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

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.

RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase

“…Half of this processed RNA was then used as the input for RNAseq library preparation via the TGIRT template-switching method developed in our laboratory ( Fig. 1B; Qin et al 2016). The remainder was stored for comparisons with a second TGIRT enzyme denoted TeI4c RT to be done later.…”

DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

“…S6A). To test whether knockout of DUSP11 affects the non-miRNA transcriptome, we used thermostable group II intron reverse transcriptase high-throughput sequencing (TGIRT-seq), which enables the analysis of full-length, highly structured RNAs (Mohr et al 2013;Katibah et al 2014;Nottingham et al 2016;Qin et al 2016). We used two different RNA-seq library preparation protocols in order to test for differences in the greatest range of host RNAs.…”

“…TGIRT-seq libraries were constructed essentially as described Qin et al 2016). Briefly, total RNA from HEK293T cells or A549 cells was first ribo-depleted by using a RiboZero Gold (human/mouse/rat) kit (Illumina).…”

“…cDNAs were synthesized via TGIRT template switching with 1 µM purified TeI4c-ΔEn-MRF reverse transcriptase for 15 min at 60°C as described (Mohr et al 2013;Qin et al 2016). During synthesis, a DNA oligonucleotide containing the complement of an Illumina Read 2 sequencing primer-binding site was seamlessly linked to the 5 ′ cDNA end.…”

Integrative single-cell and cell-free plasma RNA transcriptomics elucidates placental cellular dynamics

“…Significantly elevated levels of total cell-free DNA and selected placenta-specific RNA transcripts have also been reported in the maternal plasma of women with PE (8)(9)(10)(11), restricted fetal growth (12), and preterm birth (13)(14)(15), supporting a role for cell-free nucleic acids as a noninvasive tool for placental monitoring. Previous studies have attempted to provide a comprehensive assessment of maternal plasma nucleic acids by microarray analysis, massively parallel transcriptomic, or methylomic sequencing (16)(17)(18)(19)(20)(21)(22). Several groups have explored the use of fetal-specific DNA polymorphisms, organ-specific DNA methylation (21), nucleosome footprinting (23), DNA fragmentation patterns (24), and tissuespecific RNA transcripts (19,20) to isolate the placental signal in the pool of circulating cell-free fetal nucleic acids and obtain changes of overall placental contribution.…”