LAUSR

Abstract Background Aberrant methylation is pervasive in cancer and a promising source of diagnostic and predictive biomarkers. Bisulfite conversion, the current standard for detecting methylated cytosines, requires higher DNA input requirements due to destruction of DNA, and is problematic for the low cell-free DNA (cfDNA) recovery from plasma. Enzymatic methyl-seq (EM-Seq) uses enzymatic conversion in place of bisulfite. Here, we compare quality metrics and cancer-associated signals of whole-genome sequenced libraries with bisulfite conversion (WGBS) and EM-Seq. Methods We collected blood and isolated cfDNA from 7 healthy controls and 15 participants with cancer. Paired libraries from each participant were prepared using WGBS and EM-seq, were sequenced to a median depth of 1.7x, and processed using a custom bioinformatics pipeline. Results The quality of data produced by both methods was largely comparable, including highly correlated methylation levels across CpG islands (average Pearson R = 0.97). However, EM-seq performed better on a number of technical metrics including: higher conversion efficiency, increased alignment quality, and coverage. For library pairs where CNAs were detectable (n = 7), the assessed tumor fractions were similar between the two methods, with 6 out of 7 having estimates within 1.15 fold. WGBS libraries showed evidence of DNA damage, including consistently shorter fragments (7.9 ± 2.1 bp shorter mean length in WGBS relative to paired EM-Seq) and the requirement of more amplification cycles (12 vs 8). Like standard whole genome sequencing, control EM-seq libraries had less variability in genome-wide coverage distributions and preserved the canonical cfDNA fragment length distribution. Conclusions Our results show that both approaches produce concordant estimates of methylation levels throughout the genome. However, EM-seq demonstrates a number of advantages, including higher performance in key quality metrics. We conclude that EM-Seq is a suitable method for assessing plasma cell-free methylome in a cancer setting and avoiding bisulfite-associated DNA degradation. Legal entity responsible for the study Lexent Bio Inc. Funding Lexent Bio. Disclosure N. Lambert: Full/Part-time employment: Lexent Bio Inc. A. Robertson: Full/Part-time employment: Lexent Bio Inc. R. Srivas: Full/Part-time employment: Lexent Bio Inc. N. Peterman: Full/Part-time employment: Lexent Bio Inc. J. Close: Full/Part-time employment: Lexent Bio Inc. T. Wilson: Full/Part-time employment: Lexent Bio Inc. P. George: Full/Part-time employment: Lexent Bio Inc. H. Wood: Full/Part-time employment: Lexent Bio Inc. B. Wong: Full/Part-time employment: Lexent Bio Inc. A. Tezcan: Full/Part-time employment: Lexent Bio Inc. H. Tezcan: Full/Part-time employment: Lexent Bio Inc.