Using the pick-otus protocol, we classified the sequence reads into OTUs on the basis of sequence similarity. Sequence reads were then clustered against the February 2011 release of the Greengenes 97% reference dataset (http://greengenes.secondgenome.com) [20, 21]. Taxonomy was assigned using the Basic Local Alignment Search Tool (BLAST) [22]. The representative sequences of all OTUs were then aligned to the Greengenes reference alignment using PyNAST [18], and this alignment was used to construct a phylogenetic tree using FastTree [23] within QIIME. The
resulting tree topology with associated branch lengths was used for subsequent diversity analyses (for many downstream analyses, samples were rarefied at 6173 and 9390 sequences per sample selleck products for the homogenisation and for the water content evaluations, respectively). One sample (LO1.1) was removed from the analysis because of low count reads. Alpha diversity was estimated using the phylogenetic BAY 73-4506 clinical trial diversity metric. Beta diversity analysis was performed using the UPGMA clustering method based on weighted and unweighted UniFrac distances
[24]. Availability of supporting data Sequences have been deposited in NCBI database with the accession number SRP040438. Acknowledgements We thank Ricardo Gonzalo, Francisca Gallego, Rosa Arjona and Rosario M. Prieto from the Unit of High Technology, Vall d’Hebron Research Institute, for technical assistance. This work was performed as a part of the PhD research of Ms. Alba Santiago and Ms. Suchita Panda, students of the Universitat Autònoma de Barcelona FAD (UAB). This study was partially funded by unrestricted grants
from the Fondo de Investigacion Sanitaria (PI10/00902, CP13/00181) and in part by HENUFOOD (CEN-20101016) and by the European Community’s Seventh Framework Programme (FP7/2007-2013): International Human Microbiome Standards (IHMS), grant agreement HEALTH.2010.2.1.1-2. CIBERehd is funded by the Instituto de Salud Carlos III. Electronic supplementary material Additional file 1: Table S1: Legend of Figure 1. (XLSX 94 KB) Additional file 2: Figure S1: Alpha-diversity curves at a number of rarefaction depths. Each line represents the results of the alpha-diversity phylogenetic diversity whole tree metric (PD whole tree in QIIME) for all samples from subjects #5 and #8. (PNG 437 KB) Additional file 3: Figure S2: Kit for stool collection (see the method section). (PNG 1 MB) References 1. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA: Diversity of the human intestinal microbial flora. Science 2005,308(5728):1635–1638.PubMedCentralPubMedCrossRef 2. Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende DR, Li J, Xu J, Li S, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto JM, Hansen T, Le Paslier D, Linneberg A, Nielsen HB, Pelletier E, Renault P, et al.