Patent Publication Number: US-7214492-B1

Title: Nucleic acid arrays to monitor water and other ecosystems

Description:
RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. patent application Ser. No. 10/131,618, filed Apr. 24, 2002 now abandoned. The disclosure of U.S. patent application Ser. No. 10/131,618, is hereby incorporated by reference in its entirety. 
    
    
     FEDERAL FUNDING 
     Parts of this work were supported by federal funding in the form of a Cooperative Agreement #82946501 with the Environmental Protection Agency to C. Neal Stewart, Vincent C. Henrich and Parke A. Rublee and an Environmental Protection Agency STAR Grant #R831627 to Parke A. Rublee. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to nucleic acid arrays to monitor water and other ecosystems. 
     BACKGROUND 
     As the threat of bioterrorist activities has become evident in recent years, concern about the vulnerability of ecosystems such as municipal water supplies to deliberate contamination has grown. Additionally, human development can result in chemicals, waste, and/or agricultural runoff being introduced into the ecosystem. Increases in population may lead to over-harvesting of marine resources, landscape alterations that alter the ecosystem, and the introduction of living and non-living contaminants into the ecosystem (Mallin, J. M., et al. 2000 , Marine Pollution Bulletin,  41:56–75). For example, spills from concentrated animal feeding operations can degrade water quality and stimulate algal blooms (Mallin, M. A., 2000 , American Scientist,  88:26–37). Also, over-development of coastal areas may introduce coliform bacteria, and the introduction of nutrients and or chemical compounds may lead to eutrophication or other changes in microbial communities (Mallin, M. A., et al. 2000 , Ecological Applications,  10:1047–1056; Paerl, H. W., et al. 2003 , FEMS Microbiology Ecology,  46:233–246; DeLorenzo, M. E., et al., 1999 , Environmental Toxicology and Chemistry,  18:2824–2835). 
     Reservoirs, recreational lakes, and coastal areas can be difficult to secure against accidental or intentional contamination. Further, the contamination of a water source has the propensity to impact a relatively large population, and water filtration systems may not sufficiently alleviate the threat. Perhaps most troubling is the lack of a real-time test to detect the agents that are most likely to contaminate water supplies. The turnaround times for culturing microbes is slow enough that consumption of contaminated water may occur before the test results are known. Also, the expense involved in frequent monitoring of the water supply with currently available laboratory tests can be prohibitive. 
     As yet, there has not been a large-scale, deliberate contamination of a municipal water source. However, sporadic and relatively confined natural contaminations have demonstrated the importance in being able to monitor the water supply. The number of outbreaks attributable to contaminated drinking water supplies more than doubled in 1999–2000 over the previous two-year period, with contamination of well water also on the rise. In addition, recreational water sources have also reported significant increases in contamination (Bowman, 2002, Outbreaks of waterborne illnesses on the rise in US,  Scripps - Howard News Service , Nov. 23, 2002). These incidents of water contamination were exacerbated by the difficulty in pinpointing the cause of the outbreak and subsequent misdiagnosis of the symptoms, illustrating the importance of “early warning” diagnostics of water supplies. 
     A number of microbial genome sequencing projects have been initiated to characterize pathogenic organisms. Although identification and characterization of genomic sequence data for individual pathogens may provide for the identification of specific microbes, such targeted testing fails to provide a comprehensive, economically feasible system for monitoring ecosystems of interest, such as municipal water supplies. The accuracy of a molecular diagnostic test for a microbe may be compromised where the pathogenic agent is endemic, or possesses substantial genetic similarity to non-pathogenic organisms (Leff et al., 1995 , Appl. Environ. Microbiol.,  61:1634–1636; Xiao et al, 1999 , Appl Environ. Microbiol.,  65:3386–3391). Also, although some putative contaminants of water have been identified, anticipating all possible contaminants is not feasible, and thus, specific tests are inherently limited. 
     Thus, there is a need for devices and methods that enable real-time monitoring of water supplies and other ecosystems of interest. The monitoring system should allow for detection of known, as well as unknown, contaminants. The monitoring system should be available in a format that is accessible for routine monitoring, as well as for rapid testing in response to a specific event. 
     SUMMARY 
     The present invention provides devices and systems for monitoring water and other ecosystems. The present invention may be embodied in a variety of ways. 
     One embodiment of the present invention comprises an array for monitoring an ecosystem comprising a plurality of oligonucleotides immobilized at known locations on a substrate, such that each location on the array comprises an oligonucleotide having a sequence derived from a single, predetermined microbial operational taxonomic unit (OTU). 
     In another embodiment, the present invention may comprise a device for monitoring water quality comprising an array, wherein the array comprises a plurality of oligonucleotides immobilized at known locations on a substrate, and wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU). 
     Another embodiment of the present invention comprises a system for monitoring an ecosystem of interest. The system may comprise an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate. In one embodiment, the array may be designed such that each location on the array comprises an oligonucleotide having a sequence derived from a single, predetermined microbial operational taxonomic unit (OTU). The system may further include a device able to measure hybridization of a DNA sample to the array. Also, the system may include a DNA sample for hybridizing to the array. In one embodiment, the ecosystem may comprise a body of water. 
     Yet other embodiments of the present invention comprise methods for monitoring ecosystems. The method may comprise the step of generating an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate, such that each location on the array comprises an oligonucleotide having a sequence derived from a single predetermined microbial operational taxonomic unit (OTU). The method may also comprise the step of preparing a nucleic acid sample derived from a water sample of interest and hybridizing the nucleic acid sample to the array. Also, the method may comprise the step of measuring hybridization of the nucleic acid sample to the array, and correlating the hybridization of the nucleic acid to the array with a parameter that comprises at least part of the ecosystem. In one embodiment, the ecosystem of interest comprises a body of water. 
     In yet another embodiment, the present invention comprises a method of analyzing a pattern to evaluate the status of a biosystem. The method may comprise the step of measuring hybridization of a DNA sample to an array of oligonucleotides immobilized at known locations on a substrate, wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU), and correlating the hybridization of the DNA to the array with a parameter that comprises at least part of the ecosystem. A single change in the pattern of hybridization to the array for a first DNA sample isolated from an ecosystem of interest as compared to a second DNA sample isolated from an ecosystem of interest may be associated with a change in one parameter, or a plurality of parameters. Additionally or alternatively, a plurality of changes in the pattern of hybridization to the array for a first DNA sample isolated from an ecosystem of interest as compared to a second DNA sample isolated from an ecosystem of interest is associated with a change in one or more parameters. Samples may vary by location of the ecosystem, the time of sampling of a single ecosystem, or the location of sampling within a single ecosystem. 
     Embodiments of the present invention also comprise methods for identifying and/or generating bioindicators for ecosystems of interest. In one embodiment, a method of generating a bioindicator may comprise preparing a nucleic acid sample comprising a plurality of DNA molecules from an ecosystem of interest. The method may also comprise determining the sequence of at least some of the plurality of DNA molecules in the isolated DNA sample. The method may further comprise grouping the DNA sequences into operational taxonomic units (OTUs). Also, the method may comprise identifying at least one nucleic acid sequence that is specific to a single OTU. In one embodiment, the ecosystem of interest comprises a body of water. 
     Embodiments of the present invention also comprise bioindicators isolated using the methods and systems of the present invention. In one embodiment, the present invention comprises a bioindicator, or a collection of bioindicators, for the analysis of an ecosystem of interest, wherein a bioindicator comprises an isolated nucleic acid having a sequence derived from a single predetermined microbial operational taxonomic unit. 
     In yet another embodiment, the present invention also comprises a method to prepare a nucleic acid sample from a biosystem of interest, the nucleic acid sample comprising a plurality of bioindicator DNA sequences, comprising the step of amplifying a DNA sample isolated from a biosystem with a plurality of primers that have the ability to specifically amplify nucleic acid sequences comprising bioindicators. Also, in an embodiment, the present invention comprises an oligonucleotide primer having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. 
     In yet another embodiment, the present invention may comprise a method to identify a microbe comprising the ability to modify, or adjust to, an ecosystem. The method may comprise the step of identifying a bioindicator that is associated with a particular microbe. Also, the method may comprise the steps of identifying the bioindicator in at least one ecosystem, and correlating the presence of the microbe with a parameter specific to the ecosystem. Also, the method may comprise identifying the nature of the ability of the microbe to modify, or adjust to, the ecosystem. 
