scholarly journals HAM-TBS: High accuracy methylation measurements via targeted bisulfite sequencing

2017 ◽  
Author(s):  
Simone Röh ◽  
Tobias Wiechmann ◽  
Susann Sauer ◽  
Maik Ködel ◽  
Elisabeth B. Binder ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Cost Effective ◽  
Accurate Method ◽  
High Accuracy ◽  
Ctcf Binding ◽  
Stress System ◽  
Robust Detection ◽  
Targeted Bisulfite Sequencing ◽  
Effective Manner

AbstractBackgroundThe ability to accurately and efficiently measure DNA methylation is vital to advance the understanding of this mechanism and its contribution to common diseases. Here, we present a highly accurate method to measure methylation using bisulfite sequencing (termed HAM-TBS). This novel method is able to assess DNA methylation in multiple samples with high accuracy in a cost-effective manner. We developed this assay for the FKBP5 locus, an important gene in the regulation of the stress system and previously linked to stress-related disorders, but the method is applicable to any locus of interest.ResultsHAM-TBS enables multiplexing of up to 96 samples spanning a region of ~10 kb using the llumina MiSeq. It incorporates a triplicate bisulfite conversion step, pooled target enrichment via PCR, PCR-free library preparation and a minimum coverage of 1,000x. Furthermore, we designed and validated a targeted panel to specifically assess regulatory regions within the FKBP5 locus including the transcription start site, topologically associated domain boundaries, intergenic and proximal enhancers as well as glucocorticoid receptor and CTCF binding sites that are not covered in commercially available DNA methylation arrays.ConclusionsHAM-TBS represents a highly accurate, medium-throughput sequencing approach for robust detection of DNA methylation changes in specific target regions.

scholarly journals Performance comparison and in-silico harmonization of commercial platforms for DNA methylome analysis by targeted bisulfite sequencing

2021 ◽  
Author(s):  
Miljana Tanic ◽  
Ismail Moghul ◽  
Simon Rodney ◽  
Pawan Dhami ◽  
Heli Vaikkinen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
Bisulfite Sequencing ◽  
Epigenetic Modification ◽  
Cost Effective ◽  
Performance Comparison ◽  
Performance Criteria ◽  
Targeted Bisulfite Sequencing ◽  
Cpg Sites ◽  
Targeted Analysis

Abstract DNA methylation is a key epigenetic modification in the regulation of cell fate and differentiation, and its analysis is gaining increasing importance in both basic and clinical research. Targeted Bisulfite Sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. Here we benchmarked five commercially available TBS platforms, including three hybridization capture-based (Agilent, Roche, and Illumina) and two RRBS-based (Diagenode and NuGen), across 11 samples. A subset of these were also compared to whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed performance with respect to workflow complexity, on/off-target performance, coverage, accuracy, and reproducibility. We find all platforms able to produce usable data but with major differences for some performance criteria, especially in the number and identity of the CpG sites covered, which affects the interoperability of datasets generated on these different platforms. To overcome this limitation, we used imputation and show that it improves the interoperability from an average of 10.35% (0.8M CpG sites) to 97% (7.6M CpG sites). Our study provides cross-validated guidance on which TBS platform to use for different features of the methylome and offers an imputation-based harmonization solution for improved interoperability between platforms, allowing comparative and integrative analysis.

scholarly journals Performance comparison and in-silico harmonisation of commercial platforms for DNA methylome analysis by targeted bisulfite sequencing

2021 ◽  
Author(s):  
Miljana Tanić ◽  
Ismail Moghul ◽  
Simon Rodney ◽  
Pawan Dhami ◽  
Heli Vaikkinen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
Bisulfite Sequencing ◽  
Epigenetic Modification ◽  
Cost Effective ◽  
Performance Comparison ◽  
Performance Criteria ◽  
Targeted Bisulfite Sequencing ◽  
Cpg Sites ◽  
Targeted Analysis

AbstractDNA methylation is a key epigenetic modification in the regulation of cell fate and differentiation, and its analysis is gaining increasing importance in both basic and clinical research. Targeted Bisulfite Sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. Here we benchmarked five commercially available TBS platforms, including three hybridization capture-based (Agilent, Roche, and Illumina) and two RRBS-based (Diagenode and NuGen), across 16 samples. A subset of these were also compared to whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed performance with respect to workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. We find all platforms able to produce usable data but major differences for some performance criteria, especially in the number and identity of the CpG sites covered, which affects the interoperability of datasets generated on these different platforms. To overcome this limitation, we used imputation and show that it improves the interoperability from an average of 10.35% (0.8M CpG sites) to 97% (7.6M CpG sites). Our study provides cross-validated guidance on which TBS platform to use for different features of the methylome and offers an imputation-based harmonization solution for improved interoperability between platforms, allowing comparative and integrative analysis.

scholarly journals DeepSignal: detecting DNA methylation state from Nanopore sequencing reads using deep-learning

2019 ◽  
Vol 35 (22) ◽  
pp. 4586-4595 ◽  
Author(s):  
Peng Ni ◽  
Neng Huang ◽  
Zhi Zhang ◽  
De-Peng Wang ◽  
Fan Liang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Deep Learning ◽  
Bisulfite Sequencing ◽  
Homo Sapiens ◽  
Accurate Method ◽  
Supplementary Information ◽  
Nanopore Sequencing ◽  
Methylation State ◽  
State Prediction ◽  
Genome Level

