Name that microbe: Rapid identification of taxa responsible for individual fragments in fingerprints of microbial community structure.
Alastair Grant and Lesley A. Ogilvie 2004.
Molecular Ecology Notes 4, 133-136
Name that microbe: Rapid identification of taxa responsible for individual fragments in fingerprints of microbial community structure.Alastair Grant and Lesley A. Ogilvie 2004.Molecular Ecology Notes 4, 133-136 |
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Sequencing of cloned 16S rDNA has revolutionised our understanding of microbial ecology, revealing a previously unsuspected diversity of bacteria and archaea. However, to carry out microbial community ecology we need to quantify the abundance of microbial taxa at a number of sites or a number of time points. Sequencing of large numbers of 16S clones is too time consuming to permit these sorts of comparisons. Microbial ecology has developed a number of methods of "fingerprinting" microbial communities, making it possible to quantify communities in a large number of samples. DGGE, TGGE and other approaches based on gradient gels are attractive because individual bands from the fragment can be cut from the gel, cloned and sequenced. However, these methods typically resolve only a relatively small number of fragments; have relatively low reproducibility between laboratories and are normally qualitative or semi-quantitative in their estimates of taxon abundances. The most powerful fingerprinting, such as T-RFLP, LH-PCR and ARISA quantify the size and abundance of DNA fragments using automated DNA sequencers. These give quantitative data on taxon abundance and readily reproducible results. However, at present the only option to determine exactly which microbial taxon is responsible for a particular fragment in one of these profiles is to randomly sequence 16S clones and hope to sample most of the important taxa.
In this paper we outline a strategy to efficiently select 16S clones corresponding to a fragment of a particular length, thus greatly reducing the number of clones that must be sequenced. The methods are illustrated with a case study using TRFLP, but can also be used in the same way in studies using LHPCR or Automated RISA. Copies of reprints are available on request from Alastair Grant.
Dr Alastair Grant
Director
Centre for Ecology,
Evolution and Conservation
School of
Environmental Sciences
University of
East Anglia
Norwich
NR4 7TJ
UK
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