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mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mean) = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.