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      Fast UniFrac: Facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data

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          Abstract

          Next-generation sequencing techniques, and PhyloChip, have made simultaneous phylogenetic analyses of hundreds of microbial communities possible. Insight into community structure has been limited by the inability to integrate and visualize such vast datasets. Fast UniFrac overcomes these issues, allowing integration of larger numbers of sequences and samples into a single analysis. Its new array-based implementation offers orders of magnitude improvements over the original version. New 3D visualization of principal coordinates analysis (PCoA) results, with the option to view multiple coordinate axes simultaneously, provides a powerful way to quickly identify patterns that relate vast numbers of microbial communities. We demonstrate the potential of Fast UniFrac using examples from three data types: Sanger-sequencing studies of diverse free-living and animal-associated bacterial assemblages and from the gut of obese humans as they diet, pyrosequencing data integrated from studies of the human hand and gut, and PhyloChip data from a study of citrus pathogens. We show that a Fast UniFrac analysis using a reference tree recaptures patterns that could not be detected without considering phylogenetic relationships and that Fast UniFrac, coupled with BLAST-based sequence assignment, can be used to quickly analyze pyrosequencing runs containing hundreds of thousands of sequences, revealing patterns relating human and gut samples. Finally, we show that the application of Fast UniFrac to PhyloChip data could identify well-defined subcategories associated with infection. Together, these case studies point the way towards a broad range of applications and demonstrate some of the new features of Fast UniFrac.

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          Most cited references29

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          Pyrosequencing enumerates and contrasts soil microbial diversity.

          Estimates of the number of species of bacteria per gram of soil vary between 2000 and 8.3 million (Gans et al., 2005; Schloss and Handelsman, 2006). The highest estimate suggests that the number may be so large as to be impractical to test by amplification and sequencing of the highly conserved 16S rRNA gene from soil DNA (Gans et al., 2005). Here we present the use of high throughput DNA pyrosequencing and statistical inference to assess bacterial diversity in four soils across a large transect of the western hemisphere. The number of bacterial 16S rRNA sequences obtained from each site varied from 26,140 to 53,533. The most abundant bacterial groups in all four soils were the Bacteroidetes, Betaproteobacteria and Alphaproteobacteria. Using three estimators of diversity, the maximum number of unique sequences (operational taxonomic units roughly corresponding to the species level) never exceeded 52,000 in these soils at the lowest level of dissimilarity. Furthermore, the bacterial diversity of the forest soil was phylum rich compared to the agricultural soils, which are species rich but phylum poor. The forest site also showed far less diversity of the Archaea with only 0.009% of all sequences from that site being from this group as opposed to 4%-12% of the sequences from the three agricultural sites. This work is the most comprehensive examination to date of bacterial diversity in soil and suggests that agricultural management of soil may significantly influence the diversity of bacteria and archaea.
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            Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.

            We constructed error-correcting DNA barcodes that allow one run of a massively parallel pyrosequencer to process up to 1,544 samples simultaneously. Using these barcodes we processed bacterial 16S rRNA gene sequences representing microbial communities in 286 environmental samples, corrected 92% of sample assignment errors, and thus characterized nearly as many 16S rRNA genes as have been sequenced to date by Sanger sequencing.
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              Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection.

              The gut microbiotas of zebrafish and mice share six bacterial divisions, although the specific bacteria within these divisions differ. To test how factors specific to host gut habitat shape microbial community structure, we performed reciprocal transplantations of these microbiotas into germ-free zebrafish and mouse recipients. The results reveal that communities are assembled in predictable ways. The transplanted community resembles its community of origin in terms of the lineages present, but the relative abundance of the lineages changes to resemble the normal gut microbial community composition of the recipient host. Thus, differences in community structure between zebrafish and mice arise in part from distinct selective pressures imposed within the gut habitat of each host. Nonetheless, vertebrate responses to microbial colonization of the gut are ancient: Functional genomic studies disclosed shared host responses to their compositionally distinct microbial communities and distinct microbial species that elicit conserved responses.
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                Author and article information

                Journal
                101301086
                33338
                ISME J
                The ISME journal
                1751-7362
                1751-7370
                18 August 2009
                27 August 2009
                January 2010
                1 July 2010
                : 4
                : 1
                : 17-27
                Affiliations
                [1 ]Department of Computer Science, University of Colorado, Boulder, CO 80309, USA
                [2 ]Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA
                [3 ]Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108, USA
                [4 ]Howard Hughes Medical Institute
                Author notes
                [*]

                The first two authors contributed equally.

                Article
                nihpa135997
                10.1038/ismej.2009.97
                2797552
                19710709
                8e79c170-fb2b-4125-a271-e275298e5f36

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                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Award ID: T32 GM065103-08 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Award ID: P01 DK078669-03 ||DK
                Categories
                Article

                Microbiology & Virology
                multiplex pyrosequencing of 16s rdna,beta diversity,community ecology,phylochips

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