The Opportunities and Challenges of Molecular Tagging Next-Generation Sequencing in Liquid Biopsy

Liquid biopsy (LB) is a promising tool that is rapidly evolving as a standard of care in early and advanced stages of cancer settings. Next-generation sequencing (NGS) methods have become essential in molecular diagnostics and clinical laboratories dealing with LB analytes, i.e., cell-free DNA and RNA. The sensitivity and high-throughput capacity of NGS enable us to overcome technical issues that are mainly attributable to low-abundance (below 1% mutated allelic frequency) tumour genetic material circulating within biological fluids. In this context, the introduction of unique molecular identifiers (UMIs), also known as molecular barcodes, applied to various NGS platforms greatly improved the characterization of rare genetic alterations, as they resulted in a drastic reduction in background noise while maintaining high levels of positive predictive value and sensitivity. Different UMI strategies have been developed, such as single (e.g., safe-sequencing system, Safe-SeqS) or double (duplex-sequencing system, Duplex-Seq) strand-based labelling, and, currently, considerable results corroborate their potential implementation in a routine laboratory. Recently, the US Food and Drug Administration approved the clinical use of two comprehensive UMI-based NGS assays (FoundationOne Liquid CDx and Guardant360 CDx) in cfDNA mutational assessment. However, to definitively translate LB into clinical practice, UMI-based NGS protocols should meet certain feasibility requirements in terms of cost-effectiveness, wet laboratory performance and easy access to web-source and bioinformatic tools for downstream molecular data.