     There may be certain advantages that may be realized with various embodiments of the present invention. By using a microarray comprising nucleic acid sequences that are specific to microbial operational taxonomic units, the array may cover a wide variety of microbial taxa. Thus, the array may provide a single test that provides substantially comprehensive information on community structure. 
     The array may, in certain embodiments, provide quantitative data. For example, by using quantifiable labels to label individual samples, or to differentially label specific sequences in a single sample, the array may provide information on the abundance of specific organisms of interest, such as key bioindicators, pathogens, or microbial contaminants in a water system. 
     Also, the array, if applied to a number of samples over time, may be able to indicate the “trajectory” of the system as either improving or degrading, where probes associated with the quality of the ecosystem of interest are known. 
     Microorganisms generally respond rapidly to environmental changes. The microarrays of the present invention may therefore provide results in near “real-time” (i.e., within hours) of an event, such as a contamination, occurring. Thus, the array may detect changes in ecosystem perturbations early, so that potential problems may be quickly rectified. 
     There may be a large number of microorganisms specific to any one ecosystem of interest, such as a specific body of water. Also, the prevalence of particular microorganisms may vary depending upon the water source. Still, by prudent selection of the nucleic acid sequences used as part of an array, the array may be diagnostic of a plurality of microorganisms that are specific to many types of ecosystems. Thus, once developed, the array can be a highly cost-effective way to monitor a variety of ecosystems. Also, the microarray platform may be easily modified and expanded to include new targets of interest as they are identified. 
     A microbial community may be affected by biological changes, physical changes, or chemical changes to the environment. Because the microarrays of the present invention provide a measure of the microbial community, the array may be sensitive to a wide variety of changes that may occur in the ecosystem of interest. 
     The present invention may be better understood by reference to the description and figures that follow. It is to be understood that the invention is not limited in its application to the specific details as set forth in the following description and figures. The invention is capable of other embodiments and of being practiced or carried out in various ways. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The present invention may be better understood by reference to the following figures. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1  shows a method for identification of a bioindicator in accordance with an example embodiment of the present invention. 
         FIG. 2  shows rank-abundance curves for 16S rDNA libraries from three different lakes (Lake Townsend, NC; City Lake, NC; and Lake Toolik, AK) in accordance with an embodiment of the present invention. The curves on left-hand side represent Lake Townsend samples taken from different stations (Station 1 or Station 2) or at different times of the year (March or June). The median for each distribution partitions the operational taxonomic units (OTUs) into two groups shown in black and white; a stippled pattern is used where the median falls within an OTU. 
         FIG. 3  shows rank-abundance curves for 18S rDNA libraries from three different lakes (Lake Townsend, NC; City Lake, NC; and Lake Toolik, AK) in accordance with an embodiment of the present invention. The curves on left-hand side represent Lake Townsend samples taken from different stations (Station 1 or Station 2) or at different times of the year (March or June). The median for each distribution partitions the operational taxonomic units (OTUs) into two groups shown in black and white; a stippled pattern is used for a median that falls within an OTU. 
         FIG. 4  shows a phylogenetic tree of 16S rDNA operational taxonomic units (OTUs) derived by an Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) analysis using DNA isolated from three different lakes in accordance with an embodiment of the present invention. Numbers in parentheses designate the size of OTUs (i.e., the number of unique sequences per OTU). Identifications by BLAST analysis using the GenBank database are shown. Water samples from which the rDNA was isolated were as follows: LT-1J: Lake Townsend, station 1, June; LT-2J: Lake Townsend, station 2, June; LT-1M: Lake Townsend, station 1, March; CL: City Lake; TL: Toolik Lake. 
         FIG. 5  shows a phylogenetic tree of 18S rDNA operational taxonomic units (OTUs) derived by an Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) analysis using DNA isolated from three different lakes in accordance with an embodiment of the present invention. Numbers in parentheses designate the size of OTUs (i.e., the number of unique sequences per OTU). Identifications by BLAST analysis using the GenBank database are shown. Water samples from which the rDNA was isolated were as follows: LT-1J: Lake Townsend, station 1, June; LT-2J: Lake Townsend, station 2, June; LT-1M: Lake Townsend, station 1, March; CL: City Lake; TL: Toolik Lake. 
         FIG. 6  shows Venn diagrams that illustrate the relative patterns of shared operational taxonomic units (OTUs) for five separate fresh water samples in accordance with an embodiment of the present invention. The size of each sample component is based on the total number of OTUs for that water sample relative to the other samples. Water samples from which the rDNA was isolated were as follows: LT-1J: Lake Townsend, station 1, June; LT-2J: Lake Townsend, station 2, June; LT-1M: Lake Townsend, station 1, March; CL: City Lake; TL: Toolik Lake. 
         FIG. 7  shows quantitative PCR (Q-PCR) product accumulation curves generated using operational taxonomic unit (OTU)-specific primers and genomic DNA isolated from lake samples in accordance with an embodiment of the present invention where Panel (A) shows the relative abundance of the OTU for an unidentified cyanobacterium LD27 initially detected in Lake Townsend Station 1, March (LT-1M), panel (B) shows the relative abundance of OTU for  Zoogloea ramigera  initially detected in City Lake (CL), and panel (C) shows the relative abundance of OTU for  Asterionella formosa  initially detected in Lake Townsend Station 1, March (LT-1M). Pos: a positive control including a mixture of the three test DNAs; Neg: a negative control having no DNA; LT-1J: Lake Townsend, station 1, June; LT-1M: Lake Townsend, station 1, March; CL: City Lake. 
         FIG. 8  shows a method for monitoring water in accordance with an example embodiment of the present invention. 
         FIG. 9  illustrates the use of pattern analysis for assessing changes in an ecosystem in accordance with an embodiment of the present invention. 
         FIG. 10  illustrates the sequences from prokaryotic rDNA (Panels A-1 and A-2), eukaryotic rDNA (Panels B-1 and B-2), prokaryotic variable regions (Panels C-1 and C-2), and prokaryotic variable regions (Panels D-1 and D-2), in accordance with an embodiment of the present invention. 
         FIG. 11  shows a schematic diagram of amplification and labeling of two DNA samples for array analysis with universal primers for 16S rDNA or 18S rDNA (Panel A); and results of one hybridization experiment (Panel B), where the eukaryotic and prokaryotic PCR products are detected on the microarray, in accordance with an embodiment of the present invention. 
         FIG. 12  shows a schematic diagram of hybridization of an array to two samples, in accordance with an embodiment of the present invention, where multiplex PCR reactions of each sample use the same primers, but the product of the amplification reaction from the first sample is labeled with Cy3 (green) and the product of the amplification reaction from the second sample is labeled with Cy5 (red). Panel A shows a schematic representation of the red (darkest arrowhead) and green (lighter arrowhead) amplified DNA hybridizing to immobilized DNA at an individual position on the array; Panel B shows a schematic representation of the color as viewed at each array position, wherein the overall color may comprise an average of the colors of the hybridizing probes; and Panel C shows an actual results of a hybridization experiment using red and green labeled probes, where the top panel shows PCR reactions using 16S primers, and the bottom panel shows the same samples amplified with 18S primers, and where prokaryotic oligonucleotides are spotted on the left side of each panel and eukaryotic oligonucleotides are spotted on the right side of each panel; green spots in lower right-hand position of each grid are for orientation. 
         FIG. 13  shows an array that contains cyanobacteria sequences probed with amplification products generated using cyanobacteria multiplex primers from two water samples (one sample labeled in green and one sample labeled in red) that each contained cyanobacteria in accordance with an embodiment of the present invention, where oligonucleotide probes to 18S rDNA are spotted in Grid A and B, probes to 16S rDNA of  Escherichia coli  are spotted in Grid C, and twenty-one 16S rDNA probes containing cyanobacteria sequences, sequences associated with arsenic-responsive microbes, and other sequences identified in collected lake samples, are spotted in Grid D. 
         FIG. 14  shows hybridization of two samples to an array, where multiplex PCR reactions for both samples were identical and reaction products were either labeled with a red dye (Cy5) or a green dye (Cy3), allowing comparison of samples in accordance with an embodiment of the present invention. Shown are results from a test of two lake samples used to hybridize first singly (top panels) (left panel: Cy3-labeled sample; right panel: Cy5-labeled sample) and then together (bottom panel) to the same array of 105 oligonucleotide probes. The array is partitioned into duplicates of eight grids. 
     
