The importance of high analytical sensitivity and specificity of 5 and 6-base assays to enhance the detection of ctDNA in liquid biopsy applications

Sensitive detection of cancer-derived DNA fragments (ctDNA) within cell-free DNA (cfDNA) in liquid biopsy is essential for identifying early-stage cancers, monitoring treatment response and minimal residual disease. A major challenge in these applications is detecting ctDNA, indicative of disease state, when this represents a small fraction of the total cfDNA. Advances in methylomic profiling, including 6-base sequencing with duet evoC which distinguishes 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), have enabled high-sensitivity ctDNA detection by analyzing methylation patterns across individual sequencing fragments (fragment level analysis) as opposed to more traditional analyses that average methylation levels at individual CpG loci. We present a comparative analysis of the background error rates in fragment-level methylation analysis between the duet suite of methylation assays from biomodal and alternative methylation sequencing technologies, showing error rates below 10 -5 for both biomodal assays compared to error rates above 10 -4 for alternative technologies. We further show how this lower error rate can enable more sensitive detection of ctDNA, and present the application of these technologies to detecting ctDNA in cfDNA from patients diagnosed with stages I-IV colorectal cancer. Our findings further emphasize the power of fragment-level analyses in cfDNA-based applications. They illustrate that it is critical to carefully consider analytical performance to achieve maximum clinical sensitivity and specificity from limited cfDNA samples.