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Long-read Sequencing of cfDNA from Patient-derived Liquid Biopsies

By Stuart P. Atkinson, Ph.D.

April 8, 2025

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Introduction: Short‐read Sequencing of Cell‐free DNA Leads the Way

Cell‐free DNA (cfDNA) represents a component of liquid biopsies (any body fluid sampled from patients) whose isolation and genetic/epigenetic analysis can support the management of diseases such as cancer, including diagnosis, prognosis, analysis of therapeutic responses, and monitoring possible disease recurrence. cfDNA analysis generally employs short‐read DNA sequencing, a commonly applied form of next-generation sequencing; however, this approach lacks portability/adaptability and speed and possesses an inherent bias towards short cfDNA fragments. This type of cfDNA analysis also requires complex and expensive sequencing platforms often shared by entire institute, which typically leads to extended waiting times between sample isolation and data visualization, with such delays likely to impact ongoing patient care.

In a recent EMBO Molecular Medicine study (van der Pol et al.), researchers from the laboratory of Florent Mouliere (University of Manchester) explored whether long-read sequencing using an Oxford Nanopore Technologies (ONT) platform could provide sufficient relevant data from cfDNA in liquid biopsies and, as such, represent a potentially quicker, cheaper, and more applicable means of managing cancer patients. ONT long-read sequencing can extract a broader range of genomic, epigenomic, and transcriptomic features from the same sequencing run compared to more specialized methods (Euskirchen et al., Katsman et al.).

The potential application of ONT long-read sequencing to cfDNA analysis had remained relatively unexplored until no due to a reported higher rate of sequencing errors compared to short‐read sequencing (Cheng et al., Marcozzi et al.); however, more recent studies have revealed that ONT sequencing of blood plasma cfDNA can generate viable data regarding genomic events such as copy number aberrations (a common type of somatic mutation in cancer involving the deletion/amplification of large contiguous genome segments) (Martignano et al.).

Excitingly, the new study of van der Pol et al. now provides support for ONT long-read sequencing as a cost‐effective, rapid, and adaptable platform for the accurate analysis of cfDNA from liquid biopsies, which may offer near real-time data to support the effective management of cancer patients in a wide range of clinical settings.

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Oxford Nanopore Technologies Long-read Sequencing of Cell-Free DNA: A Rapid and Effective Alternative?

The ONT long-read sequencing approach supported the determination of genomic copy number aberrations and cfDNA fragmentation patterns less than 24 hours after the initial collection of the liquid biopsies (including ~18 hours of computation), which comprised blood plasma samples from lung cancer patients and urine samples from bladder cancer patients (alongside healthy controls). For this analysis, the team employed the MinION ONT platform, a cutting-edge, palm-sized sequencer that enables real-time sequencing in diverse environments, alongside an aptly named and newly designed computational tool (ITSFASTR or InTegrated Sequence and Fragmentome AnalysiS Time Reduction) that reproducibly analyzes genomic and fragmentomic patterns. While speed and portability remain critical advantages, the relative cost-effectiveness of the MinION ONT sequencing platform may support the implementation of this approach in low‐ to middle‐income countries, where improved cancer management could have an outsized impact. Encouragingly, comparative experiments provided evidence for similar results between short‐ and long‐read sequencing data; additionally, the authors revealed that the integration of improved computing technology could shorten the process to around 7 hours (Wick, Judd, and Holt), a significantly shorter time compared to the theoretical (~24-84 h) and in-practice (~34 days) times required to generate data on the same samples via short‐read sequencing.

A subsequent analysis of the tumor‐derived cfDNA fraction from blood plasma (lung cancer patients) and urine (bladder cancer patients) samples correlated highly with the same values calculated via short‐read sequencing, suggesting equivalent sensitivity. Analysis of the cfDNA size profile, fragmentation pattern, fragment‐end composition, and nucleosome profiling near transcription start sites in plasma and urine samples by ONT long-read sequencing displayed features typical of cfDNA. Of note, fragmentation‐based nucleosome profiling from plasma samples represents a promising alternative to more conventional, labor‐intensive means of deconvoluting chromatin states (Snyder et al., Lo et al.), while nucleosome profiling can enable tissue‐of‐origin deconvolution from plasma samples of high‐depth whole genome sequencing data (Snyder et al., Zhu et al.). ONT long-read sequencing also recovered a high proportion of long tumor‐derived cfDNA fragments (over 300 bp) from plasma and urine samples, as confirmed in human and xenograft (human tumors in a mouse model) samples and contrary to the common belief that short and fragmented cfDNA predominates. The analysis of longer cfDNA fragments can offer a more significant amount of information when compared to shorter fragments in the context of scarce tumor signals (Suzuki et al.).

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Going Long into the Future of Cell-free DNA Analysis?

This exciting study highlights ONT long-read sequencing as a cost‐effective, rapid, and portable approach to accurately analyzing cfDNA from liquid biopsies that provides information on copy number aberrations, tumor fraction, and long cfDNA fragments, which may support cancer patient management. Moving forward, the authors hoped to validate their findings in larger cohorts of liquid biopsies, implement improvements to the recovery of single nucleotide variants from plasma cfDNA via ONT long-read sequencing (Marcozzi et al.), and perhaps explore additional ONT platforms that permit deeper sequencing coverage to track minimal residual disease.

To discover more about this exciting line of research from the Florent Mouliere laboratory, check out their recent studies that compared cfDNA and small extracellular-vesicle-associated DNA in lung cancer patients (Moldovan et al.), revealed how cell-free DNA genomic and fragmentomic data may improve cancer patient survival and recurrence analysis (Moldovan et al.), and described a new means of profiling cfDNA profiling in cancer patients (Chen et al.).

For more on how long-read sequencing of cfDNA from liquid biopsies may support improved cancer patient care, check out EMBO Molecular Medicine, December 2023.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

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