Abstract Motivation The Oxford Nanopore sequencing enables to directly detect methylation states of bases in DNA from reads without extra laboratory techniques. Novel computational methods are required to improve the accuracy and robustness of DNA methylation state prediction using Nanopore reads. Results In this study, we develop DeepSignal, a deep learning method to detect DNA methylation states from Nanopore sequencing reads. Testing on Nanopore reads of Homo sapiens (H. sapiens), Escherichia coli (E. coli) and pUC19 shows that DeepSignal can achieve higher performance at both read level and genome level on detecting 6 mA and 5mC methylation states comparing to previous hidden Markov model (HMM) based methods. DeepSignal achieves similar performance cross different DNA methylation bases, different DNA methylation motifs and both singleton and mixed DNA CpG. Moreover, DeepSignal requires much lower coverage than those required by HMM and statistics based methods. DeepSignal can achieve 90% above accuracy for detecting 5mC and 6 mA using only 2× coverage of reads. Furthermore, for DNA CpG methylation state prediction, DeepSignal achieves 90% correlation with bisulfite sequencing using just 20× coverage of reads, which is much better than HMM based methods. Especially, DeepSignal can predict methylation states of 5% more DNA CpGs that previously cannot be predicted by bisulfite sequencing. DeepSignal can be a robust and accurate method for detecting methylation states of DNA bases. Availability and implementation DeepSignal is publicly available at https://github.com/bioinfomaticsCSU/deepsignal. Supplementary information Supplementary data are available at bioinformatics online.

scholarly journals Quantification of Soluble or Insoluble Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay

2020 ◽  
Vol 2020 ◽  
pp. 1-8 ◽  
Author(s):  
Bhagya Deepachandi ◽  
Sudath Weerasinghe ◽  
Thisira Priyantha Andrahennadi ◽  
Nadira D. Karunaweera ◽  
Nadeeja Wickramarachchi ◽  
...  
Keyword(s):  
Cost Effective ◽  
Accurate Method ◽  
Research Field ◽  
Protein Quantification ◽  
Color Intensity ◽  
Resource Limited Settings ◽  
Protein Assay ◽  
Resource Limited ◽  
Effective Manner ◽  
Lowry Assay

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu’s phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10–500 μg/ml (1–50 μg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

scholarly journals Altered DNA methylation in human placenta after (suspected) preterm labor

Epigenomics ◽  
2020 ◽  
Vol 12 (20) ◽  
pp. 1769-1782
Author(s):  
Jon Schoorlemmer ◽  
Sofía Macías-Redondo ◽  
Mark Strunk ◽  
Ricardo Ramos-Ruíz ◽  
Pilar Calvo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Preterm Birth ◽  
Human Placenta ◽  
Preterm Labor ◽  
Bisulfite Sequencing ◽  
Term Birth ◽  
Targeted Bisulfite Sequencing ◽  
Per Se ◽  
Methylation Patterns

Aim: The aim of this study was to determine if alterations in DNA methylation in the human placenta would support suspected preterm labor as a pathologic insult associated with diminished placental health. Methods: We evaluated placental DNA methylation at seven loci differentially methylated in placental pathologies using targeted bisulfite sequencing, in placentas associated with preterm labor (term birth after suspected preterm labor [n = 15] and preterm birth [n = 15]), and controls (n = 15). Results: DNA methylation levels at the NCAM1 and PLAGL1 loci in placentas associated with preterm labor did differ significantly (p < 0.05) from controls. Discussion: Specific alterations in methylation patterns indicative of an unfavourable placental environment are associated with preterm labor per se and not restricted to preterm birth.

scholarly journals Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia

2015 ◽  
Vol 31 (1) ◽  
pp. 24-33 ◽  
Author(s):  
Ye Du ◽  
Meiyan Li ◽  
Jing Chen ◽  
Yonggang Duan ◽  
Xuebin Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Sperm Motility ◽  
Bisulfite Sequencing ◽  
Targeted Bisulfite Sequencing

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 Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage

2020 ◽  
Vol 120 ◽  
pp. 104784
Author(s):  
D.A. Moser ◽  
S. Müller ◽  
E.M. Hummel ◽  
A.S. Limberg ◽  
L. Dieckmann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
High Sensitivity ◽  
Targeted Bisulfite Sequencing

scholarly journals EPIC-TABSAT: analysis tool for targeted bisulfite sequencing experiments and array-based methylation studies

2019 ◽  
Vol 47 (W1) ◽  
pp. W166-W170 ◽  
Author(s):  
Julie Krainer ◽  
Andreas Weinhäusel ◽  
Karel Hanak ◽  
Walter Pulverer ◽  
Seza Özen ◽  
...  
Keyword(s):  
Bisulfite Sequencing ◽  
Cost Effective ◽  
Analysis Tool ◽  
Sequencing Data ◽  
Targeted Bisulfite Sequencing ◽  
Cost Effective Approach ◽  
Analysis Workflow ◽  
Complete Data Analysis ◽  
User Friendly ◽  
Validation Research

Abstract DNA methylation is one of the major epigenetic modifications and has frequently demonstrated its suitability as diagnostic and prognostic biomarker. In addition to chip and sequencing based epigenome wide methylation profiling methods, targeted bisulfite sequencing (TBS) has been established as a cost-effective approach for routine diagnostics and target validation applications. Yet, an easy-to-use tool for the analysis of TBS data in combination with array-based methylation results has been missing. Consequently, we have developed EPIC-TABSAT, a user-friendly web-based application for the analysis of targeted sequencing data that additionally allows the integration of array-based methylation results. The tool can handle multiple targets as well as multiple sequencing files in parallel and covers the complete data analysis workflow from calculation of quality metrics to methylation calling and interactive result presentation. The graphical user interface offers an unprecedented way to interpret TBS data alone or in combination with array-based methylation studies. Together with the computation of target-specific epialleles it is useful in validation, research, and routine diagnostic environments. EPIC-TABSAT is freely accessible to all users at https://tabsat.ait.ac.at/.

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