1. Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution
- Author
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Sriharsa Pradhan, Laura-Madison Hussong, Thomas C. Evans, Theodore B. Davis, Ivan R. Corrêa, Romualdas Vaisvila, Shengxi Guan, Chloé Baum, Lana Saleh, Zhiyi Sun, Nan Dai, Mala Samaranayake, Laurence Ettwiller, Bo Yan, New England Biolabs, Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
5-Hydroxymethylcytosine ,0303 health sciences ,[SDV]Life Sciences [q-bio] ,Deamination ,Method ,Computational biology ,Methylation ,Biology ,Bisulfite ,03 medical and health sciences ,5-Methylcytosine ,chemistry.chemical_compound ,0302 clinical medicine ,Differentially methylated regions ,chemistry ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,DNA methylation ,Genetics ,natural sciences ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,030217 neurology & neurosurgery ,Genetics (clinical) ,DNA ,030304 developmental biology - 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.
- Published
- 2021