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Abstract
Cell-free DNA (cfDNA) has emerged as a powerful minimally invasive biomarker with broad applications in oncology, non-invasive prenatal testing (NIPT), transplant monitoring, infectious disease detection, and autoimmune disorders. While short-read next-generation sequencing (NGS) provides high accuracy for single nucleotide variant detection, it is limited in resolving structural variants, repetitive regions, haplotypes, and epigenetic modifications. Long-read sequencing technologies, particularly Pacific Biosciences (PacBio) Single-Molecule Real-Time (SMRT) sequencing and Oxford Nanopore Technologies (ONT), have significantly advanced cfDNA analysis by enabling direct sequencing of native DNA molecules and simultaneous detection of genetic and epigenetic features. PacBio HiFi sequencing delivers highly accurate long reads (>99.9%), making it well suited for precision oncology and centralized clinical applications. In contrast, ONT provides real-time sequencing, portability, and flexible throughput, supporting rapid diagnostics and point-of-care testing. Recent technological improvements, including enhanced nanopore chemistries, duplex sequencing, artificial intelligence–based basecalling, and the PacBio Revio platform, have further improved sequencing accuracy and efficiency. This review compares SMRT and nanopore sequencing for cfDNA analysis, focusing on read length, accuracy, throughput, cost, structural variant detection, methylation profiling, and clinical applicability. Evidence from studies published between 2016 and 2025 indicates that these technologies are complementary rather than competitive. Integrated sequencing approaches combining the high accuracy of SMRT with the speed and accessibility of nanopore platforms may represent the future of cfDNA diagnostics. Key challenges, including cfDNA fragmentation, low-input samples, bioinformatic complexity, and the need for standardized workflows, remain critical considerations for clinical implementation.