Distinct Features of Plasma Ultrashort Single-Stranded Cell-Free DNA as Biomarkers for Lung Cancer Detection

Jordan Cheng, Neeti Swarup, Feng Li, Misagh Kordi, Chien Chung Lin, Szu Chun Yang, Wei Lun Huang, Mohammad Aziz, Yong Kim, David Chia, Yu Min Yeh, Fang Wei, David Zheng, Liying Zhang, Matteo Pellegrini, Wu Chou Su, David T.W. Wong

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


BACKGROUND: Using broad range cell-free DNA sequencing (BRcfDNA-Seq), a nontargeted next-generation sequencing (NGS) methodology, we previously identified a novel class of approximately 50 nt ultrashort single-stranded cell-free DNA (uscfDNA) in plasma that is distinctly different from 167 bp mononucleosomal cell-free DNA (mncfDNA). We hypothesize that uscfDNA possesses characteristics that are useful for disease detection. METHODS: Using BRcfDNA-Seq, we examined both cfDNA populations in the plasma of 18 noncancer controls and 14 patients with late-stage nonsmall cell lung carcinoma (NSCLC). In comparison to mncfDNA, we assessed whether functional element (FE) peaks, fragmentomics, end-motifs, and G-Quadruplex (G-Quad) signatures could be useful features of uscfDNA for NSCLC determination. RESULTS: In noncancer participants, compared to mncfDNA, uscfDNA fragments showed a 45.2-fold increased tendency to form FE peaks (enriched in promoter, intronic, and exonic regions), demonstrated a distinct end-motif-frequency profile, and presented with a 4.9-fold increase in G-Quad signatures. Within NSCLC participants, only the uscfDNA population had discoverable FE peak candidates. Additionally, uscfDNA showcased different end-motif-frequency candidates distinct from mncfDNA. Although both cfDNA populations showed increased fragmentation in NSCLC, the G-Quad signatures were more discriminatory in uscfDNA. Compilation of cfDNA features using principal component analysis revealed that the first 5 principal components of both cfDNA subtypes had a cumulative explained variance of >80%. CONCLUSIONS: These observations indicate that the distinct biological processes of uscfDNA and that FE peaks, fragmentomics, end-motifs, and G-Quad signatures are uscfDNA features with promising biomarker potential. These findings further justify its exploration as a distinct class of biomarker to augment pre-existing liquid biopsy approaches.

Original languageEnglish
Pages (from-to)1270-1282
Number of pages13
JournalClinical Chemistry
Issue number11
Publication statusPublished - 2023 Nov 1

All Science Journal Classification (ASJC) codes

  • General Medicine


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