通过广谱无细胞DNA测序（BRcfDNA-Seq），我们之前在血浆中识别出一类新颖的约50nt超短单链无细胞DNA（uscfDNA），该DNA与约167bp单核体无细胞DNA（mncfDNA）有明显差异。我们假设uscfDNA具有用于疾病检测的特性。使用BRcfDNA-Seq，我们检测了18名非癌症对照者和14名晚期非小细胞性肺癌（NSCLC）患者的血浆中的两种cfDNA群体。与mncfDNA相比，我们评估了功能元素（FE）峰值、片段组学、末端基序和G-四连体（G-Quad）特征是否对NSCLC的鉴定具有有用性。在非癌症参与者中，与mncfDNA相比，uscfDNA片段显示出形成FE峰值的趋势增加了45.2倍（富集在启动子、内含子和外显子区域），展示了独特的末端基序频率特征，并表现出4.9倍的G-四连体签名增加。在NSCLC参与者中，只有uscfDNA群体中发现了FE峰值候选物。此外，uscfDNA展示了与mncfDNA不同的末端基序频率候选物。尽管两种cfDNA群体在NSCLC中都显示出增加的片段化，但G-四连体签名在uscfDNA中具有更好的区分能力。使用主成分分析组合cfDNA特征显示，两种cfDNA亚型的前5个主成分的累计解释方差超过80%。这些观察结果表明，uscfDNA具有不同的生物学过程，并且FE峰值、片段组学、末端基序和G-四连体签名是具有有前景的生物标记物潜力的uscfDNA特征。这些发现进一步证明其作为一类独特的生物标志物的探索，以增强已有的液体活组织检查方法。© Association for Diagnostics & Laboratory Medicine 2023。保留所有权利。有关权限，请发送电子邮件至: journals.permissions@oup.com。

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.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.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%.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.© Association for Diagnostics & Laboratory Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.