# Holistic determination of ends of cfDNA molecules

## TL;DR

This paper introduces new methods to analyze both ends of cell-free DNA molecules, improving cancer detection and understanding of DNA fragmentation.

## Contribution

The study introduces 2-end and 4-end sequencing to analyze all ends of cfDNA molecules and develops new fragmentomics markers.

## Key findings

- A constellation of fragmentomics markers achieved an AUC of 0.95 for HCC detection.
- 3′ FRAGMA improved HCC detection with an AUC of 0.97.
- 4-end sequencing enhances cancer detection by analyzing all four ends of cfDNA.

## Abstract

Cell-free DNA (cfDNA) end motifs serve as fragmentomics biomarkers for cancer. Prior studies primarily focused on 5′ ends, whereas 3′ ends were overlooked due to artifactual modification in existing sequencing protocols. We utilized single-stranded library preparation (“2-end sequencing”) to assess the native 5′ and 3′ end motifs (EM5 and EM3, respectively). Additionally, we demonstrated diagnostic power from the nucleotide motifs located immediately upstream and downstream of 5′ and 3′ ends, named pre-end motifs (PREMs) and post-end motifs (POEMs). These fragmentomics markers collectively achieved an area under the curve (AUC) of 0.95 for hepatocellular carcinoma (HCC) detection. Fragmentomics-based methylation analysis of 3′ ends (3′ FRAGMA) improved detection of HCC (AUC: 0.97). We further developed “4-end sequencing” to interrogate both ends of both strands of a double-stranded cfDNA molecule, enhancing fragmentomics-based cancer detection. Holistic end profiling adds to the armamentarium of liquid biopsy and sheds light on the biology of cfDNA fragmentation.

•ssDNA sequencing library preparation enables 3′ fragmentomics analysis of cfDNA•Stem-loop-adaptor-mediated dsDNA sequencing enables holistic analysis of all four ends•A constellation of 5′, 3′, pre-, and post-end fragmentomics markers is developed•Holistic ends analysis boosts cancer detection and deepens cfDNA biological insights

ssDNA sequencing library preparation enables 3′ fragmentomics analysis of cfDNA

Stem-loop-adaptor-mediated dsDNA sequencing enables holistic analysis of all four ends

A constellation of 5′, 3′, pre-, and post-end fragmentomics markers is developed

Holistic ends analysis boosts cancer detection and deepens cfDNA biological insights

cfDNA fragmentomics is an emerging branch of liquid biopsy. Jiang et al. broke through the conventional limitation of 5′ fragmentomics to a constellation of markers involving the 3′ end, sequences flanking fragmentation sites, and all four ends of a dsDNA molecule. Holistic fragmentomics enhances diagnostic performance and deepens biological insights.