Microbial Environmental Genomics (MEG) pp 273-287

Part of the Methods in Molecular Biology book series (MIMB, volume 1399) | Cite as

Metatranscriptomics of Soil Eukaryotic Communities

  • Rajiv K. Yadav
  • Claudia Bragalini
  • Laurence Fraissinet-Tachet
  • Roland Marmeisse
  • Patricia Luis
Protocol

Abstract

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.

Key words

Metatranscriptomics Environmental RNA cDNA synthesis cDNA size fractionation cDNA libraries Sequence capture 

References

  1. 1.
    Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR et al (2000) Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl Environ Microbiol 66:2541–2547PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Ferrer M, Beloqui A, Timmis KN, Golyshin PN (2009) Metagenomics for mining new genetic resources of microbial communities. J Mol Microbiol Biotechnol 16:109–123PubMedCrossRefGoogle Scholar
  3. 3.
    Chistoserdova L (2010) Recent progress and new challenges in metagenomics for biotechnology. Biotechnol Lett 32:1351–1359PubMedCrossRefGoogle Scholar
  4. 4.
    He S, Wurtzel O, Singh K, Froula JL, Yilmaz S, Tringe SG et al (2010) Validation of two ribosomal RNA removal methods for microbial metatranscriptomics. Nat Methods 10:807–812CrossRefGoogle Scholar
  5. 5.
    Stewart FJ, Ottesen EA, DeLong EF (2010) Development and quantitative analyses of a universal rRNA-substraction protocol for microbial metatranscriptomics. ISME J 4:896–907PubMedCrossRefGoogle Scholar
  6. 6.
    Grant S, Grant WD, Cowan DA, Jones BE, Ma Y, Ventosa A, Heaphy S (2006) Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples. Appl Environ Microbiol 72:135–143PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Bailly J, Fraissinet-Tachet L, Verner MC, Debaud JC, Lemaire M, Wesolowski-Louvel M, Marmeisse R (2007) Soil eukaryotic functional diversity, a metatranscriptomic approach. ISME J 1:632–642PubMedCrossRefGoogle Scholar
  8. 8.
    Damon C, Lehembre F, Oger-Desfeux C, Luis P, Ranger J, Fraissinet-Tachet L, Marmeisse R (2012) Metatranscriptomics reveals the diversity of genes expressed by eukaryotes in forest soils. PLoS One 7:e28967PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Damon C, Vallon L, Zimmermann S, Haider MZ, Galeote V, Dequin S et al (2011) A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes. ISME J 5:1871–1880PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Lehembre F, Doillon D, David E, Perrotto S, Baude J, Foulon J et al (2013) Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes. Environ Microbiol 15:2829–2840PubMedGoogle Scholar
  11. 11.
    Kellner H, Luis P, Portetelle D, Vandenbol M (2011) Screening of a soil metatranscriptomic library by functional complementation of Saccharomyces cerevisiae mutants. Microbiol Res 166:360–368PubMedCrossRefGoogle Scholar
  12. 12.
    Bragalini C, Ribière C, Parisot N, Vallon L, Prudent E, Peyretaillade E et al (2014) Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples. DNA Res 21:685–694PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Ribière C, Beugnot R, Parisot N, Gasc C, Defois C, Denonfoux J et al (2015) Targeted gene capture by hybridization to illuminate ecosystem functioning, Methods in molecular biology. Springer, New YorkGoogle Scholar
  14. 14.
    Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30:892–897PubMedGoogle Scholar
  15. 15.
    Luis P, Kellner H, Martin F, Buscot F (2005) A molecular method to evaluate basidiomycete laccase gene expression in forest soils. Geoderma 128:18–27CrossRefGoogle Scholar
  16. 16.
    Damon C, Barroso G, Ferandon C, Ranger J, Fraissinet-Tachet F, Marmeisse R (2010) Performance of the COX1 gene as a marker for the study of metabolically active Pezizomycotina and Agaricomycetes fungal communities from the analysis of soil RNA. FEMS Microbiol Ecol 74:693–705PubMedCrossRefGoogle Scholar
  17. 17.
    Yadav RK, Barbi F, Ziller A, Luis P, Marmeisse R, Reddy MS, Fraissinet-Tachet L (2014) Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA. BMC Biotechnol 14:80PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Wellenreuther R, Schupp I, Poustka A, Wiemann S (2004) SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics 5:36PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Denonfoux J, Parisot N, Dugat-Bony E, Biderre-Petit C, Boucher D, Morgavi DP et al (2013) Gene capture coupled to high-throughput sequencing as a strategy for targeted metagenome exploration DNA. DNA Res 20:185–196PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Green MR, Sambrook J (2012) Molecular cloning, a laboratory manual, vol 1, 4th edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Rajiv K. Yadav
    • 1
  • Claudia Bragalini
    • 1
    • 2
  • Laurence Fraissinet-Tachet
    • 1
  • Roland Marmeisse
    • 1
  • Patricia Luis
    • 1
  1. 1.Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université Lyon 1Université de LyonVilleurbanne CedexFrance
  2. 2.Department of Life Sciences and Systems BiologyUniversity of TurinTurinItaly

Personalised recommendations