scholarly journals Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaogang Li ◽  
Xinxin Shi ◽  
Yin Gong ◽  
Wenting Guo ◽  
Yuanrui Liu ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cost Effective ◽  
Chemical Labeling ◽  
Whole Genome Analysis ◽  
Single Base ◽  
Chemically Modified ◽  
Sequencing Method ◽  
Single Base Resolution ◽  
Selective Chemical ◽  
Apobec Family

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C) or 5mC. Herein, we developed a method based on embryonic stem cell (ESC) whole-genome analysis, which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single-base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

scholarly journals Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

2021 ◽  
Author(s):  
Xiaogang Li ◽  
Xinxin Shi ◽  
Wenting Guo ◽  
Yuanrui Liu ◽  
Chunwei Peng ◽  
...  
Keyword(s):  
Cost Effective ◽  
Chemical Labeling ◽  
Whole Genome Analysis ◽  
Single Base ◽  
Chemically Modified ◽  
Sequencing Method ◽  
Single Base Resolution ◽  
Selective Chemical ◽  
Tet Enzymes ◽  
Apobec Family

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation (TET) enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically-modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C)or 5-methylcytosine (5mC). Herein, we developed a method based on ESC whole-genome analysis which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme APOBEC-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

Selective Chemical Labeling and Sequencing of 5-Carboxylcytosine in DNA at Single-Base Resolution

2020 ◽  
Vol 92 (18) ◽  
pp. 12710-12715
Author(s):  
Yalun Xie ◽  
Yafen Wang ◽  
Zhiyong He ◽  
Wei Yang ◽  
Boshi Fu ◽  
...  
Keyword(s):  
Chemical Labeling ◽  
Single Base ◽  
Single Base Resolution ◽  
Selective Chemical

scholarly journals MBRidge: an accurate and cost-effective method for profiling DNA methylome at single-base resolution

2015 ◽  
Vol 7 (4) ◽  
pp. 299-313 ◽  
Author(s):  
Wanshi Cai ◽  
Fengbiao Mao ◽  
Huajing Teng ◽  
Tao Cai ◽  
Fangqing Zhao ◽  
...  
Keyword(s):  
Cost Effective ◽  
Single Base ◽  
Dna Methylome ◽  
Cost Effective Method ◽  
Single Base Resolution

scholarly journals Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

2017 ◽  
Author(s):  
Carine Legrand ◽  
Francesca Tuorto ◽  
Mark Hartmann ◽  
Reinhard Liebers ◽  
Dominik Jacob ◽  
...  
Keyword(s):  
Bisulfite Sequencing ◽  
Specific Activity ◽  
Embryonic Stem ◽  
Wide Distribution ◽  
28S Rrna ◽  
Rna Methylation ◽  
Single Base ◽  
Single Base Resolution ◽  
Methylation Patterns ◽  
Whole Transcriptome

AbstractCytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing. Using RNAs from mouse embryonic stem cells we performed a comprehensive methylation analysis of mouse tRNAs, rRNAs and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA. In addition, mRNAs were found to be very sparsely methylated or not methylated at all. Finally, the tRNA-specific activity of the DNMT2 methyltransferase could be resolved at single-base resolution, which provided important further validation. Our approach can be used to profile cytosine-5 RNA methylation patterns in many experimental contexts and will be important for understanding the function of cytosine-5 RNA methylation in RNA biology and in human disease.

Xeno nucleic acid probes mediated methylation-specific PCR for single-base resolution analysis of N6-methyladenosine in RNAs

The Analyst ◽  
2021 ◽  
Author(s):  
Qinli Pu ◽  
Hongyan Yu ◽  
Xi Zhou ◽  
Junjie Li ◽  
Yu-Jun Yang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Gene Locus ◽  
Cost Effective ◽  
Specific Gene ◽  
Rna Modifications ◽  
Nucleic Acid Probes ◽  
Single Base ◽  
Specific Pcr ◽  
Resolution Analysis ◽  
Single Base Resolution

Reliable and cost-effective quantification of RNA modifications at a specific gene locus is essential to elucidate the pathogenic mechanism encoded by RNA epigenetics. Current methods to quantify N6-methyladenosine (m6A) at...

scholarly journals DeepH&M: Estimating single-CpG hydroxymethylation and methylation levels from enrichment and restriction enzyme sequencing methods

2020 ◽  
Vol 6 (27) ◽  
pp. eaba0521
Author(s):  
Yu He ◽  
Hyo Sik Jang ◽  
Xiaoyun Xing ◽  
Daofeng Li ◽  
Michael J. Vasek ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Scale Up ◽  
Cost Effective ◽  
Epigenetic Mark ◽  
Single Base ◽  
Time Points ◽  
Biological Time ◽  
Mapping Technology ◽  
High Concordance ◽  
Single Base Resolution

Increased appreciation of 5-hydroxymethylcytosine (5hmC) as a stable epigenetic mark, which defines cell identity and disease progress, has engendered a need for cost-effective, but high-resolution, 5hmC mapping technology. Current enrichment-based technologies provide cheap but low-resolution and relative enrichment of 5hmC levels, while single-base resolution methods can be prohibitively expensive to scale up to large experiments. To address this problem, we developed a deep learning–based method, “DeepH&M,” which integrates enrichment and restriction enzyme sequencing methods to simultaneously estimate absolute hydroxymethylation and methylation levels at single-CpG resolution. Using 7-week-old mouse cerebellum data for training the DeepH&M model, we demonstrated that the 5hmC and 5mC levels predicted by DeepH&M were in high concordance with whole-genome bisulfite–based approaches. The DeepH&M model can be applied to 7-week-old frontal cortex and 79-week-old cerebellum, revealing the robust generalizability of this method to other tissues from various biological time points.

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