Abstract 3739: EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA

Abstract

Abstract The study of DNA methylation from cell-free DNA (cfDNA) and DNA extracted from formalin fixed paraffin embedded (FFPE) tissues are notoriously difficult. The use of these DNA inputs is becoming increasingly important in the field of cancer diagnostics. The cytosine modifications, 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC), are key epigenetic factors that play an important role in cellular processes and their mis-regulation results in diseased states like cancer. Advances in the study of these epigenetic factors in combination with improvements in sample preparation have enabled cancer biomarker identification based on methylation profiling. The historical gold standard for detecting cytosine methylation has been bisulfite sequencing. This method uses chemical conversion of cytosines into uracils and has been used for both targeted and whole genome methylation analysis. However, bisulfite conversion leads to DNA damage which results in shorter DNA insert sizes as well as biases in the data. It is challenging to make bisulfite libraries from cfDNA and FFPE DNA as typically the DNA is of low quantity and quality. We developed an enzyme-based methylation detection technology (EM-seq) to address the flaws inherent to bisulfite sequencing. This method, primarily due to the intact nature of DNA after conversion, results in better quality libraries with longer insert sizes, lower duplication rates and minimal GC bias. EM-seq libraries prepared from cfDNA and lung FFPE DNA had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to bisulfite libraries. EM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. EM-seq’s superior sequencing metrics establish it as a standard method for robust methylation profiling, particularly for these types of challenging DNA samples. Citation Format: Louise Williams. EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3739.