    
    
     DETAILED DESCRIPTION 
     Ecogenomics is the recovery, characterization, and analysis of genomes recovered from organisms living in the natural environment. The present invention utilizes ecogenomics to develop nucleic acid based arrays for monitoring water supplies and other ecosystems. 
     Thus, the present invention provides methods, systems, and devices to monitor ecosystems. The methods, systems, and devices of the present invention may be used to monitor water. Embodiments of the present invention comprise bioindicators, and methods for isolating a bioindicator for the analysis of an ecosystem of interest. In one embodiment, the present invention comprises a method for developing bioindicators for monitoring water. The bioindicator may comprise a biological system, such as a microorganism, or a molecule, such as a nucleic acid sequence, that changes in response to a parameter that is related to the ecosystem of interest. The bioindicators may comprise known microorganisms or unknown microorganisms. Rather than measuring the microorganism itself, the present invention provides molecular bioindicators to monitor ecosystems of interest. 
     A bioindicator for the analysis of an ecosystem of interest may comprise an isolated nucleic acid having a sequence derived from a single predetermined microbial operational taxonomic unit (OTU). In one embodiment, the present invention may comprise a method for preparing a bioindicator for the analysis of a biosystem of interest comprising the steps of: preparing a nucleic acid sample comprising a plurality of DNA molecules from an ecosystem of interest; determining the sequence of at least some of the plurality of DNA molecules in the isolated DNA sample; grouping the DNA sequences into operational taxonomic units (OTUs); and identifying at least one nucleic acid sequence that is specific to a single OTU. 
     DNA from a variety of ecosystems may be used to develop bioindicators. In one embodiment, the ecosystem may comprise a body of water. For example, water from freshwater lakes may be used. Additionally or alternatively, water from estuaries may be used. Additionally or alternatively, water from other types of biosystems, such as tidal pools, wetlands, streams, rivers, and salt water may be used. 
     The method may utilize bioindicators that are shared among biosystems, and/or bioindicators that are specific to one, or a few, biosystems. Thus, a plurality of bioindicators may be developed. The bioindicators may be used as a means to analyze biosystems of interest. In one embodiment, the method may comprise the step of identifying the distribution of at least one of the OTUs in at least two ecosystems of interest. 
     Once the DNA molecules have been categorized and grouped by OTU, the sequences of the DNA molecules in each of the OTUs may be used to develop the collection of bioindicators as an assay system. Thus, the method may comprise preparing a collection of bioindicators wherein at least one of the OTUs comprises sequences that are unshared between at least two of the ecosystems of interest. Additionally and alternatively, the method may comprise preparing a collection of bioindicators wherein at least one of the OTUs comprises sequences that are shared between at least two of the ecosystems of interest. 
     The method may also include a step of immobilizing a plurality of oligonucleotide bioindicators at known locations on a substrate to form an array, wherein each oligonucleotide has a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU). In one embodiment, at least one of the OTUs comprises sequences that are shared between at least two of the ecosystems of interest. Alternatively or additionally, at least one of the OTUs may comprise sequences that are unshared between at least two of the ecosystems of interest. 
     The oligonucleotides immobilized on the array should be of sufficient length to provide specific hybridization to nucleic acid molecules isolated from various water samples that are used to probe the array. The immobilized oligonucleotides may be at least 20 nucleotides in length. In alternate embodiments, the immobilized oligonucleotides may range from about 30 to 200, or from 40 to 100 nucleotides in length. In one example embodiment, the immobilized oligonucleotides are each about 50 nucleotides in length. Specific hybridization does not require a perfect match between the oligonucleotide immobilized on the array and the ecosystem sample used to probe the array. As used herein, specific hybridization comprises hybridization such that a nucleic acid molecule isolated from the ecosystem of interest hybridizes to a single location (i.e., a single oligonucleotide sequence) on the array. As is known in the art, specific hybridization does not require that the immobilized oligonucleotide and the DNA sequence from the sample comprise perfect complementarity, but may allow for a mismatch at one or several base pairs. The level of mismatch will be determined at least in part by the sequence and length of the oligonucleotide immobilized on the array, as well as the hybridization conditions. 
     Also, the amount of the oligonucleotide immobilized on the array should be sufficient to allow detection of complementary nucleic acid sequences by the array, but in an amount such that background hybridization to unrelated sequences is avoided. In alternate embodiments, the oligonucleotides immobilized on the array range from about 1 fg to about 10 μg, or from about 50 fg to about 10 ng, or from about 0.5 pg to 1,000 pg, or from about 2 pg to 200 pg, or from about 8 pg to about 50 pg, at each location. 
     The operational taxonomic units may utilize variable ribosomal DNA (rDNA) sequences as a means to detect specific organisms. Thus, the immobilized oligonucleotides may comprise eukaryotic ribosomal DNA sequences, and/or prokaryotic ribosomal DNA sequences. Additionally, or alternatively, the immobilized oligonucleotides may comprise pathogen-specific sequences. Additionally, or alternatively, the immobilized oligonucleotides may comprise novel sequences from as yet unidentified microbes. 
     The oligonucleotides immobilized on the array may be derived from sequences found by analysis of microbes present in various sources of water. In one embodiment, oligonucleotides having sequences specific to organisms found in freshwater lakes may be used. Additionally or alternatively, oligonucleotides having sequences specific to organisms found in estuaries may be used. Additionally or alternatively, oligonucleotides having sequences specific to organisms found in other types of water systems, such as tidal pools, wetlands, streams, rivers, and salt water may be used. 
     In one embodiment, at least one immobilized oligonucleotide comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. In alternative embodiments, the array may comprise at least 10 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ. SEQ ID NO: 113 or a fragment thereof, or at least 20 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 50 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 100 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. 
     The bioindicators may be used to probe an array comprising nucleic acid sequences that are specific to organisms of interest. In one embodiment, the bioindicator may be evaluated to determine how the presence and/or absence of the bioindicator may correlate with a parameter of the ecosystem. Thus, the method may comprise using the polymerase chain reaction (PCR) to amplify a DNA sequence comprising a predetermined OTU. In one embodiment, at least one of the PCR primers comprises a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. For example, the bioindicator may be used to probe an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate, and wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU) so as to correlate the pattern of hybridization with a parameter that comprises at least part of the ecosystem. In an embodiment, the parameter comprises water quality. 
     Also, the method may use a first bioindicator from a particular microbe as an “entry point” for isolating other bioindicators from the microbe. Thus, in one embodiment, a bioindicator isolated from a portion of the genome of a microbe is used to identify a second bioindicator nucleic acid sequence from the same microbe. For example, the sequence of a first bioindicator nucleic acid molecule may be used to identify and isolate contiguous DNA sequence from the microbe that can serve as a bioindicator. 
     Embodiments of the present invention also comprise bioindicators isolated using the methods and systems of the present invention. In one embodiment, the present invention comprises a bioindicator, or a collection of bioindicators, for the analysis of an ecosystem of interest, wherein a bioindicator comprises an isolated nucleic acid having a sequence derived from a single predetermined microbial operational taxonomic unit (OTU). The bioindicator may comprise a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. Or, in alternate embodiments, the collection of bioindicators may comprise at least 2, or 5, or 10, or 20, or 40, or 50 distinct nucleic acid sequences that individually comprise a sequence identical to SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. 
     The present invention also comprises methods to monitor ecosystems. For example, one embodiment of the present invention may comprise a method for monitoring the water quality in a water sample of interest comprising measuring the hybridization of a nucleic acid sample from a water sample of interest to a plurality of oligonucleotides, and assessing at least one parameter of water quality based upon hybridization of the nucleic acid sample to the plurality of oligonucleotides. In one embodiment, an array of oligonucleotides is used. Thus, the plurality of oligonucleotides may be immobilized at known locations on a substrate, such that each location on the array comprises an oligonucleotide. 
     In one embodiment, the method may comprise the steps of: generating an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate, wherein each location on the array comprises an oligonucleotide having a sequence derived from a single, predetermined microbial operational taxonomic unit (OTU); preparing a nucleic acid sample from the ecosystem of interest; hybridizing the nucleic acid sample to the array; measuring hybridization of the nucleic acid sample to the array; and correlating hybridization of the nucleic acid sample to the array with a parameter that comprises at least part of the ecosystem. 
     In one embodiment, the ecosystem may comprise a body of water. For example, water from freshwater lakes may be used. Additionally or alternatively, water from estuaries may be used. Additionally or alternatively, water from other types of biosystems, such as tidal pools, wetlands, streams, rivers, and salt water may be used. 
