PROJECT SUMMARY/ABSTRACT Circulating tumor DNA (ctDNA) which mirrors the parental tumor, is present in the plasma of non-small cell lung carcinoma (NSCLC) patients. Liquid biopsy analysis of ctDNA allows early detection and genetic monitoring. However, current PCR-based liquid biopsy methods often have difficulty detecting ctDNA due to low copies in circulation. In contrast, the electric field-induced release and measurement liquid biopsy (eLB) platform, detects mutant EGFR (L858R, exon19del, T790M) ctDNA with a >90% tissue-genotype concordance in <50ul of unprocessed plasma of NSCLC patients. Preliminary data demonstrate that the ctDNA detected by eLB may be ultrashort single-stranded ctDNA (usssctDNA) which contrasts the typically described 160-basepair internucleosomal double-stranded ctDNA. This new subpopulation is undetectable through PCR-based liquid biopsy methods due to size-selection bias. The goal of the F99 Phase is to use usssctDNA-seq, a novel NGS pipeline, to explore if NSCLC plasma contains high quantities of ctDNA that are ultrashort and single-stranded. This pipeline consists of a usssctDNA-specific extraction, library preparation, deep sequencing, and bioinformatic analysis. To verify, 80 late-stage NSCLC EGFR-mutated plasma samples will undergo usssctDNA-seq. The discovery of usssctDNA will open up a wealth of previously unnoticed information leading to improved treatment and survival of NSCLC patients. Afterward, during the K00 Phase, the trainee hopes to explore the biological origins of biofluid ctDNA and the incorporation of usssctDNA into modern multi-analyte approaches for pan- cancer detection. This will involve training in new skills such as organoid modeling, methylation and leukocyte sequencing, and bioinformatic machine learning at a world-leading cancer-oriented institute. Ultimately, the goal of this proposed F99/K00 project will to prepare the trainee for a career in cancer research with an expertise in liquid biopsy and cell-free DNA biology.