Depletion‐assisted multiplexed cell‐free RNA sequencing reveals distinct human and microbial signatures in plasma versus extracellular vesicles

Abstract Background Cell‐free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature. Methods To investigate these fragmented cell‐free RNAs (cfRNAs), we developed a cost‐effective cfRNA sequencing method called DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing). DETECTOR‐seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements. Results Using DETECTOR‐seq, we conducted a comprehensive analysis of cell‐free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing‐related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor‐mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types. Conclusions Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA‐based studies. We envision the cost effectiveness and efficiency of DETECTOR‐seq will empower transcriptome‐wide investigations in the fields of cfRNAs and liquid biopsy. Keypoints DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma. Distinct human and microbial cell‐free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed. Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.