The present invention relates to a method for analyzing DNA including forming labeled DNA fragments by cleaving genomic DNA into DNA fragments, selectively functionalizing any non-methylated CpG sites present in the DNA with a linker including a hydrolyzable moiety, and attaching a label to the linker. The method further includes the step of separating the labeled DNA fragments from any non-labeled DNA fragments, hydrolyzing the hydrolyzable moiety of the linker of the separated labeled DNA fragments so as to release the DNA fragments from the label, and sequencing the released DNA fragments.

The present invention relates to a method for analyzing DNA including forming labeled DNA fragments by cleaving genomic DNA into DNA fragments, selectively functionalizing any non-methylated CpG sites present in the DNA with a linker including a hydrolyzable moiety, and attaching a label to the linker. The method further includes the step of separating the labeled DNA fragments from any non-labeled DNA fragments, hydrolyzing the hydrolyzable moiety of the linker of the separated labeled DNA fragments so as to release the DNA fragments from the label, and sequencing the released DNA fragments.

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

Aspects of the present disclosure relate to methods for modification and detection of methylated nucleotides. Embodiments are directed to detection of RNA methylation. Disclosed are methods and compositions for transcriptome-wide detection of N.sup.6-methyladenosine in mRNA. In some cases, methods for modifying a methylated nitrogenous base are described. Also disclosed are enzymes and other molecules useful for RNA methylation detection.

Aspects of the present disclosure relate to methods for modification and detection of methylated nucleotides. Embodiments are directed to detection of RNA methylation. Disclosed are methods and compositions for transcriptome-wide detection of N.sup.6-methyladenosine in mRNA. In some cases, methods for modifying a methylated nitrogenous base are described. Also disclosed are enzymes and other molecules useful for RNA methylation detection.

Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Provided herein are methods, systems, and compositions for determining a base in a polynucleotide. In various aspects, the methods, systems, and compositions presented herein are useful for performing 4-base, 5-base, or 6-base sequencing of polynucleotide molecules, for example, from liquid biopsy samples or wherein the base is a low frequency mutation.

Compositions and methods for carboxymethylation of cytosine containing DNA and applications thereof for direct sequencing of 5mC are disclosed.

Compositions and methods for carboxymethylation of cytosine containing DNA and applications thereof for direct sequencing of 5mC are disclosed.