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EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution
BACKGROUND: A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventiona...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9979530/ https://www.ncbi.nlm.nih.gov/pubmed/36859282 http://dx.doi.org/10.1186/s13148-023-01451-7 |
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author | Lee, Jaywon Lee, Dongin Kim, Hwang-Phill Kim, Tae-You Bang, Duhee |
author_facet | Lee, Jaywon Lee, Dongin Kim, Hwang-Phill Kim, Tae-You Bang, Duhee |
author_sort | Lee, Jaywon |
collection | PubMed |
description | BACKGROUND: A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods. RESULTS: EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples. CONCLUSIONS: EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13148-023-01451-7. |
format | Online Article Text |
id | pubmed-9979530 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-99795302023-03-03 EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution Lee, Jaywon Lee, Dongin Kim, Hwang-Phill Kim, Tae-You Bang, Duhee Clin Epigenetics Methodology BACKGROUND: A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods. RESULTS: EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples. CONCLUSIONS: EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13148-023-01451-7. BioMed Central 2023-03-01 /pmc/articles/PMC9979530/ /pubmed/36859282 http://dx.doi.org/10.1186/s13148-023-01451-7 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Lee, Jaywon Lee, Dongin Kim, Hwang-Phill Kim, Tae-You Bang, Duhee EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
title | EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
title_full | EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
title_fullStr | EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
title_full_unstemmed | EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
title_short | EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
title_sort | ebs-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9979530/ https://www.ncbi.nlm.nih.gov/pubmed/36859282 http://dx.doi.org/10.1186/s13148-023-01451-7 |
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