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DNA Res. 2014 Dec;21(6):685-94. doi: 10.1093/dnares/dsu030. Epub 2014 Oct 3.

Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.

Author information

1
Department of Life Sciences and Systems Biology, University of Turin, viale Mattioli 25, Turin 10125, Italy Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France.
2
EA 4678 CIDAM, BP 10448, Clermont Université, Université d'Auvergne, Clermont-Ferrand F-63001, France.
3
Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France.
4
Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche, Viale Mattioli 25, Turin 10125, Italy.
5
Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne 69622, France patricia.luis@univ-lyon1.fr.

Abstract

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.

KEYWORDS:

glycoside hydrolase family GH11; metatranscriptomics; sequence capture; soil RNA; soil eukaryotes

PMID:
25281543
PMCID:
PMC4263301
DOI:
10.1093/dnares/dsu030
[Indexed for MEDLINE]
Free PMC Article

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