     Where the ecosystem of interest comprises water, the parameter comprising at least part of the ecosystem may relate to the quality of the water. Thus, the parameter may relate to a quality that is important to evaluate if the water is suitable for drinking, cooking, bathing, agriculture, or other uses of water. 
     In one approach, the oligonucleotides are each selected to comprise different taxonomic units. Or, the oligonucleotides may be selected such that at least some are from the same microbial operational taxonomic unit (OTU). 
     The oligonucleotides immobilized on the array should be of sufficient length to provide specific hybridization to nucleic acid molecules isolated from various ecosystem samples that are used to probe the array. The immobilized oligonucleotides may be at least 20 nucleotides in length. In alternate embodiments, the immobilized oligonucleotides may range from about 30 to 200, or from 40 to 100 nucleotides in length. In one example embodiment, the immobilized oligonucleotides are each about 50 nucleotides in length. 
     Also, the amount of the oligonucleotide immobilized on the array should be sufficient to allow detection of complementary nucleic acid sequences by the array, but in an amount such that background hybridization to unrelated sequences is avoided. In alternate embodiments, the oligonucleotides immobilized on the array range from about 1 fg to about 10 μg, or from about 50 fg to about 10 ng, or from about 0.5 pg to 1,000 pg, or from about 2 pg to 200 pg, or from about 8 pg to about 50 pg, at each location. 
     The operational taxonomic units may utilize variable ribosomal DNA (rDNA) sequences as a means to detect specific organisms. Thus, the immobilized oligonucleotides may comprise eukaryotic ribosomal DNA sequences, and/or prokaryotic ribosomal DNA sequences. Additionally, or alternatively, the immobilized oligonucleotides may comprise pathogen-specific sequences. Additionally, or alternatively, the immobilized oligonucleotides may comprise novel sequences from as yet unidentified microbes. 
     The oligonucleotides immobilized on the array may be derived from sequences found by analysis of microbes present in various ecosystems of interest. In one embodiment, oligonucleotides having sequences specific to organisms found in freshwater lakes may be used. Additionally or alternatively, oligonucleotides having sequences specific to organisms found in estuaries may be used. Additionally or alternatively, oligonucleotides having sequences specific to organisms found in other types of water systems, such as tidal pools, wetlands, streams, rivers and salt water may be used. 
     In one embodiment, at least one immobilized oligonucleotide comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. In alternative embodiments, the array may comprise at least 10 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 20 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 50 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 100 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. 
     The array may be hybridized with a nucleic acid sample comprising at least one bioindicator that is isolated from the sample of interest. For example, PCR may be used to amplify rDNA sequences from genomic DNA from a water sample of interest, and the array used to probe those sequences. Alternatively or additionally, PCR may be used to amplify known pathogen-specific sequences from a water sample of interest, and the array used to probe those sequences. Alternatively and additionally, PCR may be used to amplify unidentified (i.e., novel) sequences specific to a water sample of interest, and the array used to probe those sequences. Alternatively or additionally, PCR may be used to amplify known non-pathogen specific sequences specific to a water sample of interest, and the array used to probe those sequences. 
     The bioindicator isolated from the ecosystem of interest may comprise nucleic acid sequences isolated from rDNA. In one embodiment, the nucleic acid sample hybridized to the array sequence comprises a plurality of rDNA sequences. For example, the nucleic acid sequence hybridized to the array may be generated using PCR primers derived at least in part from a ribosomal variable region so as to specifically amplify rDNA sequences. For example, the primers may comprise at least one oligonucleotide molecule having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. In separate and alternative embodiments, the primers used for amplification of DNA from a sample of interest may comprise at least 10 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof, or at least 20 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof, or at least 40 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. In one example embodiment, the primers used for amplification of a sample of interest include about 50 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. 
     By measuring hybridization of the array to a sample from an ecosystem of interest, changes in at least one parameter that are indicative of a change in the ecosystem may be measured. Thus, in one embodiment, the present invention comprises a method for analyzing a pattern to evaluate the status of a biosystem comprising the step of measuring hybridization of a nucleic acid sample to an array of oligonucleotides immobilized at known locations on a substrate, wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU), and correlating the hybridization of the nucleic acid to the array with a parameter that comprises at least part of the ecosystem. In one embodiment, a single change in the pattern of hybridization to the array for a first nucleic acid sample isolated from an ecosystem of interest as compared to a second nucleic acid sample isolated from an ecosystem of interest is associated with a change in one parameter. Or, a single change in the pattern of hybridization to the array for a first nucleic acid sample isolated from an ecosystem of interest as compared to a second nucleic acid sample isolated from an ecosystem of interest may be associated with a change in a plurality of parameters. Additionally or alternatively, a plurality of changes in the pattern of hybridization to the array of a first nucleic acid sample isolated from an ecosystem of interest as compared to a second nucleic acid sample isolated from an ecosystem of interest is associated with a change in one parameter. Or, a plurality of changes in the pattern of hybridization to the array of a first nucleic acid sample isolated from an ecosystem of interest as compared to a second nucleic acid sample isolated from an ecosystem of interest may be associated with a change in a plurality of parameters. In one embodiment, the pattern analysis may comprise a computer program including known patterns such that samples may be analyzed using a computer. Samples may vary by location of the ecosystem, the time of sampling of a single ecosystem, or the location of sampling within a single ecosystem. 
     Embodiments of the present invention also comprise arrays of oligonucleotides for monitoring an ecosystem of interest. For example, one embodiment of the present invention comprises a device comprising an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate, such that each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU). 
     For example, in an embodiment the present invention comprises a device for monitoring water quality comprising an array, wherein the array comprises a plurality of oligonucleotides immobilized at known locations on a substrate, and wherein each location on the array comprises an oligonucleotide having a sequence that is derived from a single, predetermined microbial operational taxonomic unit (OTU). 
     Also included as part of the present invention are systems for monitoring ecosystems of interest. Thus, another embodiment of the present invention comprises a system for monitoring an ecosystem of interest comprising an array comprising a plurality oligonucleotides immobilized at known locations on a substrate, such that each location on the array comprises an oligonucleotide having a sequence that is derived from a single predetermined microbial operational taxonomic unit (OTU), and a device able to measure hybridization of the nucleic acid sample to the array. The system may further include a DNA sample comprising a plurality of nucleic acid sequences derived from an ecosystem sample of interest. 
     In one embodiment, the ecosystem analyzed using the devices and or systems of the present invention may comprise a body of water. For example, water from freshwater lakes may be used. Additionally or alternatively, water from estuaries may be used. Additionally or alternatively, water from other types of biosystems, such as tidal pools, wetlands, streams, rivers and salt water may be used. 
     The oligonucleotides immobilized on the array should be of sufficient length to provide specific hybridization to nucleic acid molecules isolated from various water samples that are used to probe the array. The immobilized oligonucleotides may be at least 20 nucleotides in length. In alternate embodiments, the immobilized oligonucleotides may range from about 30 to 200, or from 40 to 100 nucleotides in length. In one example embodiment, the immobilized oligonucleotides are each about 50 nucleotides in length. 
     Also, the amount of the oligonucleotide immobilized on the array of the devices and/or systems of the present invention should be sufficient to allow detection of complementary nucleic acid sequences by the array, but in an amount such that background hybridization to unrelated sequences is avoided. In alternate embodiments, the oligonucleotides immobilized on the array range from about 1 fg to about 10 μg, or from about 50 fg to about 10 ng, or from about 0.5 pg to 1,000 pg, or from about 2 pg to 200 pg, or from about 8 pg to about 50 pg, at each location. 
     The operational taxonomic units may utilize variable ribosomal DNA (rDNA) sequences as a means to detect specific organisms. Thus, the immobilized oligonucleotides may comprise eukaryotic ribosomal DNA sequences, and/or prokaryotic ribosomal DNA sequences. Additionally, or alternatively, the immobilized oligonucleotides may comprise pathogen-specific sequences. Additionally, or alternatively, the immobilized oligonucleotides may comprise novel sequences from as yet unidentified microbes. 
     The oligonucleotides immobilized on the array of the devices and/or systems of the present invention may be derived from sequences found by analysis of microbes present in various sources of water. In one embodiment, oligonucleotides having sequences specific to organisms found in freshwater lakes may be used. Additionally or alternatively, oligonucleotides having sequences specific to organisms found in estuaries may be used. Additionally or alternatively, oligonucleotides having sequences specific to organisms found in other types of water systems, such as tidal pools, wetlands, streams, rivers, and salt water may be used. 
