Probe-based negative selection for underrepresented phylotypes in large environmental clone libraries
| Autor: | Jenni Hultman, Petri Auvinen, Martin Romantschuk, Miia Pitkäranta, Lars Paulin |
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| Rok vydání: | 2008 |
| Předmět: |
Microbiology (medical)
Library Molecular Sequence Data Biology Microbiology Polymerase Chain Reaction law.invention 03 medical and health sciences Nucleic acid thermodynamics Microbial ecology Phylogenetics law Environmental Microbiology Genomic library Molecular Biology Polymerase chain reaction Phylogeny 030304 developmental biology Gene Library Genetics Phylotype 0303 health sciences Bacteria 030306 microbiology Nucleic acid sequence Fungi Nucleic Acid Hybridization Nucleic Acid Probes |
| Zdroj: | Journal of microbiological methods. 75(3) |
| ISSN: | 0167-7012 |
| Popis: | Studies based on cloning and sequencing to investigate microbial diversity in a vast range of samples has become widespread in recent years. Results have revealed immense microbial diversity in many different environments, but also dominance of a few sequence types in the constructed clone libraries. Here we describe a method to enrich the clone libraries by avoiding sequencing of known, abundant sequence types, instead focusing on novel, rare ones. The protocol is based on gridding the PCR products from clone libraries on membranes and hybridisation of species-specific probes. Clones that do not give positive hybridisation results are sequenced. This method was used for fungal clone libraries from compost samples. Altogether 1536 clones were gridded and six probes used. From these clones, 59% hybridised with a probe, and therefore, only 41% of the clones were sequenced. In addition, 384 samples were sequenced to verify the hybridisation results. The numbers of false-negative (5.2%) and false-positive (3.9%) hybridisations were low. This method provides a mean of lowering the costs of sequencing projects and speeding up the process of characterising microbial diversity in environmental samples. The method is especially suitable for samples with a few dominating sequence types. |
| Databáze: | OpenAIRE |
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