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

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.

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

10.1101/2021.03.12.435105 ◽

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.

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Differential DNA Methylation of the IMMP2L Gene in Families with Maternally Inherited 7q31.1 Microdeletions is Associated with Intellectual Disability and Developmental Delay

Cytogenetic and Genome Research ◽

10.1159/000514491 ◽

2021 ◽

pp. 1-15

Author(s):

Stanislav A. Vasilyev ◽

Nikolay A. Skryabin ◽

Anna A. Kashevarova ◽

Ekaterina N. Tolmacheva ◽

Renata R. Savchenko ◽

...

Keyword(s):

Dna Methylation ◽

Intellectual Disability ◽

Bisulfite Sequencing ◽

Epigenetic Modification ◽

Copy Number Variations ◽

Incomplete Penetrance ◽

Cpg Sites ◽

Transport Chain ◽

Underlying Mechanisms ◽

Chromosome 7Q

Most copy number variations (CNVs) in the human genome display incomplete penetrance with unknown underlying mechanisms. One such mechanism may be epigenetic modification, particularly DNA methylation. The IMMP2L gene is located in a critical region for autism susceptibility on chromosome 7q (AUTS1). The level of DNA methylation was assessed by bisulfite sequencing of 87 CpG sites in the IMMP2L gene in 3 families with maternally inherited 7q31.1 microdeletions affecting the IMMP2L gene alone. Bisulfite sequencing revealed comparable levels of DNA methylation in the probands, healthy siblings without microdeletions, and their fathers. In contrast, a reduced DNA methylation index and increased IMMP2L expression were observed in lymphocytes from the healthy mothers compared with the probands. A number of genes were upregulated in the healthy mothers compared to controls and downregulated in probands compared to mothers. These genes were enriched in components of the ribosome and electron transport chain, as well as oxidative phosphorylation and various degenerative conditions. Differential expression in probands and mothers with IMMP2L deletions relative to controls may be due to compensatory processes in healthy mothers with IMMP2L deletions and disturbances of these processes in probands with intellectual disability. The results suggest a possible partial compensation for IMMP2L gene haploinsufficiency in healthy mothers with the 7q31.1 microdeletion by reducing the DNA methylation level. Differential DNA methylation of intragenic CpG sites may affect the phenotypic manifestation of CNVs and explain the incomplete penetrance of chromosomal microdeletions.

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New Targeted Approaches for Epigenetic Age Predictions

10.1101/799031 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yang Han ◽

Julia Franzen ◽

Thomas Stiehl ◽

Michael Gobs ◽

Chao-Chung Kuo ◽

...

Keyword(s):

Dna Methylation ◽

Cost Effective ◽

Amplicon Sequencing ◽

Digital Pcr ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

High Throughput Analysis ◽

Cpg Sites ◽

Targeted Analysis ◽

Epigenetic Age

AbstractAging causes epigenetic modifications, which are utilized as a biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. In this study, we utilized 4,650 Illumina BeadChip datasets of blood to select the best suited CpG sites for targeted analysis. DNA methylation analysis at these sites with either pyrosequencing or droplet digital PCR (ddPCR) revealed a high correlation with chronological age. In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower precision at individual CpGs. However, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often accompanied by a CTCF binding site at the peak. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified but reveals a stochastic pattern. Based on this, we have developed an alternative model for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. Thus, the stochastic evolution of age-associated DNA methylation patterns, which seems to resemble epigenetic drift, enables epigenetic clocks for individual DNA strands.

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

10.1101/163535 ◽

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.

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Abstract 428: DNA Methylation of ASC is Associated with Decreased ASC and IL-1β Expression in Heart Failure

Circulation Research ◽

10.1161/res.117.suppl_1.428 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Brittany Butts ◽

Javed Butler

Keyword(s):

