scholarly journals EpiHRMAssay, in tube and in silico combined approach for the scanning and epityping of heterogeneous DNA methylation

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Marco Cirilli ◽  
Ines Delfino ◽  
Emilia Caboni ◽  
Rosario Muleo
Keyword(s):  
Dna Methylation ◽  
In Silico ◽  
Large Scale ◽  
Methylation Status ◽  
Cost Effective ◽  
Melting Temperatures ◽  
Calibration Models ◽  
Dna Methylation Profile ◽  
Melting Analysis ◽  
Diversity Studies

Reliable and cost-effective assays with adequate sensitivity are required to detect the DNA methylation profile in plants for scientific and industrial purposes. The proposed novel assay, named EpiHRMAssay, allows to quantify the overall methylation status at target loci and to enable high-throughput analyses. It combines in tube High Resolution Melting Analysis on bisulphite-treated templates with the in silico prediction of the melting profile of virtual epialleles using uMELTSM software. The predicted melting temperatures (Tm-s) of a set of epialleles characterized by different numbers of methylated cytosines (#mC) or different mC configurations were obtained and used to build calibration models, enabling the quantification of methylation in unknown samples using only the in tube observed melting temperature (Tm-o). EpiHRMAssay was validated by analysing the promoter region of CMT3, DDM1, and ROS1 genes involved in the regulation of methylation/demethylation processes and chromatin remodelling within a population of peach plants. Results demonstrate that EpiHRMAssay is a sensitive and reliable tool for locus-specific large-scale research and diagnostic contexts of the regulative regions of genes, in a broad range of organisms, including mammals. EpiHRMAssay also provides complementary information for the assessment of heterogeneous methylation and can address an array of biological questions on epigenetic regulation for diversity studies and for large-scale functional genomics.

Progressive alteration of DNA methylation of Alu, MGMT, MINT2, and TFPI2 genes in colonic mucosa during colorectal cancer development

2021 ◽  
pp. 1-6
Author(s):  
Ben Kang ◽  
Hyun Seok Lee ◽  
Seong Woo Jeon ◽  
Soo Yeun Park ◽  
Gyu Seog Choi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Healthy Subjects ◽  
Large Scale ◽  
Methylation Status ◽  
Clinical Information ◽  
Field Cancerization ◽  
Aberrant Methylation ◽  
Clinical Value ◽  
Mortality And Morbidity

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity in the world. It is characterized by different pathways of carcinogenesis and is a heterogeneous disease with diverse molecular landscapes that reflect histopathological and clinical information. Changes in the DNA methylation status of colon epithelial cells have been identified as critical components in CRC development and appear to be emerging biomarkers for the early detection and prognosis of CRC. OBJECTIVE: To explore the underlying disease mechanisms and identify more effective biomarkers of CRC. METHODS: We compared the levels and frequencies of DNA methylation in 11 genes (Alu, APC, DAPK, MGMT, MLH1, MINT1, MINT2, MINT3, p16, RGS6, and TFPI2) in colorectal cancer and its precursor adenomatous polyp with normal tissue of healthy subjects using pyrosequencing and then evaluated the clinical value of these genes. RESULTS: Aberrant methylation of Alu, MGMT, MINT2, and TFPI2 genes was progressively accumulated during the normal-adenoma-carcinoma progression. Additionally, CGI methylation occurred either as an adenoma-associated event for APC, MLH1, MINT1, MINT31, p16, and RGS6 or a tumor-associated event for DAPK. Moreover, relatively high levels and frequencies of DAPK, MGMT, and TFPI2 methylation were detected in the peritumoral nonmalignant mucosa of cancer patients in a field-cancerization manner, as compared to normal mucosa from healthy subjects. CONCLUSION: This study identified several biomarkers associated with the initiation and progression of CRC. As novel findings, they may have important clinical implications for CRC diagnostic and prognostic applications. Further large-scale studies are needed to confirm these findings.

scholarly journals Identification of prostate cancer specific methylation biomarkers from a multi-cancer analysis

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yiyi Pu ◽  
Chao Li ◽  
Haining Yuan ◽  
Xiaoju Wang
Keyword(s):  
Prostate Cancer ◽  
Dna Methylation ◽  
In Silico ◽  
Prostate Cancer Screening ◽  
Methylation Status ◽  
The Cancer Genome Atlas ◽  
Translation Rate ◽  
Normal Prostate ◽  
Cancer Data ◽  
Significant Difference