     In one embodiment, at least one immobilized oligonucleotide comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. In alternative embodiments, the array may comprise at least 10 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 20 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 50 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or at least 100 immobilized oligonucleotides that individually comprises a sequence identical to any one of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof, or the complement of SEQ ID NO: 5–SEQ ID NO: 113 or a fragment thereof. 
     The array devices and/or systems of the present invention may be hybridized with a nucleic acid sample that is isolated from the sample of interest. For example, PCR may be used to amplify rDNA sequences from genomic DNA from a water sample of interest, and the array used to probe those sequences. The nucleic acid sequence for hybridizing to the array may be generated using polymerase chain reaction (PCR) primers derived at least in part from at least one of a eukaryotic or prokaryotic ribosomal variable region. In an embodiment at least one of the PCR primers comprises a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. In alternate embodiments, the PCR primers comprise at least 2, or 5, or 10, or 20, or 40, or 50 primers that individually have a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. Alternatively or additionally, PCR may be used to amplify known pathogen-specific sequences from a water sample of interest, and the array used to probe those sequences. Alternatively and additionally, PCR may be used to amplify unidentified (i.e., novel) sequences specific to a water sample of interest, and the array used to probe those sequences. Alternatively or additionally, PCR may be used to amplify known non-pathogen specific sequences specific to a water sample of interest, and the array used to probe those sequences. 
     The present invention also provides methods for isolating samples from an ecosystem of interest that may be analyzed using molecular methods. Thus, in yet another embodiment, the present invention comprises a method to prepare a nucleic acid sample from a biosystem of interest, the nucleic acid sample comprising a plurality of bioindicator DNA sequences, wherein the method comprises amplifying a DNA sample isolated from a biosystem with a plurality of primers that have the ability to specifically amplify nucleic acid sequences comprising bioindicators. In one embodiment, at least some of the primers comprise at least a portion of a variable region of a ribosomal RNA. For example, the primers may comprise at least one oligonucleotide molecule having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. In separate and alternative embodiments, the primers used for amplification of DNA from a sample of interest may comprise at least 10 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316, or a fragment thereof, or at least 20 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof, or at least 40 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316, or a fragment thereof. In one example embodiment, the primers used for amplification of a sample of interest include about 50 oligonucleotide molecules having a sequence identical to any one of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. 
     The present invention also comprises compositions for isolating samples from an ecosystem of interest that may be analyzed using molecular methods. Thus, in yet another embodiment, the present invention comprises an oligonucleotide having a sequence identical to any one sequence of SEQ ID NO: 114–SEQ ID NO: 316 or a fragment thereof. 
     The present invention may also comprise a method to identify microbes that are able to modify, or adjust to, an a particular ecosystem. In one embodiment, the method may comprise the steps of: (a) identifying a bioindicator that is associated with a particular microbe; (b) identifying the bioindicator in at least one ecosystem; and (c) correlating the presence of the microbe with a parameter specific to the ecosystem. The method may further comprise identifying the nature of the ability of the microbe to modify, or adjust to, the ecosystem. For example, the identification of a microbe whose presence shows an association with a particular toxin may indicate that the microbe has the ability to modify the toxin and/or modify the effect of the toxin on the environment. 
     Definitions 
     As used herein, the following terms shall have the definitions set out below. Also, in accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques may be used that will be apparent to those skilled in the relevant art. Such techniques are explained fully in the literature (see, e.g., Maniatis, Fritsch &amp; Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985). 
     As used herein, “taxonomic unit” is a group of organisms that are considered distinct enough to be treated as a separate unit. A taxonomic unit may comprise a family, genus or species but is not limited as such. Also as used herein, each “operational taxonomic unit (OTU)” comprises a group of one or more microorganisms that are treated as a unit based on a SSU rDNA sequence identity of ≧97.5% among all members of the group. 
     As used herein, a bioindicator is an organism or part thereof, or a biological process, whose change in numbers, structure, or function points to a change, or a plurality of changes, in the environment. Generally, a bioindicator has a relatively high and easily identifiable sensitivity to selective environmental influences. The changes in the environment may relate to various changes in the ecosystem such as changes in a single chemical or chemical combinations in either absolute abundance or relative abundance to each other, changes in temperature, changes in biological populations, and the like. Any microbe whose relative abundance depends upon a biotic and/or an abiotic aspect(s) of biosystem of interest, or whose appearance is restricted to a subset of biosystems, is a bioindicator. Moreover, a plurality of two or more bioindicators (two or more present or more abundant than in the absence of a condition, two or more absent or less abundant than in the absence of the condition, or combinations of two or more in abundance and/or presence) may together indicate a condition of the ecosystem of interest. A condition of an ecosystem may refer to either a single biotic or abiotic factor or a combination of such factors. 
     A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. 
     A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), microbes, viruses, plasmids, and chromosomes. 
     DNA molecules may be identified by their nucleic acid sequences, which are generally presented in the 5′ to 3′ direction, wherein 5′ and 3′ indicate the linkages formed between the 5′-phosphate group of one nucleotide and the 3′-hydroxyl group of the next. As used herein, for a sequence presented in the 5′-3′ direction, its complement is the DNA strand which hybridizes to that sequence according to the Watson-Crick base pairing model. Thus, the sequence of the complement is defined by the sequence of the original strand, such that adenine base-pairs with thymine, and cytosine base-pairs with guanine. 
     The term “gene” means a region of DNA encoding for the mRNA sequence that codes for a given protein/polypeptide along with elements regulating mRNA expression, or a region of DNA encoding for a ribosomal RNA (rRNA) sequence that performs a structural function as a subunit of ribosomes along with elements regulating rRNA expression. 
     “Messenger RNA” or “mRNA” shall mean a RNA molecule that encodes for a polypeptide. “Ribosomal RNA” or “rRNA” shall mean a RNA molecule that performs a structural function in ribosomes. 
     “Complementary DNA” or “cDNA” shall mean the DNA molecule synthesized by polymerization of deoxyribonucleotides from an RNA template by an enzyme with reverse transcriptase activity. 
     An “isolated nucleic acid” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as an oligonucleotide, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. 
     “Oligonucleotide”, as used herein, is defined as a molecule comprised of two or more deoxy- or ribonucleotides, preferably more than eight. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. As is known in the art, it is possible that each of the olignucleotide molecules may vary by a few bases. For example, the synthesis of the oligonucleotide may result in a portion of the oligonucleotides being less than full length. Or, a portion of the oligonucleotides immobilized at a particular location may degrade by a small percentage over time. Such oligonucleotides are considered to be “fragments” of the original oligonucleotide. Thus, an oligonucleotide sequence that is derived from, and specific to, a single, individual OTU may include fragments of that oligonucleotide. 
     A “DNA fragment” includes polynucleotides and/or oligonucleotides and refers to a plurality of joined nucleotide units formed from naturally-occurring bases and cyclofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits. “DNA fragment” also refers to purine and pyrimidine groups and moieties which function similarly but which have non naturally-occurring portions. Thus, DNA fragments may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species. They may also contain altered base units or other modifications, provided that biological activity is retained. DNA fragments may also include species that include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the cyclofuranose portions of the nucleotide subunits may also occur as long as biological function is not eliminated by such modifications. 
     The term “identical” refers to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning two sequences and refers to the number of identical residues (i.e., amino acid or nucleotide) at positions shared by the compared sequences. Sequence alignment and comparison may be conducted using the algorithms standard in the art (e.g. Smith and Waterman,  Adv. Appl. Math.  2:482 (1981); Needleman and Wunsch,  J. Mol. Biol.  48:443 (1970); Pearson and Lipman,  Proc. Natl. Acad. Sci . ( USA ), 85:2444 (1988)) or by computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wis.) publicly available as BLAST and FASTA. Also, ENTREZ, available through the National Institutes of Health, Bethesda Md., may be used for sequence comparison. In one embodiment, percent identity of two sequences may be determined using GCG with a gap weight of 1, such that each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences. 
     “Primer” shall refer to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, the source of primer and the method used. For example, for multiplex PCR amplification of genomic DNA, the oligonucleotide primer typically contains 15–30 or more nucleotides, although it may contain fewer nucleotides. 
     The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product. 
