scholarly journals Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

2017 ◽  
Author(s):  
Nelly Olova ◽  
Felix Krueger ◽  
Simon Andrews ◽  
David Oxley ◽  
Rebecca V. Berrens ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Pcr Amplification ◽  
Whole Genome ◽  
Library Preparation ◽  
Bisulfite Conversion ◽  
Global Methylation ◽  
Preparation Methods ◽  
Whole Genome Bisulfite Sequencing ◽  
Genome Bisulfite Sequencing

AbstractBackgroundWhole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.ResultsWe perform a comparative analysis of several commonly used pre-and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies.ConclusionsWe show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of BS conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

164 Genome-wide DNA Methylation Profiling in in Utero Heat-stressed Pigs as Measured by Whole-genome Bisulfite Sequencing

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 87-88
Author(s):  
Luiz F Brito ◽  
Jacob M Maskal ◽  
Shi-Yi Chen ◽  
Hinayah R Oliveira ◽  
Jason R Graham ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Cellular Response ◽  
Bisulfite Sequencing ◽  
Cholesterol Biosynthesis ◽  
In Utero ◽  
Whole Genome ◽  
Whole Genome Bisulfite Sequencing ◽  
Differentially Methylated Genes ◽  
Genome Bisulfite Sequencing

Abstract In utero heat stress (IUHS) has several postnatal consequences in pigs that compromise health, increase stress response, and reduce performance. These phenotypes may be caused by epigenetic modifications such as DNA methylation, which are heritable molecular modifications that impact gene expression and phenotypic outcomes without changing the DNA sequence. Therefore, we aimed to compare the DNA methylation profiles between in-utero thermoneutral (IUTN) and IUHS pigs to identify differentially methylated regions. Twenty-four pregnant gilts were evenly assigned to either a thermoneutral (17.5 ± 2.1°C) or heat stress (cycling 26 to 36°C) chamber from d 0 to 59 of gestation, followed by thermoneutral conditions (20.9 ± 2.3°C) for the rest of gestation and until the piglets were weaned. At 105 d of age, 10 IUTN and 10 IUHS piglets were euthanized and Longissimus dorsi muscle samples were collected and used to perform whole-genome bisulfite sequencing (WGBS). Purified genomic DNA was fragmented and bisulfite conversion was performed. Illumina platforms were used to sequence WGBS libraries. All pigs had similar proportions of methylation at CpG sites. Two-hundred-sixty-eight genomic regions were differentially methylated between IUTN and IUHS pigs. These identified regions are located across all pig chromosomes and ranged from 2 (SSC18) to 40 (SSC10). Eighty-five unique differentially-methylated genes were identified. These genes have been reported to be involved in key biological processes such as transcriptional repressor activity and tRNA processing (e.g., SKOR2,TRMT6, TSEN2), cellular response to heat stress (e.g.,CCAR2), placental vascularization (e.g.,FZD5), central nervous system (e.g.,VEPH1), cholesterol biosynthesis (e.g., CYB5R1), insulin receptor substrate (e.g.,IRS2), synaptic transmission (e.g.,RIMBP2), neurotrophic factor receptor activity (e.g.,LIFR), immune response (e.g., CD84), DNA repair (e.g., CHD1L), and cell proliferation and endocrine signaling (e.g., SSTR1, CYB5R1). These findings contribute to a better understanding of the epigenomic mechanisms underlying postnatal consequences of IUHS in pigs.

scholarly journals Efficient and accurate determination of genome-wide DNA methylation patterns in Arabidopsis thaliana with enzymatic methyl sequencing

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Suhua Feng ◽  
Zhenhui Zhong ◽  
Ming Wang ◽  
Steven E. Jacobsen
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Accurate Determination ◽  
Gc Content ◽  
Epigenetic Mark ◽  
Whole Genome ◽  
Whole Genome Bisulfite Sequencing ◽  
Genome Wide ◽  
A Genome ◽  
Genome Bisulfite Sequencing

Abstract Background 5′ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing. Results Using an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions. Conclusion We suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

scholarly journals How to Design a Whole-Genome Bisulfite Sequencing Experiment

Epigenomes ◽  
2018 ◽  
Vol 2 (4) ◽  
pp. 21 ◽  
Author(s):  
Claudius Grehl ◽  
Markus Kuhlmann ◽  
Claude Becker ◽  
Bruno Glaser ◽  
Ivo Grosse
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Alternative Methods ◽  
Transcriptional Gene Silencing ◽  
Whole Genome ◽  
Reduced Representation ◽  
Genome Region ◽  
Whole Genome Bisulfite Sequencing ◽  
Sequencing Experiment ◽  
Genome Bisulfite Sequencing

Aside from post-translational histone modifications and small RNA populations, the epigenome of an organism is defined by the level and spectrum of DNA methylation. Methyl groups can be covalently bound to the carbon-5 of cytosines or the carbon-6 of adenine bases. DNA methylation can be found in both prokaryotes and eukaryotes. In the latter, dynamic variation is shown across species, along development, and by cell type. DNA methylation usually leads to a lower binding affinity of DNA-interacting proteins and often results in a lower expression rate of the subsequent genome region, a process also referred to as transcriptional gene silencing. We give an overview of the current state of research facilitating the planning and implementation of whole-genome bisulfite-sequencing (WGBS) experiments. We refrain from discussing alternative methods for DNA methylation analysis, such as reduced representation bisulfite sequencing (rrBS) and methylated DNA immunoprecipitation sequencing (MeDIPSeq), which have value in specific experimental contexts but are generally disadvantageous compared to WGBS.

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