Heart Failure ◽

Dna Methylation ◽

Inflammatory Cytokines ◽

Epigenetic Modification ◽

Cpg Islands ◽

Inflammasome Activation ◽

Cpg Sites ◽

Exon 1 ◽

Vital Component ◽

Intron Region

Introduction: Heart failure (HF) is associated with formation and activation of inflammasome, a complex of intracellular interaction proteins that trigger maturation of inflammatory cytokines to initiate inflammatory response. ASC, a vital component of the inflammasome, is controlled through epigenetic modification via methylation of CpG islands surrounding exon 1. Methods: To assess the relationships between DNA methylation of ASC, ASC expression, and inflammatory cytokines IL-1β and IL-18 in HF, stored samples from 155 chronic HF patients (age 56.9±12.0 yr, 64% male, 47% black, and ejection fraction 29.9±14.9) were analyzed. DNA extracted from PMBCs were analyzed by pyrosequencing for percent methylation of seven CpG sites in the intron region preceding exon 1 of the ASC gene. ASC mRNA was quantified via real-time PCR and analyzed as the ratio ASC:GAPDH. Serum ASC, IL-1β, and IL-18 were measured by ELISA. Results: Higher ASC methylation was associated with lower ASC mRNA (r=0-.328, p<0.001) and protein (r=-.464, p<0.001) expression. Lower ASC mRNA expression was associated with lower ASC protein expression (r=0.494, p<0.001). Decreased IL-1β expression was associated with higher ASC methylation (r=-.424, p=0.005) and lower ASC mRNA (r=.619, p<0.001) and ASC protein (r=.433, p<0.001). IL-18 expression was not significantly associated with ASC methylation or expression. Conclusions: Increased ASC methylation was associated with lower IL-1β, likely via decreased ASC gene expression. As ASC is required for inflammasome activation of IL-1β, this study implicates the inflammasome pathway as a driver of inflammation in HF, proving a potential target for novel interventions.

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Genome-Wide DNA Methylation Profiling in the Lotus (Nelumbo nucifera) Flower Showing its Contribution to the Stamen Petaloid

Plants ◽

10.3390/plants8050135 ◽

2019 ◽

Vol 8 (5) ◽

pp. 135 ◽

Cited By ~ 5

Author(s):

Zhongyuan Lin ◽

Meihui Liu ◽

Rebecca Njeri Damaris ◽

Tonny Maraga Nyong’a ◽

Dingding Cao ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Bisulfite Sequencing ◽

Epigenetic Modification ◽

Nelumbo Nucifera ◽

Flower Formation ◽

Relationship Analysis ◽

Genome Wide ◽

Dna Methylation Profiling ◽

Methylation Profiling

DNA methylation is a vital epigenetic modification. Methylation has a significant effect on the gene expression influencing the regulation of different physiological processes. Current studies on DNA methylation have been conducted on model plants. Lotus (Nelumbo nucifera) is a basic eudicot exhibiting variations during development, especially in flower formation. DNA methylation profiling was conducted on different flower tissues of lotuses through whole genome bisulfite sequencing (WGBS) to investigate the effects of DNA methylation on its stamen petaloid. A map of methylated cytosines at the single base pair resolution for the lotus was constructed. When the stamen was compared with the stamen petaloid, the DNA methylation exhibited a global decrease. Genome-wide relationship analysis between DNA methylation and gene expression identified 31 different methylation region (DMR)-associated genes, which might play crucial roles in floral organ formation, especially in the stamen petaloid. One out of 31 DMR-associated genes, NNU_05638 was homolog with Plant U-box 33 (PUB33). The DNA methylation status of NNU_05638 promoter was distinct in three floral organs, which was confirmed by traditional bisulfite sequencing. These results provide further insights about the regulation of stamen petaloids at the epigenetic level in lotus.

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The Impact of DNA Methylation Dynamics on the Mutation Rate During Human Germline Development

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401511 ◽

2020 ◽

Vol 10 (9) ◽

pp. 3337-3346

Author(s):

Yijia Zhou ◽

Funan He ◽

Weilin Pu ◽

Xun Gu ◽

Jiucun Wang ◽

...

Keyword(s):

Dna Methylation ◽

Mutation Rate ◽

Germline Mutation ◽

Developmental Stages ◽

Rare Variants ◽

Epigenetic Modification ◽

Genome Project ◽

Germline Development ◽

Cpg Sites ◽

The Impact

Abstract DNA methylation is a dynamic epigenetic modification found in most eukaryotic genomes. It is known to lead to a high CpG to TpG mutation rate. However, the relationship between the methylation dynamics in germline development and the germline mutation rate remains unexplored. In this study, we used whole genome bisulfite sequencing (WGBS) data of cells at 13 stages of human germline development and rare variants from the 1000 Genome Project as proxies for germline mutations to investigate the correlation between dynamic methylation levels and germline mutation rates at different scales. At the single-site level, we found a significant correlation between methylation and the germline point mutation rate at CpG sites during germline developmental stages. Then we explored the mutability of methylation dynamics in all stages. Our results also showed a broad correlation between the regional methylation level and the rate of C > T mutation at CpG sites in all genomic regions, especially in intronic regions; a similar link was also seen at all chromosomal levels. Our findings indicate that the dynamic DNA methylome during human germline development has a broader mutational impact than is commonly assumed.