     “Probe” shall refer to any oligonucleotide that can be immobilized at individual addresses on the array. Such probes are single-stranded DNA molecules. The act of “probing” as used herein describes the step of hybridizing a nucleic acid sample with a probe having a known sequence, or a plurality of probes having known sequences (i.e., an array), to determine whether any of the sequences in the sample are complementary to the probe sequence(s). 
     As used herein, the term “hybridization” refers generally to a technique wherein denatured RNA or DNA is combined with complementary nucleic acid sequence that is either free in solution or bound to a solid phase. As recognized by one skilled in the art, complete complementarity between the two nucleic acid sequences is not a pre-requisite for hybridization to occur. Hybridization conditions can be described as ranging from low to high stringency. Hybridization that occurs under high stringency conditions is specific in that a large percentage of complementarity between two nucleotide molecules is required for hybridization to occur under stringent conditions. Hybridization that occurs under low stringency conditions is less specific in that a lower percentage of complementarity between two nucleotide molecules is required for hybridization to occur under stringent conditions. Even under highly stringent conditions, there may not be perfect complementarity between two oligonucleotide molecules that hybyridize. Generally, highly stringent conditions refer to washing hybrids in low salt buffer at high temperatures. Hybridization may be to filter bound DNA using hybridization solutions standard in the art such as 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS) at 65° C., and washing in 0.25 M NaHPO 4 , 3.5% SDS followed by washing 0.1×SSC/0.1% SDS at a temperature ranging from room temperature to 68° C. depending on the length of the probe (see e.g. Ausubel, F. M. et al.,  Short Protocols in Molecular Biology,  4 th  Ed., Chapter 2, John Wiley &amp; Sons, N.Y). For example, a high stringency wash comprises washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. for a 14 base oligonucleotide, or at 48° C. for a 17 base oligonucleotide, or at 55° C. for a 20 base oligonucleotide, or at 60° C. for a 25 base oligonucleotide, or at 65° C. for a nucleotide probe about 250 nucleotides in length. Equivalent stringencies may be obtained with other wash solutions by varying the temperature as is known in the art. Nucleic acid probes may be labeled with radionucleotides by end-labeling with, for example, [γ- 32 P]ATP, or by incorporation of radiolabeled nucleotides such as [α- 32 P]dCTP by random primer labeling. Alternatively, probes may be labeled by incorporation of biotinylated or fluorescein labeled nucleotides, and the probe detected using antibodies to the label, or by chemical incorporation of cyanin dyes as described herein. 
     As used herein, “restriction endonucleases” and “restriction enzymes” shall refer to bacterial enzymes that cleave double-stranded DNA at or near a specific nucleotide sequence. 
     A polypeptide refers to any peptide generated from a protein or the full-length protein itself. A polypeptide may include the full-length protein or a fragment generated by proteolytic cleavage, chemical cleavage, or other means. 
     As used herein, an array or microarray is a solid-state grid containing short sequences of nucleic acid (usually DNA) of known sequence fixed at a particular position (i.e., location or address) on the grid. DNA arrays are usually termed microarrays due to the small size of the grid and the small amounts of nucleotide (e.g., μM or nM amounts) present at each address. 
     As used herein, a computer program comprises a computer-encoded language that encodes the steps required for the computer to perform a specific task or tasks. 
     Also, as used herein, software comprises the computer program(s) used in conjunction with any other operating systems required for computer function. 
     As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. 
     Water Surveillance Using Nucleic Acid Based Arrays 
     The present invention recognizes that the abundance of certain microorganisms may be affected by local water conditions. These conditions may vary for individual species and strains. For example, whereas one species might proliferate in phosphate-rich water, another species may prefer low-phosphate water (Kilham et al, 1986 , Limnol. Oceanogr.,  31:1169–1181; Siver et al., 1999 , Limnol. Oceanograph.,  44:1928–1955). Also, phytoplankton and benthic algae may be used as indicators of eutrophication (Shubert, L. E. (ed.), 1984 , Algae as ecological indicators . Academic Press, N.Y; Stoermer, E. F. and J. P. Smol, 1999 , The Diatoms: applications for the environmental and earth sciences , Cambridge Univ. Press, Cambridge, UK). Or, the abundance of known microbial species can vary as a function of pollution levels (Lemke et al., 1997 , Microb. Ecol.,  34:224–231). It has been shown that the presence of high levels of heavy metals in water may be reflected in the tissue of the marine limpet (Pérez-López, M., et al., 2003 , Journal of Environmental Health, Part A—Toxic/Hazardous Substances and Environmental Engineering,  38:2845–2856). Also, coliform bacteria may be used as an indicator of the presence of human sewage in aquatic systems. 
     Over the past decade, direct observation and culturing of microbes has been complemented by emerging molecular approaches, including: in situ hybridization (Schöhuber, W., et al. 1991 , Appl. Environ. Microbiol.  65:1258–1267), selective PCR detection of individual taxa (e.g. Oldach, D. W., et al. 2000 , Proc. Natl. Acad. Sci. USA,  97:4303–4308; Rublee, P. A., et al. 2001 , Environ. Health Perspectives,  109 [Supplement 5]:765–767), community assays such as Differential Gradient Gel Electrophoresis (DGGE) analyses (e.g., Díez, B., et al. 2001 , Appl. Environ. Microbiol.,  67:2942–2951) and filter macroarray hybridization (e.g., Rudi, K., et al. 2000 , Appl. Environ. Microbiol.,  66:4004–4011). In situ hybridization allows taxon-specific identification and enumeration of target organisms. Although highly specific, the method is time consuming as it generally involves microscopic observation of the sample. PCR, the polymerase chain reaction, may detect a targeted organism that exists in low abundance in the natural environment. Selective PCR detection of individual microbes is highly specific, rapid, and may even be quantitative (e.g., real-time quantitative PCR), but may be limited in that primers specific to the sequence to be amplified must be available. DGGE analysis has become a relatively common approach to community assessment of prokaryotic or even eukaryotic communities, but is limited in that it relies on the assumption that different nucleic acid sequences will display differential mobility in a gradient gel, which is not always the case. 
     From the standpoint of environmental investigations, recent microarray development efforts have increasingly focused on the identification of genes from specific microbial organisms associated with environmental processes, such as nitrogen fixation, or with the detection of specific microbes (Wu et al., 2001 , Appl Environ. Microbiol.,  67:5780–5790). Most of the microbes that exist in freshwater sources, however, are unknown and/or unculturable (Kaeberlein et al., 2002 , Science,  296:1127–1129; Hiorns et al., 1997 , Appl. Evnviron. Microbiol.,  63: 2957–2960; Lopez-Garcia et al., 2001 , Nature,  409:603–607). 
     A first step of developing methods and systems to monitor water may be the identification of suitable bioindicator molecules. For example, one aspect of developing an array to monitor a water supply is to develop nucleic acid sequences that are diagnostic of the aqueous microbial population.  FIG. 1  shows an embodiment of a method of the present invention that may be used to identify potential ecosystem biomarkers. As shown in  FIG. 1 , the method  2  may comprise a first step of isolating a sample from an ecosystem of interest  4 . The ecosystem may comprise a body of water. In alternate embodiments, water from a freshwater lake, an estuary, a tidal pool, wetlands, a stream, river, or salt water may be used. 
     The bioindicator may comprise the microorganism itself, or a molecule that provides information about the microorganism. The bioindicator may comprise a nucleic acid molecule. Nucleic acid molecules may be useful as bioindicators as nucleic acid molecules comprise a source of qualitative and quantitative information. By analyzing the sequence of the DNA molecules in the water sample, information about the genetic make-up of the microbes present in the sample may be obtained. Also, by measuring the amount of DNA molecules in the water sample, information about the amount of specific microbes in the sample may be obtained. As described herein, nucleic acid molecules, such as DNA, may be used to identify and classify microorganisms into operational taxonomic units (OTUs). Thus, the method may comprise the step of isolating nucleic molecules from the water sample  6 . 
     The method may next comprise the step of determining the sequence of DNA molecules isolated from the sample of interest  8 . In an embodiment, rDNA may be used as diagnostic sequences. Thus, to make an array for monitoring water, a first step may comprise the isolation and sequence characterization of 16S rDNA (prokaryotic) and 18S (eukaryotic) rDNA species from selected water reservoir samples. The DNA molecules selected for analysis may comprise small subunit (SSU) ribosomal RNA genes (SSU rDNA). In this way, recovery of DNA sequences may be based on the endogenous abundance of individual microbes, and is not restricted to the recovery of known microorganisms. 
     The DNA sequences isolated from the ecosystem of interest may be used to generate bioindicator probes. In one embodiment, the bioindicator probes are categorized into operational taxonomic units (OTUs)  10 . Both prokaryotic and eukaryotic SSU rDNA can be useful targets for determination of operational taxonomic units because SSU rDNA sequences contain highly conserved nucleotide regions interspersed with variable regions. The conserved sequences provide an anchor by which a plurality of different rDNA sequences may be isolated from a sample. By using primers that hybridize to the conserved regions of either eukaryotic or prokaryotic rRNA genes, a library of amplified rDNA sequences that are different in the variable regions may be isolated from a single sample. Primers that may be used for amplification of prokaryotic rDNA sequences may comprise SEQ ID NOS: 1 and 2 (Table 1). Primers that may be used for amplification of eukaryotic rDNA sequences may comprise SEQ ID NOS: 3 and 4 (Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sequences and target positions of primers used to amplify rRNAs 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
               
