Abstract B026: NEBNext® enzymatic solutions for DNA methylation profiling at picogram scale

Abstract DNA methylation is an important epigenetic regulator of gene expression. 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) play distinct roles in epigenetic regulation, however the ability to distinguish between 5mC and 5hmC is often lost as they are frequently read as a combined signal in traditional sequencing approaches. NEBNext Enzymatic Methyl-seq v2 (EM-seq v2) and NEBNext Enzymatic 5hmC-seq (E5hmC-seq) are enzymatic approaches used to investigate cytosine methylation. EM-seq v2 detects both 5mC and 5hmC whereas E5hmC-seq detects only 5hmC. The first step for each method protects either 5mC and/or 5hmC from deamination by APOBEC enzyme. Cytosines are deaminated to uracil in EM-seq v2 whereas for E5hmC-seq, 5mC and cytosine bases are converted to thymine and uracil respectively. Using a subtractive data analysis approach, whereby E5hmC-seq data is subtracted from EM-seq data, the locations of 5mC and 5hmC can be decoupled at single base resolution. EM-seq v2 libraries and E5hmC-seq libraries were prepared using 0.1 ng to 200 ng of genomic DNA. Both methods leave DNA intact and result in superior sequencing libraries with longer insert sizes, lower duplication rates and minimal GC bias as well as accurately detecting methylation down to 0.1 ng DNA. These methods are streamlined due to reduced library preparation time as well as the removal of a cleanup step. Coverage of genomic features such as CpG islands and enhancers, for both methods were maintained to 1 ng but dropped as expected with the 0.1 ng inputs. EM-seq v2 and E5hmC-seq provides consistent methylation metrics for inputs ranging from 0.1 ng to 200 ng. EM-seq v2 and E5hmC-seq libraries provide accurate measurements across inputs from 0.1 ng – 200 ng with expected methylation, insert sizes and minimal GC bias. In addition, combining E5hmC-seq and EM-seq data to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease. Citation Format: Vaishnavi panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Matthew Campbell, Laura Blum, Bradley Langhorst, Louise Williams. NEBNext® enzymatic solutions for DNA methylation profiling at picogram scale [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B026.