilized bioinformatics analysis and conducted clinical experiments to demonstrate that IFI44L methylation can also serve as a promising blood biomarker for cSLE. The findings of this study can facilitate the diagnosis of cSLE and broaden our understanding of its molecular mechanisms, with a particular focus on those related to type I interferons.

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.

Circulating tumor DNA methylation: a promising clinical tool for cancer diagnosis and management.

Cancer continues to pose significant challenges to the medical community. Early detection, accurate molecular profiling, and adequate assessment of treatment response are critical factors in improving the quality of life and survival of cancer patients. Accumulating evidence shows that circulating tumor DNA (ctDNA) shed by tumors into the peripheral blood preserves the genetic and epigenetic information of primary tumors. Notably, DNA methylation, an essential and stable epigenetic modification, exhibits both cancer- and tissue-specific patterns. As a result, ctDNA methylation has emerged as a promising molecular marker for noninvasive testing in cancer clinics. In this review, we summarize the existing techniques for ctDNA methylation detection, describe the current research status of ctDNA methylation, and present the potential applications of ctDNA-based assays in the clinic. The insights presented in this article could serve as a roadmap for future research and clinical applications of ctDNA methylation.

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%.