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

Abstract

Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel templateswitching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5′ to 3 ′ gene coverage and identify more splice junctions, particularly near the 5 ′ ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq.

Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response

“…Their relative distribution inside the nucleus and the cytoplasm was calculated for each replicate: tRNA Lys , tRNA Gly , tRNA Glu , and tRNA Asp were the most abundant species, covering more than 50% of all tRNA reads. This result matches a recently published study that used TGIRT to quantify tRNA isoacceptor abundance in the Universal Human Reference RNA (Nottingham et al., 2016). In our samples, the least abundant tRNAs were tRNA Phe , tRNA Tyr , tRNA Cys , and tRNA Trp .…”

Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications

“…The initial template-primer substrate used for template switching reactions was the same as that used for RNA-seq adapter addition in RNA-seq protocols (Nottingham et al, 2016). It consists of a 34-nt RNA oligonucleotide containing an Illumina R2 sequence (R2 RNA; Table S1) with a 3′-blocking group (3SpC3; Integrated DNA Technologies) annealed to a 35-nt 5′ 32 P-labeled DNA primer ([γ-P 32 ]-ATP, Perkin Elmer), which contains the reverse complement of the R2 sequence and leaves a single nucleotide 3′ G overhang (R2RG DNA; Table S1).…”

“…As a result, group II intron RTs have higher fidelity, processivity and strand displacement activity than retroviral RTs, along with a highly proficient end-to-end template switching activity that is minimally dependent upon base pairing (Mohr et al, 2013). Recently, new methods for producing group II intron RTs in soluble form with high yield and activity have enabled their use for biotechnological applications, including new approaches for next-generation RNA sequencing (RNA-seq), identification of RNA post-transcriptional modifications, and RNA structure mapping (Clark et al, 2016; Nottingham et al, 2016; Zheng et al, 2015; Zubradt et al, 2016). …”

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.…”