Focussing reduced representation CpG sequencing through judicious restriction enzyme choice

Kirschner, Sophie A.; Hunewald, Oliver; Mériaux, Sophie B.; Brunnhoefer, Regina; Turner, Jonathan D.

doi:10.1016/j.ygeno.2016.03.001

Cited by 8 publications

(6 citation statements)

References 34 publications

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“…HinP1 I (G ˇ CGC) sites as well as other methylation-sensitive restriction enzymes, such as Hpa II (C ˇ CGG), Hha I (GCG ˇ C), and BstU I (CG ˇ CG), are clustered in CpG islands. It has been reported that by using the 21 chromosomes of the mouse genome assembly (mm10, GRCm38, Dec2011 build UCSC), in silico prediction of enzymatically digested fragments revealed that Hpa II site and Hha I sites covered the most UCSC-annotated CpG island with 94.8 and 93.4%, respectively [ 14 ]. This indicates that the short HinP1 I- HinP1 I DNA fragments should be predominantly from in CpG islands.…”

Section: Resultsmentioning

confidence: 99%

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end

2018

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Environmental factors stimulate alteration of DNA methylation level. Investigation of the genome-wide DNA methylation status is important for environmental health studies. We here designed a genomic DNA amplification and labeling protocol using a methylation-sensitive restriction enzyme HinP1 I. This method can specifically amplify genomic DNA fragments possessing methyl-CpG at the end. The fragments are a relatively short size and dominantly located on CpG-islands. By using the samples prepared by this method, a dioxin-induced change in the methylation level of the mouse Cyp1a1 promoter was successfully evaluated using oligonucleotide probes covalently bound onto a glass plate. The method developed in this paper would be useful for other genome-wide analysis platforms for the large scale epigenome-wide association studies (EWAS) including human epidemiological samples.

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Section: Resultsmentioning

confidence: 99%

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end

2018

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“…Previous studies have tested the potential of other restriction enzymes and enzyme combinations to expand the range of CpG sites that can be targeted in a genome ( 8 – 11 , 28 , 30 , 56 , 57 ). However, to our knowledge, there is currently no computational method that systematically explores the capacity of all commercially-available restriction enzymes to generate ‘personalised’ reduced-representations of the genome whilst minimising the experimental cost ( Supplementary Figure S2E ).…”

Section: Resultsmentioning

confidence: 99%

“…To address these cost inefficiencies, many methods have been developed to reduce the number of fragments that need to be sequenced for a given biological system or WGBS validation experiment ( 8 – 25 ). These methods can be broadly split into those that positively select for genomic fragments of interest and those that deplete for fragments that are not of interest.…”

Section: Introductionmentioning

confidence: 99%

cuRRBS: simple and robust evaluation of enzyme combinations for reduced representation approaches

Martin-Herranz

Ribeiro

Krueger

et al. 2017

Nucleic Acids Research

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DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show in silico that cuRRBS-defined restriction enzymes consistently out-perform MspI digestion in many biological systems, considering both CpG and CHG contexts. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets.

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“…Fragments are then size-selected and sequenced to reveal the location of non-methylated sites [ 129 ]. However, since only around 20% of the CpG islands are dynamically regulated, MRE-sequencing lacks efficiency genome-wide and is more suited for specific loci [ 130 ]. A luminometric variation of this assay (LUMA: Luminometric Methylation Assay) has been used to show a decrease in global DNA methylation in both the PFC and amygdala following six months of peripheral nerve injury in mice [ 115 ].…”

Section: Dna Methylation and Painmentioning

confidence: 99%

DNA Methylation and Non-Coding RNAs during Tissue-Injury Associated Pain

Irfan

Febrianto

Sharma

et al. 2022

IJMS

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While about half of the population experience persistent pain associated with tissue damages during their lifetime, current symptom-based approaches often fail to reduce such pain to a satisfactory level. To provide better patient care, mechanism-based analgesic approaches must be developed, which necessitates a comprehensive understanding of the nociceptive mechanism leading to tissue injury-associated persistent pain. Epigenetic events leading the altered transcription in the nervous system are pivotal in the maintenance of pain in tissue injury. However, the mechanisms through which those events contribute to the persistence of pain are not fully understood. This review provides a summary and critical evaluation of two epigenetic mechanisms, DNA methylation and non-coding RNA expression, on transcriptional modulation in nociceptive pathways during the development of tissue injury-associated pain. We assess the pre-clinical data and their translational implication and evaluate the potential of controlling DNA methylation and non-coding RNA expression as novel analgesic approaches and/or biomarkers of persistent pain.

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Focussing reduced representation CpG sequencing through judicious restriction enzyme choice

Cited by 8 publications

References 34 publications

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end

cuRRBS: simple and robust evaluation of enzyme combinations for reduced representation approaches

DNA Methylation and Non-Coding RNAs during Tissue-Injury Associated Pain

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