Short Communication Pyrosequence Read Length of 16S rRNA Gene Affects Phylogenetic Assignment of Plant-associated Bacteria

Pyrosequence targeting of the 16S rRNA gene has been adopted for microbial communities associated with field-grown plants. To examine phylogenetic drifts according to read length and bioinformatic tools, original and chopped sequences (250–570 bp) covering the V1–V4 regions of 16S rRNA genes were compared using pyrosequence and Sanger reads of rice root microbiomes. The phylogenetic assignment at genus level depended on read length, especially in the genus Bradyrhizobium, which is one of the ecologically important bacterial genera associated with plants. We discuss the methodology of phylogenetic assignments of plant-associated bacteria by 16S rRNA pyrosequence. Key words: Bacterial community, phylogenetic analysis, 16S rRNA gene, plant-associated bacteria Diverse microorganisms live in and on plants. Microbial communities of field-grown plants have been surveyed by the use of 16S rRNA sequence-based methods in many studies. In most cases, clone libraries were constructed by PCR amplification of 16S rRNA gene segments (7, 13, 14). The GS-FLX Titanium pyrosequencer (454 Life Sciences, Branford, CT, USA) has now been used to analyze plant-associated bacterial communities (Ikeda et al. unpublished). Pyrosequencing yields more than half a million sequences per run, and enables analysis of many more samples simultaneously at considerably lower cost; however, its limitations are short and variable read lengths, and a lower sequence quality than those of Sanger dideoxy sequencing (5). Generally, for 400-base partial 16S rRNA sequences, the Ribosomal Database Project (RDP