Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics

Terminal restriction fragment length polymorphism (T-RFLP) is increasingly being used to examine microbial community structure and accordingly, a range of approaches have been used to analyze data sets. A number of published reports have included data and results that were statistically flawed or lacked rigorous statistical testing. A range of simple, yet powerful techniques are available to examine community data, however their use is seldom, if ever, discussed in microbial literature. We describe an approach that overcomes some of the problems associated with analyzing community datasets and offer an approach that makes data interpretation simple and effective. The Bray-Curtis coefficient is suggested as an ideal coefficient to be used for the construction of similarity matrices. Its strengths include its ability to deal with data sets containing multiple blocks of zeros in a meaningful manner. Non-metric multi-dimensional scaling is described as a powerful, yet easily interpreted method to examine community patterns based on T-RFLP data. Importantly, we describe the use of significance testing of data sets to allow quantitative assessment of similarity, removing subjectivity in comparing complex data sets. Finally, we introduce a quantitative measure of sample dispersion and suggest its usefulness in describing site heterogeneity.