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DNA methylation patterns of β-globin cluster in β-thalassemia patients

Clinical Epigenetics ◽

10.1186/s13148-020-00987-2 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Xiuqin Bao ◽

Yangjin Zuo ◽

Diyu Chen ◽

Cunyou Zhao

Keyword(s):

Dna Methylation ◽

Cord Blood ◽

Methylation Level ◽

Epigenetic Modification ◽

Fetal Hemoglobin ◽

Cpg Sites ◽

Hbf Level ◽

Globin Promoter ◽

Methylation Patterns ◽

Normal Controls

Abstract Background Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in β-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. Results Here, we evaluated DNA methylation patterns of the β-globin cluster from peripheral bloods of 105 β0/β0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and β-globin promoters displayed hypomethylation in β0/β0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of β0/β0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among β0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls. Conclusions Our findings revealed methylation patterns in β-globin cluster associated with β0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in β0 thalassemia patients and provided candidate targets for the therapies of β-hemoglobinopathies.

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1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab1 ◽

2019 ◽

Vol 31 (1) ◽

pp. 126

Author(s):

J. E. Duan ◽

Z. Jiang ◽

F. Alqahtani ◽

I. Mandoiu ◽

H. Dong ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Epigenetic Regulation ◽

Bisulfite Sequencing ◽

Whole Genome ◽

Differentially Methylated Regions ◽

Whole Genome Bisulfite Sequencing ◽

Cpg Sites ◽

Genome Wide ◽

Genome Bisulfite Sequencing

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

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Use of DNA methylation to distinguish gallbladder carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e15605 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e15605-e15605

Author(s):

Xiaoqing Jiang ◽

Bin Li ◽

Zhiquan Qiu ◽

Yong Yu ◽

Zhishuai Li ◽

...

Keyword(s):

Dna Methylation ◽

Malignant Disease ◽

Gallbladder Disease ◽

White Blood Cells ◽

Stage Iv ◽

P Value ◽

Targeted Bisulfite Sequencing ◽

Gallbladder Diseases ◽

Cpg Sites ◽

A Genome

e15605 Background: Gallbladder cancer (GBC), an uncommon malignancy with a high mortality rate, is often diagnosed late due to lack of early symptoms and the relative hidden nature of the gallbladder. Despite the advancements in imaging technologies, there is no reliable screening test for GBC. The role of aberrant DNA methylation in the process of tumorigenesis both at individual genes and a genome-wide scale has been well elucidated. It occurs very early in cancer development, thus capable of serving as a screening marker. Methods: Panel Design: Methylation data of tumor samples (12 types, n = 4,772), adjacent normal (8 types, n = 411), and normal white blood cells (n = 656) from TCGA and GSE were compared. Differentially methylated sites were derived using a Bayesian hierarchical model-DSS with an adjusted p-value < 0.05. Our panel covers 80,672 CpG sites, spanning 1.05Mb of human genome. This panel contains 12,196 GBC relevant CpG sites. We performed targeted bisulfite sequencing on 23 GBC patients (6 stage II-III, 17 stage IV) and 13 patients with non-malignant gallbladder diseases (cholecystitis and gallstones). Of the 23 GBC patients, we obtained adjacent normal tissue from 7 of them. Basic clinical features such as age, gender, of patients with GBC and patients with non-malignant gallbladder diseases were comparable. Results: Among the 12,196 GBC relevant CpG sites, 10,216 sites were statistically significantly hypermethylated and 275 sites were statistically significantly hypomethylated comparing to patients with non-malignant gallbladder disease as well as adjacent normal gallbladder tissues. Subsequently, we used the derived differentially methylated CpG sites to construct a linear regression model, achieving an area under curve of 99%. Collectively, the methylation levels were comparable between tissues with non-malignant disease and adjacent normal. Interestingly, when considering the 275 hypomethylated markers alone, we observed that the methylation level of adjacent normal tissues is significantly higher than tissues with non-malignant disease. Conclusions: Collectively, our panel can effectively distinguish GBC samples from non-cancerous samples, demonstrating the potential of DNA methylation in GBC screening. Furthermore, hypomethylation markers can be used to distinguish non-malignant disease from the healthy.

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