Journal: Clinical Epigenetics

Article Title: Single-molecule quantification of 5-hydroxymethylcytosine for diagnosis of blood and colon cancers

doi: 10.1186/s13148-017-0368-9

Figure Lengend Snippet: Schematic representation of the two-step 5hmC labelling reaction. a First, T4 β-GT enzymatic glucosylation of 5hmC with UDP-6-N 3 -Glu is performed. b Next, click reaction between the N 3 group and the fluorescently labelled alkyne DBCO-Cy5 is performed. This two-step reaction results in fluorescently labelled 5hmC

Article Snippet: Three hundred nanograms of genomic DNA was mixed with 3 μl of buffer 4 (NEB), 0.5 μl of UDP-6-N 3 -Glu (0.3 mM, [ ]), 2 μl of T4 bacteriophage β-glucosyltransferase (T4-BGT, NEB) and ultrapure water to a final volume of 30 μl.

Techniques:

Journal: Genome Research

Article Title: Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution

doi: 10.1101/gr.265306.120

Figure Lengend Snippet: 5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC [CC], CpG [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.

Article Snippet: Enzymatic deamination for 5hmC detection For 5hmC detection, 200 ng of mouse E14 genomic DNA was mixed with 10 ng unmethylated lambda DNA, 10 ng of T4gt phage DNA, and 1 ng of CpG methylated pUC19 DNA and then incubated with 20 U of BGT (NEB) in 1× NEBuffer 2 for 2 h at 37°C.

Techniques: Amplification, Sequencing, Methylation, Binding Assay, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Genome Research

Article Title: Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA

doi: 10.1101/gr.266551.120

Figure Lengend Snippet: Enzymes involved in the detection of 5mC and 5hmC. ( A ) TET2 catalyzes the oxidization of 5mC to 5hmC, 5fC, and 5caC in three consecutive steps. ( B ) The T4-phage enzyme T4-BGT glucosylates pre-existing genomic 5hmC as well as 5hmC formed by the action of TET2 to 5-(β-glucosyloxymethyl)cytosine (5gmC). ( C ) APOBEC3A deaminates cytosine, 5mC, and, to a lesser extent, 5hmC.

Article Snippet: The enzymes mTET2CDΔ (TET2) , APOBEC3A (NEB), and T4-BGT (NEB) were characterized to determine their suitability for detecting cytosine methylation.

Techniques:

Journal: Genome Research

Article Title: Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA

doi: 10.1101/gr.266551.120

Figure Lengend Snippet: Enzymatic methyl-seq (EM-seq) mechanism of action and workflow. ( A ) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using EM-seq. The actions of TET2 (blue) and T4-BGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC, and 5caC are shown. The red cross represents no APOBEC3A activity. T4-BGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Supplemental Fig. 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. ( B ) DNA is sheared to ∼300 bp, end repaired, and 3′-A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-BGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.

Article Snippet: The enzymes mTET2CDΔ (TET2) , APOBEC3A (NEB), and T4-BGT (NEB) were characterized to determine their suitability for detecting cytosine methylation.

Techniques: Activity Assay, Sequencing, Amplification