Project Summary
Liquid biopsy is a potentially powerful, non-invasive approach for detecting, classifying and
monitoring tumors by capturing cell-free DNA molecules found circulating in blood and other
bodily fluids. Any next-generation sequencing (NGS) based approach requires an efficient
protocol for converting cell-free DNA into a sequencing library. Nearly all currently available NGS
library preparation methods target exclusively double-stranded DNA. However, the benefits of a
single-stranded DNA library approach are well described: higher efficiency and fidelity, less length
bias, recovery of nicked double-stranded fragments, and retention of the native ends of each
molecule, i.e. no DNA end repair. Despite the benefits, single-stranded library generation has not
been widely adopted because the only existing protocols are expensive, complicated, and time-
consuming. Claret Bioscience has developed an innovative new single-stranded approach
(SRSLY) that is fast, efficient, simple to follow, and cost-effective. The protocol is carried out in a
single tube and can convert cell-free DNA into a sequence-ready product in less than three hours.
We propose to develop the SRSLY protocol for clinical analysis by optimizing the reaction
conditions for cell-free DNA isolated from blood plasma. With data from our sequencing libraries,
we will investigate the added utility of retaining the native ends of cell-free DNA molecules for
reconstructing higher resolution nucleosome ‘maps’, as well as recovering populations of short
cell-free DNA – either nicked fragments lost to other methods or linker molecules thought to be
associated with regulatory elements with potential significance in disease processes. In
collaboration with medical oncologists at UCSF, we will analyze cell-free DNA from a study of GI
cancer patients through their course of treatment and integrate our sequencing-based metrics
with other metrics of tumor progression to learn the utility of our method in a clinical setting. We
aim to develop a scalable, cost-effective protocol that supports all NGS liquid biopsy analyses in
current use and provide data suitable for new discovery.