New generation of reverse transcriptase for RNA-Seq
TGIRT-III stand-alone enzyme and TGIRT template-switching RNA-seq kit.
TGIRT® III enzyme is a new generation of reverse transcriptase with several advantages :
- Simultaneously reverse transcribing and adding an RNA-Seq adapter to RNAs of all sizes and structures
- Less biased manner than other methods
- Make it possible to obtain full-length reads of tRNAs and other structured non-coding RNAs
2 different variations of the method
- One for RNA-Seq of small RNAs : PAGE-purified cDNAs of selected sizes are circularized with CircLigaseII
- The other for RNA-Seq of total RNAs of all size classes
Available in 2 formats : enzyme alone and kit
Advantages of the Enzyme:
1. Comprehensive strand-specific transcriptome profiling.
TGIRT®-seq of ribodepleted, fragmented Universal Human Reference RNA samples recapitulates the relative abundance of human transcripts and spike-ins comparably to non-strand-specific TruSeq v2 and better than strand-specific Tru-Seq v3. TGIRT®-seq is significantly more strand-specific than TruSeq v3 and eliminates sampling biases from random hexamer priming that are inherent to TruSeq. TGIRT®-seq shows more uniform 5' to 3' gene coverage and identifies more splice junctions than TruSeq. TGIRT®-seq enables simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq as structured small ncRNAs, including tRNAs, which are essentially absent from TruSeq datasets.
2. RNA-seq of whole-cell, exosomal, plasma, and other extracellular RNAs.
Fast processing time (<5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); comprehensive transcript profiles including mRNAs and lncRNAs together with small ncRNAs, including full-length reads of tRNAs, pre-miRNAs, and other structured small ncRNAs; less bias and greater strand specificity than conventional methods.
3. RNA-seq library construction via TGIRT® template-switching in methods like RIP-seq, HITS-CLIP, irCLIP, CRAC, ribosome profiling.
Fast processing time (< 5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); does not require RNA ligase, is less biased and more efficient by having fewer steps in the procedure.
4. Higher thermostability, processivity and strand-displacement activity than retroviral RTs.
Enables construction of RNA-seq libraries of polyadenylated RNAs using an anchored oligo(dT) primer with more uniform 5' to 3' coverage than retroviral RTs without a ribodepletion step.
Enables RNA-structure mapping via capillary electrophoresis-based methods like SHAPE or DMS structure mapping with significantly longer read lengths and fewer premature stops than for retroviral RTs.
Enables analysis of RNA templates containing GC-rich repeat expansions.
Enables synthesis of full-length, end-to-end cDNAs from tRNAs and other small structured/modified ncRNAs, which are refractory to retroviral RTs.
5. ssDNA-seq of human plasma and E. coli genomic DNAs.
Captures precise DNA ends with a simpler workflow by initiating DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin.
Enzyme Properties and Novel Activity:
- Higher thermostability, processivity, and fidelity than retroviral reverse transcriptases, allowing full-length, end-to-end cDNA synthesis from highly structured or heavily modified RNAs (e.g., tRNAs), and RNAs containing GC-rich repeat expansions.
- Novel end-to-end template-switching activity that enables attachment of RNA-seq or PCR adapters during reverse transcription and eliminates the need for a separate RNA 3'-adapter ligation step.1 This template-switching activity greatly facilitates strand-specific RNA-seq library construction with less bias than procedures employing random hexamer primer or using RNA ligase for adapter ligation.
- Efficient cDNA synthesis from annealed primers. The annealed primer should have a predicted Tm of >60oC. Pre-incubation of enzyme with substrate in reaction mix for 30 min at room temperature and initiation of reaction by addition of dNTPs are recommended. Optimal conditions for new applications should be determined by testing a range of salt concentrations from 25 to 450 mM NaCl.
Mohr, S., Ghanem, E., Smith, W., Sheeter, D., Qin, Y., King, O., Polioudakis, D., Iyer, V.R., Hunicke-Smith, S. Swamy, S., Kuersten, S., and Lambowitz, A.M. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958-970, 2013.
Collins, K. and Nilsen, T. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology. RNA 19, 1017-1018, 2013.
Enyeart, P.J., Mohr, G., Ellington, A.D., and Lambowitz A.M. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. Mobile DNA 5: 2, 2014.
Katibah, G.E., Qin, Y., Sidote, D.J., Yao, J., Lambowitz, A.M. and Collins, K. Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5. Proc. Natl. Acad. Sci., USA, 111, 12025-12030, 2014.
Shen, P.S., Park, J., Qin, Y., Li, X., Parsawar, K., Larson, M.H., Cox, J., Chen, Y., Lambowitz, A.M., Weissman, J.S., Brandman, O., and Frost, A. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347, 75-78, 2015.
Zheng, G., Qin, Y., Clark, W.C., Yi, C., He, C., and Lambowitz, A.M. and Pan, T. Efficient and quantitative high-throughput transfer RNA sequencing. Nat. Methods 12, 835-837, 2015.
Qin,Y., Yao,J., Wu,D., Nottingham, R., Mohr, S, Hunicke-Smith, S., Lambowitz, A.M., High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases. RNA 22, 111-128, 2016.
Nottingham, R.M., Wu, D.C., Qin, Y., Yao, J., Hunicke-Smith, S., and Lambowitz, A.M. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. RNA 22, 597-613, 2016.
Burke, J.M., Kincaid, R.P., Nottingham, R.M., Lambowitz, A.M., and Sullivan, C.S. DUSP11 activity on tri-phosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady state levels of cellular non-coding RNAs. Genes & Dev. 30, 2071-2092, 2016.
Zarnegar, B.J., Flynn, R.A., Shen, Y., Do, B.T., Chang, H.Y. and Khavari, P.A. irCLIP platform for characterization of protein-RNA interactions. Nat. Methods 13, 489-492, 2016.
Haque, N. and Hogg, J.R. Easier, better, faster, stronger: Improved methods for RNA-protein interaction studies, Mol. Cell, 2016 http//dx.doi.org/10.1016/j.molcel.2016.05.019
Bazzini, A.A., del Viso, F., Moreno-Mateos, M.A., Johnstone, T.G., Vejnar, C.E., Qin, Y., Yao, J., Khokha, M.K., and Giraldez, A.J. Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition. EMBO J. 35, 2087-2103, 2016.
Clark, W.C., Evans, M.E., Dominissini, D., Zheng, G., and Pan, T. tRNA base methylation identification and quantification via high-throughput sequencing. RNA 22, 1771-1784, 2016.
Liu et al. ALKBH-mediated tRNA demethylation regulates translation. Cell 167, 816-828, 2016,
Shurtleff, M.J., Yao, J., Qin, Y., Nottingham, R.M., Temoche-Diaz, M., Schekman, R., and Lambowitz, A.M. A broad role for YBX1 in defining the small non-coding RNA composition of exosomes. Proc. Natl. Acad. Sci. U.S.A. 114, 8987-8995, 2017.
Zubradt, M., Gupta, P., Persad, S., Lambowitz, A.M., Weissman, J.S., and Rouskin, S. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nature Methods 10.1038/nmeth.4057, 2016.
Wu, D.C., and Lambowitz, A.M. Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching. Scientific Reports 7, 8421, 2017.
Carrell, S.T., Tang, Z., Mohr, S., Lambowitz, A.M, and Thorton, C.A. Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase. Nucleic Acids Res. 46, e1, 2018.