The Introduction of DNA 6mA Sequencing
DNA methylation is implicated as an epigenetic mark in various important processes in eukaryotes. 5-methylcytosine (5mC) is the most predominant DNA methylation modification in eukaryotes and has been acknowledged as the best-characterized epigenetic markers. N6-methyladenine (6mA) was previously believed to exist only in prokaryotes, unicellular eukaryotes, and plants. In recent years, 6mA in DNA has been defined as another important epigenetic and epitranscriptomic marker in higher eukaryotes. A significant decrease in 6mA levels has also been reported in a variety of cancer cells (unpublished data).
CD Genomics uses multiple strategies to validate genome-wide methylation status and abundance of individual 6mA sites. Long read sequencing developed by Pacbio and Nonapore can be used to detect 6mA in different genomes. 6mA DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is another key tool o identify regions in the genome that contain 6mA.
Long read sequencing has contributed significantly to identify the genome-wide distribution of 6mA and can achieve single-nucleotide-resolution. As the absolute abundance of 6mA is relatively low, sufficient coverage higher than 100x is necessary. SMRT sequencing allows direct detection of modified nucleotides in the DNA template, including 6mA, 5mC, and 5-hydroxymethylcytosine, and does not adversely affect the determination of primary DNA sequence. Base modifications (6mA) are identified using RS Modification and Motif Analysis.
Figure 1. Simultaneous collection of data including DNA modifications
6mA DNA immunoprecipitation followed by deep sequencing (6mA DIP-Seq) was developed from existing approaches used to detect adenosine methylation in RNA. We developed this protocol and adapted it for the detection of 6mA in DNA. In this protocol, genomic DNA is extracted, fragmented and then DNA containing 6mA is pulled down with an antibody that recognizes 6mA in genomic DNA. After subsequent washes, DNA fragments that do not contain 6mA are eliminated, and the 6mA containing fragments are eluted from the antibody in order to be processed further for subsequent analyses.
Figure 2. Illustration of 6mA DNA immunoprecipitation
- The original sequencing data
- Experimental results
- Data analysis report
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- Koziol MJ, et al. Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation. Bio Protoc. 2016 Nov 5;6(21).
- Chuan-Le Xiao, et al. Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome. Nucleic Acids Res. 2018 Dec 14;46(22):11659-11670.