Abstract Background Detecting prostate cancer at a non-aggressive stage is the main goal of prostate cancer screening. DNA methylation has been widely used as biomarkers for cancer diagnosis and prognosis, however, with low clinical translation rate. By taking advantage of multi-cancer data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), we aimed to identify prostate cancer specific biomarkers which can separate between non-aggressive and aggressive prostate cancer based on DNA methylation patterns. Results We performed a comparison analysis of DNA methylation status between normal prostate tissues and prostate adenocarcinoma (PRAD) samples at different Gleason stages. The candidate biomarkers were selected by excluding the biomarkers existing in multiple cancers (pan-cancer) and requiring significant difference between PRAD and other urinary samples. By least absolute shrinkage and selection operator (LASSO) selection, 8 biomarkers (cg04633600, cg05219445, cg05796128, cg10834205, cg16736826, cg23523811, cg23881697, cg24755931) were identified and in-silico validated by model constructions. First, all 8 biomarkers could separate PRAD at different stages (Gleason 6 vs. Gleason 3 + 4: AUC = 0.63; Gleason 6 vs. Gleason 4 + 3 and 8–10: AUC = 0.87). Second, 5 biomarkers (cg04633600, cg05796128, cg23523811, cg23881697, cg24755931) effectively detected PRAD from normal prostate tissues (AUC ranged from 0.88 to 0.92). Last, 6 biomarkers (cg04633600, cg05219445, cg05796128, cg23523811, cg23881697, cg24755931) completely distinguished PRAD with other urinary samples (AUC = 1). Conclusions Our study identified and in-silico validated a panel of prostate cancer specific DNA methylation biomarkers with diagnosis value.

scholarly journals MINI REVIEW: Statistical methods for detecting differentially methylated loci and regions

2014 ◽  
Author(s):  
Mark D Robinson ◽  
Abdullah Kahraman ◽  
Charity W Law ◽  
Helen Lindsay ◽  
Malgorzata Nowicka ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Statistical Methods ◽  
Empirical Bayes ◽  
Large Scale ◽  
Rapid Development ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Data Types ◽  
Cpg Dinucleotides ◽  
Reversible Addition

DNA methylation, and specifically the reversible addition of methyl groups at CpG dinucleotides genome-wide, represents an important layer that is associated with the regulation of gene expression. In particular, aberrations in the methylation status have been noted across a diverse set of pathological states, including cancer. With the rapid development and uptake of large scale sequencing of short DNA fragments, there has been an explosion of data analytic methods for processing and discovering changes in DNA methylation across diverse data types. In this mini-review, we aim to condense many of the salient challenges, such as experimental design, statistical methods for differential methylation detection and critical considerations such as cell type composition and the potential confounding that can arise from batch effects, into a compact and accessible format. Our main interests, from a statistical perspective, include the practical use of empirical Bayes or hierarchical models, which have been shown to be immensely powerful and flexible in genomics and the procedures by which control of false discoveries are made. Of course, there are many critical platform-specific data preprocessing aspects that we do not discuss here. In addition, we do not make formal performance comparisons of the methods, but rather describe the commonly used statistical models and many of the pertinent issues; we make some recommendations for further study.

scholarly journals METHimpute: Imputation-guided construction of complete methylomes from WGBS data

2017 ◽  
Author(s):  
Aaron Taudt ◽  
David Roquis ◽  
Amaryllis Vidalis ◽  
René Wardenaar ◽  
Frank Johannes ◽  
...  
Keyword(s):  
Large Scale ◽  
Bisulfite Sequencing ◽  
Hidden Markov ◽  
Methylation Status ◽  
Population Level ◽  
Cost Effective ◽  
High Accuracy ◽  
Whole Genome ◽  
Effective Solution ◽  
Genome Bisulfite Sequencing

AbstractWhole-genome Bisulfite sequencing (WGBS) has become the standard method for interrogating plant methylomes at base resolution. However, deep WGBS measurements remain cost prohibitive for large, complex genomes and for population-level studies. As a result, most published plant methylomes are sequenced far below saturation, with a large proportion of cytosines having either missing data or insufficient coverage. Here we present METHimpute, a Hidden Markov Model (HMM) based imputation algorithm for the analysis of WGBS data. Unlike existing methods, METHimpute enables the construction of complete methylomes by inferring the methylation status and level of all cytosines in the genome regardless of coverage. Application of METHimpute to maize, rice and Arabidopsis shows that the algorithm infers cytosine-resolution methylomes with high accuracy from data as low as 6X, compared to data with 60X, thus making it a cost-effective solution for large-scale studies. Although METHimpute has been extensively tested in plants, it should be broadly applicable to other species.

