PROJECT SUMMARY/ABSTRACT Lung cancer remains the leading cause of cancer-related deaths in the U.S. and worldwide. Non–small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Early detection of indeterminate pulmonary nodules (IPNs) with chest low dose computed tomography (LDCT) screening followed by effective treatments can reduce mortality by 20% relative to chest radiography. However, the high false positive rate finding as >95% limits the application. The unmet clinical need for early diagnosis is the lack of a noninvasive test that can be applied to individuals with CT-detected lung nodules and reliably discriminates between malignant or benign nodules. Liquid biopsy focused on the characterization of tumor-associated genetic alterations in cell free circulating tumor DNA can non-invasively profile the molecular landscape of solid tumors. However, the low biological concentrations of ctDNA, low frequency of somatic mutations and the confounding impact of clonal hematopoiesis-related variants in early-stage lung cancer limit the sensitivity of ctDNA-based liquid biopsy assays. It would be essential if additional cell free DNA biomarkers could be included to allow the development of more sensitive molecular diagnostics approaches for the early assessment of lung cancer. We have discovered a distinct population of ultra-short single-stranded cell-free DNA (uscf/ctDNA) with a size of 40-70nt in healthy and NSCLC plasma. Our preliminary data showed that the fragmentomic features, including functional element profile, fragmentation patterns and end motifs of uscfDNA molecules, can distinguish NSCLC patients from healthy donors. This R21 application is to explore and test our hypothesis that the fragmentomic features of uscfDNA molecules can serve as novel biomarkers to differentiate NSCLC patients from non-cancer subjects with IPNs and allow more sensitive liquid biopsy molecular diagnostics for early NSCLC detection. Two specific aims are in place for hypothesis testing. Aim 1 is to develop a predictive model using uscfDNA-seq assay on uscfDNA fragmentomic analysis for liquid biopsy of NSCLC. Aim 2 is to pre-validate uscfDNA-Seq test for the early detection of NSCLC. Together, the translational and pre-validation, targeting uscfDNA for early detection of NSCLC can break new ground and extend previous discoveries towards impactful new directions and clinical applications.