               
                   
                   
                 ID 
                   
               
               
                 Primer 
                 Sequence 
                 NO: 
                 Location 
               
               
                   
               
               
                 16S 
                 AGAGTTTGATCCTGGCTCAG 1   
                 1 
                 8–27 2   
               
               
                 Forward 
               
               
                 16S 
                 AAGGAGGTGATCCAGCCGCA 1   
                 2 
                 1541–1522 2   
               
               
                 Reverse 
               
               
                 18S 
                 AACCTGGTTGATCCTGCCAGT 3   
                 3 
                 1–21 4   
               
               
                 Forward 
               
               
                 18S 
                 TGATCCTTCTGCAGGTTCACCTAC 3   
                 4 
                 1795–1772 4   
               
               
                 Reverse 
               
               
                   
               
               
                   1 Primers from Edwards et al., 1989, Nucleic Acids Res., 17, 7843–7853; Bruce et al., 1992, Appl. Environ. Microbiol., 58, 3413–3416. 
               
               
                   2 Position in  E. coli  (Brosius et al., 1981, J. Mol. Biol., 148, 107–127). 
               
               
                   3 Primers from Medlin et al., 1988, Gene 71, 491–499. 
               
               
                   4 Position in  S. cerevisiae  (Rubstov et al., 1980, Nucl. Acids Res., 8, 5779–5794). 
               
            
           
         
       
