ena-STUDY-CNRS - Universite Lyon 1-07-07-2014-10:19:33:769-55
Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples
Species-rich eukaryotic microbial communities play key functional roles in soil biology and also potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate and characterise 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. In this study, we developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional cDNAs synthesized from soil-extracted polyadenylated mRNA. This protocol was implemented on the Glycoside Hydrolase 11 gene family encoding endoxylanases 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, more than 90% of the resulting cDNAs were GH11 sequences, of which about 70% were full-length. Several of them were successfully expressed in Saccharomyces cerevisiae. Sequencing of PCR-amplified GH11 gene fragments showed that capture selected hundreds of phylogenetically diverse sequences, all of them different from those deposited in databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families represented by numerous alleles within eukaryotic microbial communities thriving in any type of environment.
Species-rich eukaryotic microbial communities play key functional roles in soil biology and also potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate and characterise 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. In this study, we developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional cDNAs synthesized from soil-extracted polyadenylated mRNA. This protocol was implemented on the Glycoside Hydrolase 11 gene family encoding endoxylanases 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, more than 90% of the resulting cDNAs were GH11 sequences, of which about 70% were full-length. Several of them were successfully expressed in Saccharomyces cerevisiae. Sequencing of PCR-amplified GH11 gene fragments showed that capture selected hundreds of phylogenetically diverse sequences, all of them different from those deposited in databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families represented by numerous alleles within eukaryotic microbial communities thriving in any type of environment.