5mC and 5hmC methylation sequencing: the power of 6-base sequencing in a multiomic era

The integration of genetic and epigenetic information is essential for a comprehensive understanding of genome function and regulation. Traditional sequencing methods often fall short in capturing both genetic variants and epigenetic modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) simultaneously. Recent advances in 6-base sequencing have enabled the simultaneous, base-resolution detection of canonical bases and key cytosine modifications in a single workflow. This review explores the biological significance of 5mC and 5hmC, discusses current methods to achieve 6-base sequencing, and highlights recent applications in academic and clinical settings.