scholarly journals Association Between miR-148a and DNA Methylation Profile in Individuals Exposed to Lead (Pb)

2021 ◽  
Vol 12 ◽  
Author(s):  
Marília Ladeira de Araújo ◽  
Bruno Costa Gomes ◽  
Paula Pícoli Devóz ◽  
Nathália de Assis Aguilar Duarte ◽  
Diego Luis Ribeiro ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Status ◽  
Epidemiologic Studies ◽  
Methylation Profile ◽  
Taqman Assay ◽  
Inverse Association ◽  
Dna Methylation Profile ◽  
Lead Levels ◽  
Mirnas Expression ◽  
The Impact

Experimental and epidemiologic studies have shown that lead (Pb) is able to induce epigenetic modifications, such as changes in DNA methylation profiles, in chromatin remodeling, as well as the expression of non-coding RNAs (ncRNAs). However, very little is known about the interactions between microRNAs (miRNAs) expression and DNA methylation status in individuals exposed to the metal. The aim of the present study was to investigate the impact of hsa-miR-148a expression on DNA methylation status, in 85 workers exposed to Pb. Blood and plasma lead levels (BLL and PLL, respectively) were determined by ICP-MS; expression of the miRNA-148a was quantified by RT-qPCR (TaqMan assay) and assessment of the global DNA methylation profile (by measurement of 5-methylcytosine; % 5-mC) was performed by ELISA. An inverse association was seen between miR-148a and % 5-mC DNA, as a function of BLL and PLL (β = −3.7; p = 0.071 and β = −4.1; p = 0.049, respectively) adjusted for age, BMI, smoking, and alcohol consumption. Taken together, our study provides further evidence concerning the interactions between DNA methylation profile and miR-148a, in individuals exposed to Pb.

scholarly journals Genetic species identification of ecologically important planthoppers (Prokelisia spp.) of coastal Spartina saltmarshes using High Resolution Melting Analysis (HRMA)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
G. Janelle Espinoza ◽  
Jaime R. Alvarado Bremer
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Sympatric Species ◽  
High Resolution Melting Analysis ◽  
Individual Species ◽  
Base Pairs ◽  
Melting Temperatures ◽  
Melting Analysis ◽  
Cost Effective Alternative

AbstractPhloem-feeding planthoppers of the genus Prokelisia rank among the most abundant and ecologically important browsers of coastal saltmarsh grasses of eastern North America and the Caribbean. Along the Spartina marshes of the northern Gulf of Mexico, the sympatric species P. marginata and P. dolus are the most abundant, but are difficult to distinguish from each other based solely on morphology. This study seeks to design a molecular assay based on High Resolution Melting Analysis (HRMA) as a fast, cost-effective alternative to differentiate these species. A 450 base pairs (bp) segment of cytochrome c oxidase subunit I (COI) was amplified and sequenced for representative samples of both species, and a short amplicon (SA) HRMA was designed based on the presence of fixed nucleotide differences between species found along a 60 bp segment of COI. The unambiguous identification of individual specimens of P. marginata or P. dolus was possible due to easily discernable differences in the melting temperatures of the two species along this mini barcode. This assay may prove useful for future genetic studies involving these species by preventing the overestimation of genetic diversity via inclusion of conspecifics, and in ecological studies by improving data on the effects of individual species of Prokelisia.

scholarly journals Whole-blood DNA Methylation Markers for Risk Stratification in Colorectal Cancer Screening: A Systematic Review

Cancers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 912 ◽  
Author(s):  
Raut ◽  
Guan ◽  
Schrotz-King ◽  
Brenner
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Risk Stratification ◽  
Colorectal Neoplasms ◽  
Whole Blood ◽  
Large Scale ◽  
Methylation Status ◽  
Population Characteristics ◽  
Risk Scores ◽  
Study Population

