Abstract
Human plasma contains > 40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from > 19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised intron RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.
Highlights
Extracellular RNAs in human plasma have been avidly pursued as potential biomarkers for cancer and other human diseases [1,2,3,4,5,6,7]
This study showed that Thermostable group II intron reverse transcriptase (TGIRT)-seq enables simultaneous sequencing of chemically fragmented mRNAs together with tRNAs and other structured sncRNAs, which were poorly represented in the TruSeq datasets, even after chemical fragmentation [24]
The TGIRT-seq datasets in this study were obtained from commercial human plasma pooled from multiple healthy individuals and prepared by apheresis with EDTA as the anticoagulant (IPLA-N-K2E; Innovative Research)
Summary
Extracellular RNAs in human plasma have been avidly pursued as potential biomarkers for cancer and other human diseases [1,2,3,4,5,6,7]. High-throughput RNA-sequencing (RNA-seq) has identified virtually all known RNA biotypes in human plasma, studies aimed at identifying disease biomarkers have focused mostly on plasma mRNAs or miRNAs. mRNAs in plasma and blood have been profiled by RNA-seq methods that enrich for or selectively reverse transcribed poly(A)-containing RNAs [10, 17] or by sequencing mRNA fragments with [13, 14, 18, 19] or without [4, 20] size selection, and it remains unclear which methods might be optimal for biomarker identification. Retroviral RTs have inherently low fidelity and processivity and even those that are highly engineered have difficulty reverse transcribing through stable RNA secondary structures or post-transcriptional modifications, resulting in under-representation of 5'-RNA sequences and aborted reads that can be mistaken for RNA fragments [24]. We still have an incomplete understanding of the biology of plasma RNAs and how to optimize their identification as biomarkers for human diseases
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