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Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series

Authors

  • Mark V. Brown,

    Corresponding author
    1. Department of Biological Science and Wrigley Institute for Marine Studies, University of Southern California, 3616 Trousdale Parkway, AHF B4, Los Angeles, CA, 90089, USA.
      *E-mail mbrown@ifa.hawaii.edu; Tel. (1) 808 956 6880; Fax (1) 808 956 7264.
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  • Michael S. Schwalbach,

    1. Department of Biological Science and Wrigley Institute for Marine Studies, University of Southern California, 3616 Trousdale Parkway, AHF B4, Los Angeles, CA, 90089, USA.
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  • Ian Hewson,

    1. Department of Biological Science and Wrigley Institute for Marine Studies, University of Southern California, 3616 Trousdale Parkway, AHF B4, Los Angeles, CA, 90089, USA.
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  • Jed A. Fuhrman

    1. Department of Biological Science and Wrigley Institute for Marine Studies, University of Southern California, 3616 Trousdale Parkway, AHF B4, Los Angeles, CA, 90089, USA.
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*E-mail mbrown@ifa.hawaii.edu; Tel. (1) 808 956 6880; Fax (1) 808 956 7264.

Summary

We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising > 1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities < 98%, most taxa displayed differences in ARISA fragment lengths > 10 bp, readily detectable and suggesting ARISA resolution is near the ‘species’ level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r 2 = 0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.

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