Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution
- Zhiyi Sun1,3,
- Romualdas Vaisvila1,3,
- Laura-Madison Hussong1,
- Bo Yan1,
- Chloé Baum1,2,
- Lana Saleh1,
- Mala Samaranayake1,
- Shengxi Guan1,
- Nan Dai1,
- Ivan R. Corrêa Jr.1,
- Sriharsa Pradhan1,
- Theodore B. Davis1,
- Thomas C. Evans Jr.1 and
- Laurence M. Ettwiller1
- 1New England Biolabs, Incorporated, Ipswich, Massachusetts 01938, USA;
- 2Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Université d'Evry, Université Paris-Saclay, 91000 Évry, France
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↵3 These authors contributed equally to this work.
Abstract
The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.265306.120.
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Freely available online through the Genome Research Open Access option.
- Received April 29, 2020.
- Accepted December 11, 2020.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
