Analysis of Ultrashort Single-Stranded Cell-Free DNA in Patient Saliva for Disease Detection (UCLA Cases 2022-292, 2022-043)

UCLA researchers have developed a novel next-generation sequencing (NGS) pipeline designed specifically to detect ultrashort single-stranded cell-free DNA (uscfDNA), providing significant advancements in sensitivity, clinical utility, and genomic research applications.

Background: Cell-free DNA (cfDNA) fragments circulate in body fluids and have become vital biomarkers for a wide array of diagnostic and prognostic applications, especially in oncology, prenatal testing, and organ transplantation. Traditional NGS methods efficiently capture double-stranded and longer cfDNA, but often fail to detect and analyze ultrashort single-stranded fragments, which are increasingly recognized as crucial diagnostic signals for studying tumor heterogeneity, early disease states, and physiological changes.

Innovation:

Researchers led by Professor David T. Wong in the UCLA School of Dentistry have developed a specialized NGS pipeline tailored for ultrashort single-stranded cell-free DNA. Key components and processes include:

• Optimized extraction protocols that preserve the integrity of ultrashort ssDNA fragments.
• Molecular tagging and adaptor ligation that efficiently prepare ultrashort sscfDNA for sequencing, overcoming issues that plague standard library preparation methods.
• Advanced amplification and sequencing steps, designed to maintain fidelity and maximize recovery of these fragments, leveraging modified enzymes and bespoke reagents to prevent loss and bias.

Advantages:

• Enhanced sensitivity for detecting disease-associated DNA fragments, especially in early-stage cancers or low-volume samples, allowing earlier and more accurate diagnoses.
• Expanded clinical utility, opening new pathways for minimally invasive liquid biopsies, monitoring of disease progression, and personalized medicine strategies by enabling detection of previously inaccessible molecular markers.
• Superior specificity and data quality in genomic research, leading to deeper insights into cell-free DNA biology, disease mechanisms, and novel biomarker discovery.

Conclusion: This innovation advances the field of molecular diagnostics and genomics by enabling robust detection and sequencing of ultrashort single-stranded cell-free DNA. Its specialized pipeline addresses longstanding technical barriers, delivers substantial improvements in sensitivity and specificity, and paves the way for broader adoption of liquid biopsy and precision medicine applications.