Solution hybrid selection capture for the recovery of functional full-length eukaryotic cDNAs from complex environmental samples.
Bottom Line: 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.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.
Affiliation: 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.Show MeSH
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Mentions: To address the phylogenetic diversity of the captured sequences, we first produced an amino acid sequence alignment of 62 known GH11 proteins representative of the phylogenetic diversity of this gene family. To this alignment, we added the GH11 sequences obtained by the random sequencing of plasmid inserts, the sequences producing a functional enzyme in yeast and the sequences representative of the most abundant Illumina sequence clusters before (H0) or after (H1 and H2) SHS capture. The GH11 family is a highly diversified and fast-evolving gene family and phylogenies based either on full-length protein sequence alignments or on partial alignments, as in the present case, clearly do not reflect the species phylogenies and comprise very few well-supported internal branches (Fig. 4). Phylogenetic trees obtained for sequences from the four studied soils (Fig. 4; Supplementary Fig. S6) all clearly showed that the captured sequences were distributed over the entire reference tree.Figure 4.
Affiliation: 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.