Single-Molecule Sequencing Enables Long Cell-Free DNA Detection and Direct Methylation Analysis for Cancer Patients

Abstract

Background Analysis of circulating tumor DNA has become increasingly important as a tool for cancer care. However, the focus of previous studies has been on short fragments of DNA. Also, bisulfite sequencing, a conventional approach for methylation analysis, causes DNA degradation, which is not ideal for the assessment of long DNA properties and methylation patterns. This study attempted to overcome such obstacles by single-molecule sequencing. Methods Single-molecule real-time (SMRT) sequencing was used to sequence plasma DNA. We performed fragment size and direct methylation analysis for each molecule. A methylation score concerning single-molecule methylation patterns was used for cancer detection. Results A substantial proportion of plasma DNA was longer than 1 kb with a median of 16% in hepatocellular carcinoma (HCC) patients, hepatitis B virus carriers and healthy individuals. The longest plasma DNA molecule in the HCC patients was 39.8 kb. Tumoral cell-free DNA (cfDNA) was generally shorter than nontumoral cfDNA. The longest tumoral cfDNA was 13.6 kb. Tumoral cfDNA had lower methylation levels compared with nontumoral cfDNA (median: 59.3% versus 76.9%). We developed and analyzed a metric reflecting single-molecule methylation patterns associated with cancer, named the HCC methylation score. HCC patients displayed significantly higher HCC methylation scores than those without HCC. Interestingly, compared to using short cfDNA (Area-Under-the-Curve, AUC: 0.75), the use of long cfDNA molecules greatly enhanced the discriminatory power (AUC: 0.91). Conclusions A previously unidentified long cfDNA population was revealed in cancer patients. The presence and direct methylation analysis of these molecules open new possibilities for cancer liquid biopsy.

Sequence-Based Platforms for Discovering Biomarkers in Liquid Biopsy of Non-Small-Cell Lung Cancer

“…Long-read sequencing enables the analysis of structural properties and the fragmentation patterns of cfDNA (including fragment size, nucleosome footprinting and methylation patterns), which can give information about their tissues of origin. This is known as “fragmentomics” [ 128 , 128 , 129 , 130 ], which we discuss further below. Long-read sequencing technologies (PacBio, ONT) were recently compared for real-time detection of long cfDNA in plasma.…”

Cell-Free DNA Fragmentomics: The Novel Promising Biomarker

“…Recently, the team of Dennis Lo revealed a large number of long fragments (fragment length > 500 bp) of cfDNA in maternal plasma using single-molecule sequencing technology [ 51 ]. In 2022, their team also examined long cfDNA in cancer patients using SMRT-seq and found the longest tumor cfDNA was 13.6 kb [ 52 ]. In addition to the discovery of long cfDNA, Hudecova et al identified a large number of single-stranded cfDNA of about 50 bp using the combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA).…”

New Perspectives on the Importance of Cell-Free DNA Biology

“…The presence of these size populations is generally ascribed to apoptotic cleavage of chromatin. However, there is a growing number of studies reporting on the presence of HMW cfDNA present in body fluids [ 449 , 450 , 451 , 452 , 453 , 454 , 455 , 456 , 457 ] and cell cultures [ 305 , 414 , 415 , 416 , 417 , 430 , 458 , 459 ]. These HMW fragments may originate from apoptosis but may also be derived from many other release mechanisms, such as accidental cell lysis, necrosis, NETosis, regulated release through extracellular vesicles, and so forth.…”