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RNA. 2016 Jan;22(1):111-28. doi: 10.1261/rna.054809.115. Epub 2015 Nov 9.

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases.

Author information

1
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA.
2
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.

Abstract

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling.

KEYWORDS:

diagnostics; next-generation sequencing; noncoding RNA; tRNA; transcriptome profiling

PMID:
26554030
PMCID:
PMC4691826
DOI:
10.1261/rna.054809.115
[Indexed for MEDLINE]
Free PMC Article
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