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| Status |
Public on Apr 24, 2023 |
| Title |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution [1] |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes as a lineage-defining mark dynamically altered in development and disease. The ideal method for 5mC localization would be both non-destructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here, we present Direct Methylation Sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution, using nanogram quantities of input DNA. DM-Seq employs two key DNA modifying enzymes: a neomorphic DNA methyltransferase engineered to generate the unnatural base 5-carboxymethylcytosine, and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities requires a novel adapter strategy employing 5-propynylcytosine, ultimately resulting in the accurate and direct detection of only 5mC via a C-to-T transition in sequencing. In performing comparisons to DM-Seq, we uncover a systematic bias in 5mC detection seen with the hybrid enzymatic-chemical TAPS sequencing approach. Furthermore, by applying DM-Seq to a human glioblastoma tumor, we demonstrate that DM-Seq, unlike bisulfite-sequencing, detects 5mC at prognostically-important CpGs, without confounding by 5-hydroxymethylcytosine. DM-Seq thus leverages unnatural DNA modifications to create the first method for direct 5mC profiling entirely using enzymes rather than chemical reagents.
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| Overall design |
DNA (no deamination), BS, DM, TAPS, or TAPS-beta sequencing experiments
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| Contributor(s) |
Wang T, Wu H, Kohli RM |
| Citation(s) |
37322153 |
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| Submission date |
Feb 23, 2023 |
| Last update date |
Jul 25, 2023 |
| Contact name |
Rahul M. Kohli |
| E-mail(s) |
kohlilab@gmail.com
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| Phone |
215-573-7523
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| Organization name |
University of Pennsylvania
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| Department |
Department of Medicine
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| Lab |
Rahul M. Kohli
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| Street address |
3610 Hamilton Walk, 502B Johnson Pavilion
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (2) |
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| This SubSeries is part of SuperSeries: |
| GSE225975 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution |
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| Relations |
| BioProject |
PRJNA938114 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE225964_RAW.tar |
282.3 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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