Small RNA-seq: The RNA 5

Authors

  • Lodoe Lama The City College of New York, and City University of New York Graduate Center
  • Jose Cobo The City College of New York, and City University of New York Graduate Center
  • Diego Buenaventura The City College of New York, and City University of New York Graduate Center
  • Kevin Ryan The City College of New York, and City University of New York Graduate Center

DOI:

https://doi.org/10.14440/jbm.2019.269

Keywords:

small RNA-seq, coligo, TS2126, CircLigase, RNA ligase bias

Abstract

The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5’ recessed ends are poor substrates for enzymatic adapter ligation, but this 5’ adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5’ RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5’ ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5’ adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, calling into question the benefit of this workaround. Using the TS2126 RNA ligase 1 (a.k.a. CircLigase) as the circularizing enzyme, we devised a bias test for the circularization of first strand cDNA. All possible dinucleotides were circle-ligated with similar efficiency. To re-linearize the first strand cDNA in the ribosome profiling approach, we introduce an improved method wherein a single ribonucleotide is placed between the sequencing primer binding sites in the reverse transcriptase primer, which later serves as the point of re-linearization by RNase A. We incorporate this step into the ribosomal profiling method and describe a complete improved library preparation method, Coligo-seq, for the sequencing of small RNA with secondary structure close to the 5’ end. This method accepts a variety of 5’ modified RNA, including 5’ monophosphorylated RNA, as demonstrated by the construction of a HeLa cell microRNA cDNA library.

Author Biography

Kevin Ryan, The City College of New York, and City University of New York Graduate Center

Professor, Department of Chemistry and Biochemistry

References

Berezikov E, Cuppen E, Plasterk RHA. Approaches to microRNA discovery. Nat Genet. Nature Publishing Group; 2006 Jun;38(6s):S2–S7.

Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C, et al. Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods. 2008 Jan;44(1):3–12.

Lau NC, Lim LP, Weinstein EG, Bartel DP. An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans. Science. 2001 Oct 26;294(5543):858–62.

Honda S, Morichika K, Kirino Y. Selective amplification and sequencing of cyclic phosphate–containing RNAs by the cP-RNA-seq method. Nature Protocols. Nature Publishing Group; 2016 Feb 11;11(3):476–89.

Jayaprakash AD, Jabado O, Brown BD, Sachidanandam R. Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res. 2011 Sep 2;39(21):e141–1.

Lee C, Harris RA, Wall JK, Mayfield RD, Wilke CO. RNaseIII and T4 Polynucleotide Kinase sequence biases and solutions during RNA-seq library construction. Biology Direct. BioMed Central; 2013 Jul 4;8(1):1105.

Hafner M, Renwick N, Brown M, Mihailovic A, Holoch D, Lin C, et al. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA. 2011 Aug 17;17(9):1697–712.

Cozen AE, Quartley E, Holmes AD, Hrabeta-Robinson E, Phizicky EM, Lowe TM. ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments. Nature Methods. Nature Publishing Group; 2015 Aug 3;12(9):879–84.

Zheng G, Qin Y, Clark WC, Dai Q, Yi C, He C, et al. Efficient and quantitative high-throughput tRNA sequencing. Nature Methods. Nature Publishing Group; 2015 Jul 27;12(9):835–7.

Liu X, Zheng Q, Vrettos N, Maragkakis M, Alexiou P, Gregory BD, et al. A MicroRNA Precursor Surveillance System in Quality Control of MicroRNA Synthesis. Molecular Cell. 2014 Sep;55(6):868–79.

Seidl CI, Lama L, Ryan K. Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells. Nucleic Acids Res. 3rd ed. 2013 Feb 1;41(4):2552–64.

Burke JM, Bass CR, Kincaid RP, Sullivan CS. Identification of tri-phosphatase activity in the biogenesis of retroviral microRNAs and RNAP III-generated shRNAs. Nucleic Acids Res. 2014 Nov 26;42(22):13949–62.

Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS. Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science. 2009 Apr 10;324(5924):218–23.

Kwok CK, Ding Y, Sherlock ME, Assmann SM, Bevilacqua PC. A hybridization-based approach for quantitative and low-bias single-stranded DNA ligation. Analytical Biochemistry. 2013 Apr;435(2):181–6.

Zhelkovsky AM, McReynolds LA. Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase. Nucleic Acids Res. 2011 Sep 20;39(17):e117–7.

Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003 Jul 1;31(13):3406–15.

Seidl CI, Ryan K. Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA. Darlix J-L, editor. PLoS ONE. 2011 Feb 16;6(2):e16925.

Lama L, Seidl CI, Ryan K. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III. Transcription. Taylor & Francis; 2014 Mar 12;5(2):e27913.

Chiu Y-H, MacMillan JB, Chen ZJ. RNA Polymerase III Detects Cytosolic DNA and Induces Type I Interferons through the RIG-I Pathway. Cell. 2009 Aug;138(3):576–91.

Chen S, Songkumarn P, Liu J, Wang G-L. A versatile zero background T-vector system for gene cloning and functional genomics. PLANT PHYSIOLOGY. American Society of Plant Biologists; 2009 Jul;150(3):1111–21.

Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018 Jul 2;46(W1):W537–44.

Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. EMBO Press; 2011 Oct 11;7(1):539–9.

Lama L, Ryan K. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I. RNA. 2015 Dec 16;22(1):155–61.

Hafner M, Renwick N, Farazi TA, Mihailović A, Pena JTG, Tuschl T. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods. 2012 Oct;58(2):164–70.

Sorefan K, Pais H, Hall AE, Kozomara A, Griffiths-Jones S, Moulton V, et al. Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence 2012 3:1. BioMed Central; 2012 Dec 1;3(1):4.

Ingolia NT. Genome-Wide Translational Profiling by Ribosome Footprinting. In: Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analysis. Elsevier; 2010. pp. 119–42. (Methods in Enzymology; vol. 470).

Guttman M, Russell P, Ingolia NT, Weissman JS, Lander ES. Ribosome Profiling Provides Evidence that Large Noncoding RNAs Do Not Encode Proteins. Cell. 2013 Jul;154(1):240–51.

Tate CM, Nuñez AN, Goldstein CA, Gomes I, Robertson JM, Kavlick MF, et al. Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis. Forensic Science International: Genetics. 2012 Mar;6(2):185–90.

Blondal T, Thorisdottir A, Unnsteinsdottir U, Hjorleifsdottir S, Ævarsson A, Ernstsson S, et al. Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties. Nucleic Acids Res. 2005 Jan 7;33(1):135–42.

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Published

2019-02-20

How to Cite

1.
Lama L, Cobo J, Buenaventura D, Ryan K. Small RNA-seq: The RNA 5. J Biol Methods [Internet]. 2019Feb.20 [cited 2021Dec.2];6(1):e108. Available from: https://jbmethods.org/jbm/article/view/269

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