target region selection is a critical determinant of community fingerprints generated by 16s pyrosequencing目标区域选择是一个关键的行列式社区16 s焦磷酸测序所产生的指纹.pdfVIP

Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing 1 1 2 3 Purnima S. Kumar *, Michael R. Brooker , Scot E. Dowd , Terry Camerlengo 1 Division of Periodontology, College of Dentistry, The Ohio State University, Columbus, Ohio, United States of America, 2 Research Testing Laboratory, Lubbock, Texas, United States of America, 3 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America Abstract Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1–V3 region was targeted, while Streptococ