Deep Sequencing Data Analysis pp 213-231 | Cite as
Detection of Reverse Transcriptase Termination Sites Using cDNA Ligation and Massive Parallel Sequencing
Abstract
Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5′ ends (5′ RACE), cap analysis of gene expression, and detection of RNA modifications and protein–RNA cross-links. The throughput of these methods can be increased by applying massive parallel sequencing technologies.
Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3′ end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells.
Key words
Reverse transcription Termination Sequencing TS2l26 RNA ligase CAGE GalaxyNotes
Acknowledgments
The research was funded by the Danish Council for Strategic Research, the Lundbeck Foundation and the Novo Nordisk Foundation. Morten Lindow and Susanna Obad, Santaris Pharma, provided mouse liver samples and RIKEN/Piero Carninci provided the updated CAGE protocol as well as advice ahead of publication.
References
- 1.Takahashi H, Kato S, Murata M et al (2012) CAGE (cap analysis of gene expression): a protocol for the detection of promoter and transcriptional networks. In: Deplancke B, Gheldof N (eds) Gene regulatory networks, vol 786. Humana, Totowa, NJ, pp 181–200CrossRefGoogle Scholar
- 2.Motorin Y, Muller S, Behm‐Ansmant I et al (2007) Identification of modified residues in RNAs by reverse transcription‐based methods. Methods Enzymol 425:21–53. doi: 10.1016/s0076-6879(07)25002-5 PubMedCrossRefGoogle Scholar
- 3.Mortimer SA, Weeks KM (2009) Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution. Nat Protoc 4(10):1413–1421. doi:nprot.2009.126 [pii]10.1038/nprot.2009.126PubMedCrossRefGoogle Scholar
- 4.König J, Zarnack K, Rot G et al (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17(7):909–915. doi: 10.1038/nsmb.1838 PubMedCrossRefGoogle Scholar
- 5.Shibata Y, Carninci P, Watahiki A et al (2001) Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method. Biotechniques 30(6):1250–1254PubMedGoogle Scholar
- 6.Li TW, Weeks KM (2006) Structure-independent and quantitative ligation of single-stranded DNA. Anal Biochem 349(2):242–246. doi: 10.1016/j.ab.2005.11.002 PubMedCrossRefGoogle Scholar
- 7.Hirzmann J, Luo D, Hahnen J et al (1993) Determination of messenger RNA 5'-ends by reverse transcription of the cap structure. Nucleic Acids Res 21(15):3597–3598PubMedCrossRefGoogle Scholar
- 8.Zhu YY, Machleder EM, Chenchik A et al (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30(4):892–897PubMedGoogle Scholar
- 9.Carninci P, Kasukawa T, Katayama S et al (2005) The transcriptional landscape of the mammalian genome. Science 309(5740):1559–1563. doi: 10.1126/science.1112014 PubMedCrossRefGoogle Scholar
- 10.Shiraki T, Kondo S, Katayama S et al (2003) Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc Natl Acad Sci U S A 100(26):15776–15781. doi: 10.1073/pnas.2136655100 PubMedCrossRefGoogle Scholar
- 11.Weeks KM, Mauger DM (2011) Exploring RNA structural codes with SHAPE chemistry. Acc Chem Res 44(12):1280–1291. doi: 10.1021/ar200051h PubMedCrossRefGoogle Scholar
- 12.Lucks JB, Mortimer SA, Trapnell C et al (2011) Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci U S A 108(27):11063–11068. doi: 10.1073/pnas.1106501108 PubMedCrossRefGoogle Scholar
- 13.Giardine B, Riemer C, Hardison RC et al (2005) Galaxy: a platform for interactive large-scale genome analysis. Genome Res 15(10):1451–1455. doi: 10.1101/Gr.4086505 PubMedCrossRefGoogle Scholar
- 14.Goecks J, Nekrutenko A, Taylor J et al (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11(8):R86. doi: 10.1186/Gb-2010-11-8-R86 PubMedCrossRefGoogle Scholar
- 15.Blankenberg D, Gordon A, Von Kuster G et al (2010) Manipulation of FASTQ data with Galaxy. Bioinformatics 26(14):1783–1785. doi: 10.1093/bioinformatics/btq281 PubMedCrossRefGoogle Scholar
- 16.Blankenberg D, Von Kuster G, Coraor N et al. (2010) Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol Chapter 19:Unit 19.10.11–21Google Scholar
- 17.Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. doi: 10.1186/Gb-2009-10-3-R25 PubMedCrossRefGoogle Scholar
- 18.Hannon-Lab, Gordon A (2010) FASTX-toolkit: FASTQ/A short-reads pre-processing tools. http://hannonlab.cshl.edu/fastx_toolkit/
- 19.R Foundation for Statistical Computing (2012) R: A language and environment for statistical computing, 2151st edn. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar
- 20.Gentleman RC, Carey VJ, Bates DM et al (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5(10):R80PubMedCrossRefGoogle Scholar
- 21.Aird D, Ross MG, Chen WS et al (2011) Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 12(2):R18. doi: 10.1186/gb-2011-12-2-r18 PubMedCrossRefGoogle Scholar