Ecological Archives E096-082-A6
Cyrus A. Mallon, Franck Poly, Xavier Le Roux, Irene Marring, Jan Dirk Van Elsas, and Joana Falcão Salles. 2015. Resource pulses can alleviate the biodiversity–invasion relationship in soil microbial communities. Ecology 96:915–926. http://dx.doi.org/10.1890/14-1001.1
Appendix F. Processing 16S pyrosequencing data.
Raw pyrosequencing data was treated with the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (Caporaso et al. 2010). 16S rRNA partial bacterial sequences were trimmed to a minimum and maximum length of 300 and 900 basepairs, respectively, with a threshold quality score of >25. Sequences with ambiguous or mismatched bases were thrown out, as were sequences exceeding 6 homopolymer bases. The data were denoized with Denoiser (Reeder and Knight, 2010). The resulting sequences were then clustered into operational taxonomic units (OTUs) at 97% sequence similarity using UCLUST, and a representative sequence for each OTU was selected (Edgar 2010). Chimeric sequences were identified and removed using ChimeraSlayer (Haas et al. 2010). Representative OTUs were aligned using PyNast (Caporaso et al. 2010) and then classified using Greengenes taxonomy via RDP classifier (Wang et al. 2007). Gaps in the sequence alignment were filtered, and a phylogenetic tree was constructed de novo using FastTree (Price et al. 2007). In order to minimize the effects of sequencing depth between samples when estimating species richness, our samples were rarefied to a depth of 2000 sequences per sample. Finally, QIIME was used to assess species richness, which was the number of unique OTUs per sample.
Caporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. D. Bushman, E. K. Costello, N. Fierer, A. G. Pena, J. K. Goodrich, J. I. Gordon, G. A. Huttley, S. T. Kelley, D. Knights, J. E. Koenig, R. E. Ley, C. A. Lozupone, D. McDonald, B. D. Muegge, M. Pirrung, J. Reeder, J. R. Sevinsky, P. J. Tumbaugh, W. A. Walters, J. Widmann, T. Yatsunenko, J. Zaneveld, and R. Knight. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7:335336.
Reeder, J., and R. Knight. 2010. Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat.Methods, 7:668669.
Edgar, R. C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:24602461.
Haas, B. J., D. Gevers, A. M. Earl, M. Feldgarden, D. V. Ward, G. Giannoukos, D. Ciulla, D. Tabbaa, S. K. Highlander, E. Sodergren, B. Methe, T. Z. DeSantis, J. F. Petrosino, R. Knight, B. W. Birren, and Human Microbiome Consortium. 2011. Chimeric 16S rRNA sequence formation and detection in sanger and 454-pyrosequenced PCR amplicons. Genome Res. 21:494504.
Price, M. N., P. S. Dehal, and A. P. Arkin. 2009. FastTree: Computing large minimum evolution trees with profiles instead of a distance matrix. Mol.Biol.Evol. 26:1641-1650.
Wang, Q., G. M. Garrity, J. M. Tiedje, and J. R. Cole. 2007. Naive bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl.Environ.Microbiol. 73:52615267.
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