Huiwen Che1,2,3, Peiyong Jiang1,2,3,4, L.Y. Lois Choy1,2,3,4, Suk Hang Cheng1,2,3, Wenlei Peng1,2,3, Rebecca W.Y. Chan1,2,3, Jing Liu1,2,3, Qing Zhou1,2,3, W.K. Jacky Lam1,2,3,4, Stephanie C.Y. Yu1,2,3, So Ling Lau5, Tak Y. Leung5, John Wong6, Vincent Wai-Sun Wong7, Grace L.H. Wong7, Stephen L. Chan4,8, K.C. Allen Chan1,2,3,4 and Y.M. Dennis Lo1,2,3,4 1Centre for Novostics, Hong Kong Science Park, Pak Shek Kok, Hong Kong SAR, China; 2Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; 3Department of Chemical Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; 4State Key Laboratory of Translational Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; 5Department of Obstetrics and Gynecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; 6Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; 7Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China; 8Department of Clinical Oncology, Sir Y.K. Pao Centre for Cancer, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Corresponding author: loymcuhk.edu.hk Abstract

Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's r = 0.71 vs. −0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in Dffb-deficient mice but higher in Dnase1l3-deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.278556.123.

Freely available online through the Genome Research Open Access option.

Received September 26, 2023. Accepted February 14, 2024.