Abstract 2099: EM-seq enables accurate and precise methylome analysis of challenging DNA samples

Abstract

Abstract DNA isolated from blood draws (cell-free DNA (cfDNA)) or from archival material like formalin fixed paraffin embedded (FFPE) tissues have advanced the field of cancer genetics. DNA methylation (5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC)) is a key epigenetic factor that plays an important role in cellular processes and it's misregulation results in diseased states like cancer. Advances in the field of sample preparation from biological matrices and genomics have enabled cancer biomarker identification based on methylation profiling. Bisulfite sequencing has been the gold standard for detection of methylation and is employed for both targeted and whole genome methylation analysis. However, the chemical based bisulfite conversion of cytosines to uracils leads to DNA damage which subsequently translates to shorter DNA insert sizes as well as biases in the data. DNA obtained from cfDNA and FFPE samples is typically of low quality and quantity making DNA methylation profiling using the harsh bisulfite conversion challenging. Robust biomarker detection relies primarily on the ability to profile methylation accurately. To overcome the drawbacks of bisulfite sequencing, we developed an enzyme based methylation detection technology, called NEBNext® Enzymatic Methyl-Seq (EM-Seq™), that minimizes damage to DNA enabling longer insert sizes, lower duplication rates and minimal GC bias resulting in more accurate quantification of methylation in the sample DNA. Using EM-Seq, we profiled cfDNA and lung FFPE DNA. Results for these challenging DNA types showed that the EM-Seq libraries had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to WGBS libraries. EM-seq libraries also identified a higher number of CpG's with even coverage facilitating accurate methylation calling. In conclusion, EM-Seq libraries have superior sequencing metrics resulting in robust methylation profiling for these types of challenging DNA samples. Citation Format: V K Chaithanya Ponnaluri, Louise Williams, Matthew A. Campbell, Vaishnavi Panchapakesa, Romualdas Vaisvila, Bradley Langhorst, Eileen Dimalanta, Theodore B. Davis. EM-seq enables accurate and precise methylome analysis of challenging DNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2099.