DNA methylation profiles within whole-blood samples have been reported to be associated with colorectal cancer (CRC) occurrence and might enable risk stratification for CRC. We systematically reviewed and summarized studies addressing the association of whole-blood DNA methylation markers and risk of developing CRC or its precursors. We searched PubMed and ISI Web of Knowledge to identify relevant studies published until 12th November 2018. Two reviewers independently extracted data on study population characteristics, candidate genes, methylation measurement methods, methylation levels of patients in comparison to healthy controls, p-values, and odds ratios of the markers. Overall, 19 studies reporting 102 methylation markers for risk assessment of colorectal neoplasms met our inclusion criteria. The studies mostly used Methylation Specific Polymerase Chain Reaction (MS-PCR) for assessing the methylation status of a defined set of genes. Only two studies applied array-based genome-wide assays to assess the methylation levels. Five studies incorporated panels consisting of 2–10 individual methylation markers to assess their potential for stratifying the risk of developing colorectal neoplasms. However, none of these associations was confirmed in an independent cohort. In conclusion, whole-blood DNA methylation markers may be useful as biomarkers for risk stratification in CRC screening, but reproducible risk prediction algorithms are yet to be established by large scale epigenome-wide studies with thorough validation of results in prospective study cohorts including large screening populations. The possibilities of enhancing predictive power by combining methylation data with polygenetic risk scores and environmental risk factors need to be explored.

scholarly journals Alcohol consumption is associated with widespread changes in blood DNA methylation: analysis of cross-sectional and longitudinal data

2018 ◽  
Author(s):  
Pierre-Antoine Dugué ◽  
Rory Wilson ◽  
Benjamin Lehne ◽  
Harindra Jayasekara ◽  
Xiaochuan Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Alcohol Consumption ◽  
Large Scale ◽  
Alcohol Intake ◽  
Methylation Status ◽  
Repeated Measurements ◽  
Cross Sectional ◽  
Blood Samples ◽  
Melbourne Collaborative Cohort Study ◽  
Risk Of Disease

ABSTRACTBackground:DNA methylation may be one of the mechanisms by which alcohol consumption is associated with the risk of disease. We conducted a large-scale, cross-sectional, genome-wide DNA methylation association study of alcohol consumption and a longitudinal analysis of repeated measurements taken several years apart.Methods:Using the Illumina Infinium HumanMethylation450 BeadChip, DNA methylation measures were determined using baseline peripheral blood samples from 5,606 adult Melbourne Collaborative Cohort Study (MCCS) participants. For a subset of 1,088 of them, these measures were repeated using blood samples collected at follow-up, a median of 11 years later. Associations between alcohol intake and blood DNA methylation were assessed using linear mixed-effects regression models adjusted for batch effects and potential confounders. Independent data from the LOLIPOP (N=4,042) and KORA (N=1,662) cohorts were used to replicate associations discovered in the MCCS.Results:Cross-sectional analyses identified 1,414 CpGs associated with alcohol intake at P<10-7, 1,243 of which had not been reported previously. Of these 1,243 novel associations, 1,078 were replicated (P<0.05) using LOLIPOP and KORA data. Using the MCCS data, we also replicated (P<0.05) 403 of 518 associations that had been reported previously. Interaction analyses suggested that associations were stronger for women, non-smokers, and participants genetically predisposed to consume less alcohol. Of the 1,414 CpGs, 530 were differentially methylated (P<0.05) in former compared with current drinkers. Longitudinal associations between the change in alcohol intake and the change in methylation were observed for 513 of the 1,414 cross-sectional associations.Conclusion:Our study indicates that, for middle-aged and older adults, alcohol intake is associated with widespread changes in DNA methylation across the genome. Longitudinal analyses showed that the methylation status of alcohol-associated CpGs may change with changes in alcohol consumption.

scholarly journals Data quality of Whole Genome Bisulfite Sequencing on Illumina platforms

2017 ◽  
Author(s):  
Amanda Raine ◽  
Ulrika Liljedahl ◽  
Jessica Nordlund
Keyword(s):  
Dna Methylation ◽  
Data Quality ◽  
Large Scale ◽  
Cost Effective ◽  
Bisulfite Treatment ◽  
Real Time Analysis ◽  
Genome Bisulfite Sequencing ◽  
Software Upgrade ◽  
Impact Data

AbstractThe powerful HiSeq X sequencers with their patterned flowcell technology and fast turnaround times are instrumental for many large-scale genomic and epigenomic studies. However, assessment of DNA methylation by sodium bisulfite treatment results in sequencing libraries of low diversity, which may impact data quality and yield. In this report we assess the quality of WGBS data generated on the HiSeq X system in comparison with data generated on the HiSeq 2500 system and the newly released NovaSeq system. We report a systematic issue with low basecall quality scores assigned to guanines in the second read of WGBS when using certain Real Time Analysis (RTA) software versions on the HiSeq X sequencer, reminiscent of an issue that was previously reported with certain HiSeq 2500 software versions. However, with the HD.3.4.0/RTA 2.7.7 software upgrade for the HiSeq X system, we observed an overall improved quality and yield of the WGBS data generated, which in turn empowers cost-effective and high quality DNA methylation studies.

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