A method of comprehensive sequencing analysis of the small RNA fragmentome (RiboMarker)

Circulating cell-free nucleic acids (cfDNA and cfRNA) found in blood and other biofluids are promising biomarkers for cancer. However, current methods exploiting tumor-derived cfDNA (ctDNA) are not sensitive enough in detecting minimal residual disease and early stages of cancer when it is more treatable. Small RNAs and RNA fragments (sRNA) can potentially provide higher detection sensitivity and specificity than ctDNA. Sequencing analysis of the variety of sRNAs representing the entire RNA fragmentome would improve our understanding of their roles in cancer development and help to discover novel sRNA biomarkers for cancer diagnostics and personalized treatments. However, conventional methods of sRNA-Seq library preparation are limited to detection of sRNA with 5-P and 3-OH ends that represent only less than 10% of the whole RNA fragmentome, whereas sRNAs having different phosphorylation statuses (P or OH) of their termini are hidden. Although recently developed sRNA-Seq methods allow detection of most sRNA (including the hidden ones) simultaneously, these methods cannot both detect and distinguish among the individual RNAs with differing termini combinations (RNA Types). Here we describe the RiboMarker(R) platform for preparation of sRNA sequencing libraries that addresses these shortcomings. It uses distinctive enzymatic pretreatment(s) of RNA samples that can both detect all and enrich for individual sRNA Types upfront of sequencing library preparation. The RiboMarker(R) platform has the potential capability to both identify and detect with enhanced sensitivity low abundance sRNA biomarkers of specific RNA classes and their termini.