Project description:To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild.

Project description:The Baltic Sea is one of the largest brackish water bodies in the world. Redoxclines that form between oxic and anoxic layers in the deepest sub-basins are a semi-permanent character of the pelagic Baltic Sea. The microbially mediated nitrogen removal processes in these redoxclines have been recognized as important ecosystem service that removes large proportion of the nitrogen load originating from the drainage basin. However, nitrification, which links mineralization of organic nitrogen and nitrogen removal processes, has remained poorly understood. To gain better understanding of the nitrogen cycling in the Baltic Sea, we analyzed the assemblage of ammonia oxidizing bacteria and archaea in the central Baltic Sea using functional gene microarrays and measured the biogeochemical properties along with potential nitrification rates. Overall, the ammonia oxidizer communities in the Baltic Sea redoxcline were very evenly distributed. However, the communities were clearly different between the eastern and western Gotland Basin and the correlations between different components of the ammonia oxidizer assemblages and environmental variables suggest ecological basis for the community composition. The more even community ammonia oxidizer composition in the eastern Gotland Basin may be related to the constantly oscillating redoxcline that does not allow domination of single archetype. The oscillating redoxcline also creates long depth range of optimal nitrification conditions. The rate measurements suggest that nitrification in the central Baltic Sea is able to produce all nitrate required by denitrification occurring below the nitrification zone. Two color array (Cy3 and Cy5): the universal standard 20-mer oligo is printed to the slide with a 70-mer oligo (an archetype). Environmental DNA sequences (fluoresced with Cy3) within 15% of the 70-mer conjugated to a 20-mer oligo (fluoresced with Cy5) complementary to the universal standard will bind to the oligo probes on the array. Signal is the ratio of Cy3 to Cy5. Three replicate probes were printed for each archetype. Two replicate arrays were run on duplicate targets.

Project description:To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. The expanded genome proxy array was designed as in (Rich et al., 2008). Briefly, each genotype was targeted using suites of ~20 70-mer oligonucleotide probes designed using the program ArrayOligoSelector (Zhu et al., 2003). Probes had approximately the same %GC (40%) and were distributed across the target genome or genome fragment, with no more than one probe per gene and avoiding 16S and 23S rRNA genes. The array included positive and negative control probes designed using the same method, to Halobacterium salinarum NRC-1 and a random genome sequence, respectively. The expanded array had a broader scope than the prototype of Rich et al., 2008 (268 target genotypes, as opposed to the prototype’s 14) and included a co-spot oligo for spot alignment and gridding purposes (using the “alien” oligo sequence of (Urisman et al., 2005). The targets were selected from fully-sequenced marine microbial genomes, publicly-available marine-derived BAC and fosmid clone sequences, and fully-sequenced clones from the lab’s Monterey Bay and Hawaii environmental BAC- and fosmid-based genomic libraries. Targeted genotypes are detailed in Table S1, summarized in Table S2, and presented in a schematic phylogenetic overview in Fig. 1. Previously-unpublished sequences used for array design were submitted to Genbank under accession numbers GU474833-GU474949. This submission presents the analysis of 57 samples, each hybridized to >= 3 arrays. In addition to negative control probes, for each hyrbdization date and run a negative control array was hyrbdized for which the input DNA was TE, taken through the entire aplification, labeling, and hybridization process.

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