Computer-Assisted Annotation of Small RNA Transcriptomes

Part of the Methods in Molecular Biology book series (MIMB, volume 1218)


Small noncoding RNAs (sncRNAs) are widely expressed in the cell of almost all known species. Most sncRNAs appear to have regulatory roles, ranging from facilitating RNA production and modifications (e.g., snoRNAs) to control of mRNA stability and translational efficiency (e.g., miRNAs and endo-siRNA) and to transposon silencing (e.g., piRNAs). The affordability and efficiency of next-generation RNA deep sequencing (RNA-Seq) technologies have made sncRNA deep sequencing (sncRNA-Seq) analyses a routine in biomedical research. SncRNA-Seq analyses generate millions of reads and gigabytes of data; annotation of sncRNA-Seq data remains challenging due to a lack of comprehensive sncRNA annotation pipelines. To solve this problem, we have developed a computer-assisted sncRNA annotation pipeline, which uses open-source software and allows for not only proper classification of known sncRNAs, but also discovery of novel sncRNA species. In this chapter, we describe our sncRNA annotation protocol in detail.

Key words

Noncoding RNAs Next-generation sequencing High throughput Deep sequencing Software 



This work was supported by NIH grants (HD060858, HD071736, and HD074573) to W.Y. Software was developed in the Imaging Core (Core D) with support by the COBRE grant P20 RR-18751 from the NIH.


  1. 1.
    Pfeffer S, Zavolan M, Grasser FA, Chien M, Russo JJ, Ju J, John B, Enright AJ, Marks D, Sander C, Tuschl T (2004) Identification of virus-encoded microRNAs. Science 304: 734–736. doi: 10.1126/science.1096781 PubMedCrossRefGoogle Scholar
  2. 2.
    Bourc’his D, Voinnet O (2010) A small-RNA perspective on gametogenesis, fertilization, and early zygotic development. Science 330: 617–622. doi: 10.1126/science.1194776 PubMedCrossRefGoogle Scholar
  3. 3.
    Czech B, Hannon GJ (2011) Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 12:19–31. doi: 10.1038/nrg2916 PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Eddy SR (2001) Non-coding RNA genes and the modern RNA world. Nat Rev Genet 2:919–929PubMedCrossRefGoogle Scholar
  5. 5.
    Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10:126–139PubMedCrossRefGoogle Scholar
  6. 6.
    Siomi H, Siomi MC (2009) On the road to reading the RNA-interference code. Nature 457:396–404PubMedCrossRefGoogle Scholar
  7. 7.
    Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355. doi: 10.1038/nature02871nature02871 [pii] PubMedCrossRefGoogle Scholar
  8. 8.
    Ro S, Ma HY, Park C, Ortogero N, Song R, Hennig GW, Zheng H, Lin YM, Moro L, Hsieh JT, Yan W (2013) The mitochondrial genome encodes abundant small noncoding RNAs. Cell Res 23:759–774. doi: 10.1038/cr.2013.37 PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Smith CM, Steitz JA (1997) Sno storm in the nucleolus: new roles for myriad small RNPs. Cell 89:669–672PubMedCrossRefGoogle Scholar
  10. 10.
    Ganot P, Bortolin ML, Kiss T (1997) Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs. Cell 89:799–809PubMedCrossRefGoogle Scholar
  11. 11.
    Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854PubMedCrossRefGoogle Scholar
  12. 12.
    Lai EC (2002) Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 30:363–364. doi: 10.1038/ng865 PubMedCrossRefGoogle Scholar
  13. 13.
    Morozova O, Marra MA (2008) Applications of next-generation sequencing technologies in functional genomics. Genomics 92:255–264. doi: 10.1016/j.ygeno.2008.07.001 PubMedCrossRefGoogle Scholar
  14. 14.
    Kozomara A, Griffiths-Jones S (2013) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 39:D152–D157. doi: 10.1093/nar/gkt1181
  15. 15.
    Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157. doi: 10.1093/nar/gkq1027 PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Ortogero N, Hennig GW, Langille C, Ro S, McCarrey JR, Yan W (2013) Computer-assisted annotation of murine sertoli cell small RNA transcriptome. Biol Reprod 88:3. doi: 10.1095/biolreprod.112.102269 PubMedCrossRefGoogle Scholar
  17. 17.
    Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMB J 17:10–12CrossRefGoogle Scholar
  18. 18.
    Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25. doi: 10.1186/gb-2009-10-3-r25 PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Chen X, Fau-Li Q, Li Q, Fau-Wang J, Wang J, Fau-Guo X, Guo X, Fau-Jiang X, Jiang X, Fau-Ren Z, Ren Z, Fau-Weng C, Weng C, Fau-Sun G, Sun G, Fau-Wang X, Wang X, Fau-Liu Y, Liu Y, Fau-Ma L, Ma L, Fau-Chen J.-Y, Chen Jy Fau-Wang J, Wang J, Fau-Zen K, Zen K, Fau-Zhang J, Zhang J, Fau-Zhang C.-Y.C.Y. Z (2009) Identification and characterization of novel amphioxus microRNAs by Solexa sequencing. Genome Biol 10:17Google Scholar
  20. 20.
    Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Hertel J, Hofacker IL, Stadler PF (2008) SnoReport: computational identification of snoRNAs with unknown targets. Bioinformatics 24:158–164. doi: 10.1093/bioinformatics/ btm464 PubMedCrossRefGoogle Scholar
  22. 22.
    Chan PP, Lowe TM (2009) GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res 37:D93–D97. doi: 10.1093/nar/gkn787 PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Physiology and Cell BiologyUniversity of Nevada School of MedicineRenoUSA

Personalised recommendations