BRIEF RESEARCH REPORT article
Front. Genet.
Sec. Human and Medical Genomics
Fragmenting the Future with FLARE: A Comprehensive Fragmentomics Pipeline Based on Long-read Nanopore Sequencing
Marica Ficorilli
Marta Lucchetta
Deborah Lenoci
Ilenia Rolli
Nicole Farina
Valentina Cristofaro
Lorenzo Giannini
Arianna Ottini
Alberto Deganello
Stefano Cavalieri
Lisa Francesca Licitra
Loris De Cecco
National Cancer Institute Foundation (IRCCS), Milan, Italy
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Abstract
Purpose: Cell-free DNA (cfDNA) fragmentation patterns carry biological information beyond fragment length, revealing nuclease activity, chromatin organization, and tissue of origin. Fragmentomics has emerged as a powerful approach to improve circulating tumor DNA (ctDNA) detection, particularly at low tumor fractions. However, most current methods are designed for short-read sequencing, limiting their applicability to third-generation technologies. Here, we present FLARE (Fragmentation and Long-read Analysis of Regulatory Epigenetics), an integrated fragmentomics pipeline optimized for Oxford Nanopore long-read sequencing. Methods: FLARE preserves native cfDNA fragment ends and integrates copy number profiling, tumor fraction estimation, sequence-specific end-motif analysis, and methylation-based features to enable comprehensive characterization of cfDNA fragmentation. Plasma cfDNA from six patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) treated with nivolumab was analyzed at baseline (C1D1) and during therapy (C5D1). Results: Genome-wide copy number analysis revealed recurrent chromosomal alterations consistent with HNSCC biology, with ichorCNA-derived tumor fractions ranging from 0% to 12.8%. Tumor fraction estimates derived from methylation-based fragmentomic features showed concordant trends, providing an independent measure of tumor burden and correlating with clinical response. End-motif analysis based on 5′ 4-mer frequencies, combined with non-negative matrix factorization (NMF), identified predominant A-end and G-end patterns, consistent with apoptosis-associated nuclease activity. Conclusion: FLARE provides a robust and scalable framework for fragmentomic analysis using long-read sequencing, enabling simultaneous investigation of structural and sequence-level cfDNA features. This approach demonstrates the technical feasibility of integrated fragmentomic analyses on Nanopore cfDNA and supports future integration of native methylation and transcription factor binding site analyses.
Summary
Keywords
cfDNA fragmentation, Copy number profiling, Flare, Fragmentation and Long-read Analysis of Regulatory Epigenetics, fragmentomics pipeline, Head and neck squamous cell carcinoma, HNSCC, long-read nanopore sequencing
Received
22 January 2026
Accepted
18 February 2026
Copyright
© 2026 Ficorilli, Lucchetta, Lenoci, Rolli, Farina, Cristofaro, Giannini, Ottini, Deganello, Cavalieri, Licitra and De Cecco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Loris De Cecco
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