The advent of next-generation sequencing (NGS) not only significantly brings down the cost of sequencing various genomes, but also enables many clinical and research applications, such as detection of genetic aberrations, noninvasive prenatal testing, bisulfite sequencing, single-cell RNA sequencing, ChiP-sequencing, etc. In my PhD work, two innovative applications of sequencing and molecular techniques were developed to address biomedical needs: profiling of ultra-short single- stranded DNA (ussDNA) and high-throughput in-vivo material screening using molecular barcoding. Cell-free DNA (cfDNA) that has a modal size of 167 base pairs has become a noninvasive alternative to solid tumor biopsy. Although recent cfDNA studies have suggested the presence of ultra-short DNA populations with non-canonical structure (termed as ussDNA), they remained an unexplored domain due to the lack of an efficient recovery method. Here, we developed a direct capture and sequencing (DCS) method using rationally designed degenerate capture probe and single strand-based library preparation to retain and characterize ussDNA from biofluids. Evaluation of the size distribution and abundance of ussDNA in biofluids manifested generality of its presence in human, animal species, and plants. My thesis focused on characterization of human plasma- and red blood cell-derived ussDNA, and yet more work is awaiting to study ussDNA’s generation mechanism, tissue of origin, disease implications, etc. In the second application, a high-throughput workflow was developed for in vivo screening of immune-protective biomaterials. This approach aims to renovate conventional material screening that is costly and less efficient. A cellular barcoding strategy enables simultaneous screening of a mixture of different biomaterials by encoding each candidate with a distinct donor cell line via encapsulation. With 20 donor cell lines, this workflow could screen 20 materials in a mouse model; and dual barcoding allows expansion of barcoding capacity to 400 materials, which makes larger-scale screening in a large animal feasible. Combining cellular barcoding and high-throughput sample processing and sequencing produces a fast track to evaluate biomaterial candidates more efficiently and thus significantly increase the success of finding materials with superior immunomodulating performance that could enable more translational applications.