     
     The amplified rDNA products may be used to provide a foundation for phylogenetic classification and comparison of both prokaryotic and eukaryotic microbial species isolated from the water samples of interest (see e.g., McCaig, A. E., et al., 1999 , Appl. Environ. Microbiol.,  65:1721–173036, Reysenbach, A. L., et al., 1992 , Appl. Environ. Microbiol.,  58:3417–3418; Pace et al., 1986 , Adv. Microb. Ecol.,  9:1–55; Sogin and Gunderson, 1987 , Annals. NY Acad. Sci.  503:125–139). A level of 97.5% sequence identity is a generally accepted criterion by which rDNAs may be placed in the same operational taxonomic unit (OTU). Because multiple small subunit rDNAs may reside within a genome for any single species (Farrelly et al., 1995 , Appl. Environ. Microbiol.  61:2798–2801), a 97.5% level of sequence identity generally allows for the possibility that sequences in the same species are recovered. In performing this type of analysis, it may be necessary to check for artifactual sequences resulting from the amplification protocol (e.g., CHIMERA-CHECK; Kopzcysnski et al., 1994 , Appl. Environ. Microbiol.,  60:746–748; Wang and Wang, 1995 , Appl. Environ. Microbiol.,  63:4645–4650; Qui et al., 2001, App. Environ. Microbiol., 58: 2717–2722). 
     The present invention recognizes that the sequences of small subunit rRNAs in prokaryotes and eukaryotes may allow for phylogenetic classification of known and novel species as operational taxonomic units (OTUs). For example,  FIGS. 2 and 3  show rank-abundance curves for 16S rDNA sequences, and 18S sequences, respectively, isolated from five separate samplings from three different lakes that were organized as operational taxonomic units. The water samples were taken from the following lakes: Lake Townsend, NC, Station 1, in June; Lake Townsend, NC, Station 2 in June; Lake Townsend, Station 1, in March; City Lake, NC; and Lake Toolik, AK. It can be seen that some of the OTUs have multiple members (i.e., # clones &gt;1), whereas many of the OTUs were represented by only one sequence. In one embodiment, a sequence from a defined OTU will recognize (i.e., hybridize to) other members of the OTU under the conditions used for hybridization of the array. Thus, in one embodiment, an array may be made using unique OTUs as isolated from the various samples. 
     The sequences of rDNAs from isolated samples may be used to generate phylogenetic trees. In one embodiment, the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) may be used to compare the sequence data from OTUs and to generate a phylogenetic tree. This type of analysis may be used to confirm the relationship between known sequences, and to order newly identified sequences and OTUs. For example,  FIG. 4  shows a prokaryote tree constructed using 111 different 16S rDNA OTUs, including 40 OTUs that are based on known rDNA sequences.  FIG. 5  shows a eukaryote tree constructed using 109 18S rDNA OTUs, and including 22 known rDNA sequences. In  FIGS. 4 and 5 , Lake Townsend, NC, samples are denoted LT (1J, Station 1 in June; 2J, Station 2 in June; 1M, Station 1 in March), City Lake, NC samples are denoted CL, and Toolik Lake, AK samples are denoted TL. 
     The sequence information may also permit the development of species specific primers. Species-specific primers may be used to characterize a variety of prokaryotic and eukaryotic microbes such as cyanobacteria,  Mycobacterium, Pfiesteria piscicida , and other types of microbial species (see e.g., Edwards, U., et al., 1989 , Nucleic Acids Res.,  17:7843–7853; Reysenbach, A. L., et al., 1992 , Appl. Environ. Microbiol.,  58:3417–3418; Shi, W., et al., 2002 , Appl. Environ. Microbiol.,  68:3859–3866; and O&#39;Brien, W. J., et al., 1997, The Limnology of Toolik Lake, p. 61–106, In: Freshwaters of Alaska—ecological syntheses, A. M. Milner and M. W. Oswood (eds), Springer-Verlag Publishers, New York, N.Y.). 
     Bioindicators may be unique to a specific biosystem, or may be shared among a plurality of biosystems. A diversity of microbial species may be readily retrievable even from a single body of water. Also, while any one body of water may have several unique OTUs, it is highly likely that the sample will include OTUs that are common to other biosystems. Such common OTUs may represent 10% or more of the rDNAs analyzed in the sample, and may be shared across water samples. Also, an environmental event that occurs in an environmental community, such as a contamination, may alter the abundance of individual microbial species and related bioindicators in that community. The method may therefore comprise the step of determining whether an operational taxonomic unit (OTU) and/or an individual or species-specific DNA sequence is specific to a particular ecosystem, or varies in abundance between ecosystems. For example, a bioindicator for assessing freshwater microbial communities may comprise nucleic acid sequences that are characteristic of the freshwater system, or that are diagnostic of the response of microbes to certain changes in the fresh water environment. Or, a bioindicator for assessing marsh water microbial communities may comprise nucleic acid sequences that are characteristic of the marsh environment, or that are diagnostic of the response of microbes found in the marsh environment to certain changes in a marsh water environment. Thus, referring back to  FIG. 1 , the method may therefore comprise identifying bioindicators or OTUs that vary among ecosystems 12. The method may also comprise identifying bioindicators or OTUs that are shared among ecosystems 13. 
     To determine which OTUs, if any, appear in more than one ecosystem, the sequences from a library of DNA sequences isolated from a biosystem of interest may be compared to those sequences in every other library from biosystems of interest in pairwise library comparisons, and a similarity coefficient may be calculated for each pairing. For example, in an analysis of five different samplings of DNA molecules from three different lakes (Lake Townsend, NC; City Lake, NC; and Toolik Lake, AK) there was some overlap between every pair of samples. There were also notable differences between the lakes. For example, none of the prokaryotic sequences were shared between Lake Townsend, a mesotrophic lake in North Carolina, and Toolik Lake, an oligotrophic lake in Alaska. 
     Venn diagrams may be used depict sample comparisons at different spatial and temporal scales to identify sequences that may differ between biosystems. Referring now to  FIG. 6 , in a fine-scale spatial comparison of samples taken in June from stations  1  and  2  at Lake Townsend, NC (LT-1J and LT-2J), it can be seen that even between samples that may be expected to be highly similar, differences in sequences may be found. For example, for LT-1 J and LT-2J, there were two rDNA sequences that occurred four times as often in LT-2J as in LT-1J: an unidentified 16S rDNA OTU, and an 18S rDNA from  Geminigera cryophila  (not shown). Such unequal occurrences of an OTU between samples may signify a detectable difference in the relative abundance of a specific microbial population between samples, and OTUs that consistently vary in frequency among samples are potential bioindicators. 
     Once the nucleic acid sequences isolated from various ecosystems have been identified and compared, it may be possible to compile OTUs as putative bioindicators  14  ( FIG. 1 ). For example, for five samplings from three different lakes, 26 different eukaryotic OTUs represented by multiple copies, including 11 that are associated with known species, may be identified. Another 79 eukaryotic OTUs may be obtained as single copy clones, with many of the single-copy OTUs representing unidentified species (Marshall, 2002, Masters Thesis, University of North Carolina at Greensboro). Also, 45 different prokaryotic OTUs may be detected in multiple copies. Of these, 10 species are associated with a known species, and 19 species display substantial homology to reported sequences for as yet unidentified species. Another 92 single copy rDNA sequences, most from unidentified prokaryotes, may be recovered (Amos, 2002, Masters Thesis, University of North Carolina at Greensboro). As described in more detail herein, all of the sequences identified from the ecosystems of interest (e.g., water samples), whether derived from known or previously unidentified rDNA sequences, may be used as probes printed on an array of the present invention. 
     The method may next comprise the step of organizing a nucleic acid array that can reflect microorganisms that are common to multiple communities, as well as organisms that are specific to one or only a few communities to thereby monitor the effect of an ecological change  16 . Thus, the ability to assess environmental parameters of water quality may require a sufficient number of bioindicator species that have unique profiles in different ecosystems, but may also require species that are shared among samples to allow for general application. 
     The array may comprise nucleic acids that are specific to known organisms. Additionally or alternatively, the array may comprise nucleic acids that are grouped to provide information about various taxonomic groups. For example, the array may comprise a plurality of prokaryotic and/or eukaryotic nucleic acid sequences derived from specific rDNAs. Or, the array may comprise a plurality of nucleic acid sequences organized by operational taxonomic unit. Notably, there is no requirement that the environmental DNA samples used to develop the array are the same as the environmental communities to be analyzed. 
     Samples that are derived from very different environmental communities may be expected to vary more than samples that are derived from similar communities, or the same community. Still, samples taken from a single lake, but either at a different location, or a different season, may also show variation (e.g.,  FIG. 6 ). The variation may be qualitative, in that specific OTUs are either absent from, or present in, a water sample. Or, it may be that a certain OTU is much less abundant in one sample than another sample. For example,  FIG. 7  illustrates a quantitative PCR determination of the relative amounts of three nucleic acid sequences found in each of three lake samples tested, but in highly varied amounts. For each of the curves shown in  FIGS. 7  ( 7 A,  7 B, and  7 C), the second curve from the left represents the sample from which the nucleic acid of interest was detected in high abundance, and the third and fourth curves from the left represent other lake samples tested. Also, results for a positive control, including each of the test samples, and a negative control with no DNA, are shown. Such skewed amplification curves may be found where DNAs vary in quantity between samples. In alternate embodiments, the template DNAs may vary 10-fold, or 20-fold, or 50-fold, or more than 200-fold, between samples. 
       FIG. 8  provides a schematic representation of a method that may be used to monitor water in accordance with an embodiment of the present invention. The method may comprise the first step 16 of generating an array comprising a plurality of oligonucleotides immobilized at known locations on a substrate, wherein each location on the array comprises an oligonucleotide having a sequence derived from a single, predetermined microbial operational taxonomic unit (OTU) as is described herein for  FIG. 1 . 
     Next, the method may comprise the step of isolating a sample from an ecosystem of interest  22 . In one embodiment, the ecosystem may comprise water. Samples may be collected from one body of water at different levels, or at different locations, and/or during different times of the year. Also, samples from different bodies of water may be used. For example, samples may be collected from different lakes, pools, estuaries, or marshes. Or, bodies of water comprising different types of growth levels may be used (e.g., eutrophic, mesotrophic, or oligotrophic). Or, samples may be obtained from a body of water because there is a reason to suspect that the body of water has been contaminated in some way. 
     The method may next comprise the step of preparing a DNA sample from the ecosystem of interest  24 . For example, the polymerase chain reaction (PCR) may be used to amplify DNA sequences from a water sample of interest. The PCR amplification may use primers designed to amplify sequences that, if present in the sample and amplified, are complementary to the sequences on the array. In that way, if a sequence on the array is present in the sample of interest, it may be detected. In one example, a multiplex (i.e., multiple primer) PCR amplification is used to amplify multiple sequences from the sample of interest that are complementary to sequences immobilized on the array. Primer pairs that may be used for multiplex PCR may include a sequence selected from the group of SEQ ID NO: 114–SEQ ID NO: 316. 
     The amplified DNA molecules from the sample of interest may be then labeled in some manner  26 . For example, the amplified DNA molecules may be labeled by the chemical incorporation of a fluorescent dye(s) as described herein. 
     Next, the method may comprise the step of hybridizing the labeled DNA sample from the ecosystem of interest to the array  28 . In one embodiment, a high stringency hybridization is used. For example, the hybridization conditions may comprise the conditions as described herein. 
     The presence of sequences in the sample of interest that are complementary to putative bioindicator sequences on the array may be determined by measuring locations on the array that exhibit hybridization to the labeled probe  30 . In this way, the identity, and in some cases, the relative amounts, of sequences that are in the sample may be determined. 
     The method may then comprise the step of correlating hybridization of the DNA sample to the array with a parameter that comprises at least part of the ecosystem. For example, if two samples of water (e.g., sample A and sample B) are hybridized to the same array, and different hybridization patterns result, than the oligonucleotide(s) on the array that displays a change in hybridization may correspond to a DNA sequence(s) that is diagnostic of a difference, or a plurality of differences between the two samples. Thus, the method may comprise the step of determining whether the pattern of hybridization at any one position or at a plurality of positions on the array changes  32 . 
     In one embodiment, no change in the pattern of hybridization at any one position or at a plurality of positions on the array is detected upon hybridization with two different samples. In this, case, no change in water quality is detected, and no action is required  34 . Alternatively, a change in the pattern of hybridization at any one position or at a plurality of positions on the array is detected upon hybridization with two different samples. In this case the change in the water quality may be recorded  36  and the nature of the change determined  38  such that appropriate action may be taken. 
     An example of the application of pattern analysis to the evaluation of a biosystem is shown in  FIG. 9 . As a first step, the method may include determining the pattern of hybridization on the array  32 . Methods for measuring hybridization may include use of a radiolabeled dNTP during amplification of the sample DNA such that DNA sequences that hybridize to the array may be detected by autoradiography. Alternatively, the amplified PCR products may be labeled with a fluorescent dye and hybridization detected using a fluorometer. Once the pattern of hybridization has been measured and recorded, the pattern may be compared with the pattern of hybridization produced by a separate sample, such that any changes in hybridization may be documented. At this point, a determination may be made as to whether the new hybridization pattern has a change that is associated with a known hybridization pattern  39 . If it is determined that the pattern of hybridization is not associated with a known biological change  52 , the sample may be analyzed to determine the nature of the change  54 . Once the nature of the change is known, the pattern may be added to a database  40  of patterns associated with known biological changes  56 . 
     Still referring to  FIG. 9 , in some cases, it may be possible to correlate the pattern of hybridization with known a biological change(s)  40 . This may be possible even where at least some of the sequences on the array, or the changes in the hybridization pattern, arise from novel, or previously uncharacterized, organisms. For example, there may be a single change in the pattern of hybridization to an array for two samples that is diagnostic of a single change between the two samples. Thus, as shown in  FIG. 9 , hybridization at the position corresponding to an oligonucleotide having the nucleic acid sequence of A may be associated with the presence of chromium  42 . Again, it may not be necessary to identify or characterize the organism from which sequence A is derived as such information may be ascertained merely by prior characterization that sequence A is associated with chromium in a water sample. Alternatively, a single change in the pattern of hybridization to an array for two samples may be diagnostic of a plurality of changes between the two samples. For example, a change in hybridization at sequence B may be associated with a change in the ratio of nitrogen to phosphate in a water sample  44 . Or, there may be multiple changes in the pattern of hybridization to an array for two samples that are diagnostic of a single change between the two samples. For example, the presence of hybridization at oligonucleotides having sequences C, D, and E and the absence of hybridization at oligonucleotide corresponding to sequence F may be diagnostic of the introduction of arsenic into the water system  46 . In yet another embodiment, there may be multiple changes in the pattern of hybridization to an array for two samples that are diagnostic of multiple changes between the two samples. For example, the presence of hybridization at oligonucleotides corresponding to sequences G, H, and I, in combination with the lack of hybridization at oligonucleotides corresponding to sequence J, may be diagnostic of a loss of multiple eukaryotes from the system. The information provided by the array may be comprehensive and allow for any remedial steps that may be required to be taken. The analysis may be formatted as part of a computer program so as to be run on a computer. In one embodiment, the known patterns may be included as a part of the computer program. Alternatively, the information provided by the array may be considered to provide a preliminary screening which may then be verified by chemical and/or microbiological analysis of the sample  60 . 
     As described herein, primers that may be used for amplification of bioindicator nucleic acid molecules from an ecosystem may be designed to amplify sequences from the variable regions of rDNA. Small subunit rRNA genes (SSU rDNA) are especially useful targets for the molecular identification of microbial species because these sequences contain highly conserved nucleotide regions interspersed with variable regions. The variable regions may be used as a foundation for phylogenetic classification and comparison of both prokaryotic and eukaryotic microbial species (e.g., Sogin and Gunderson, 1987 , Annals. NY Acad. Sci.,  503:125–139). The sequence information also permits detection and quantification of microbial species by PCR amplification using species-specific primers. 
     Thus, in one embodiment, ribosomal DNA that includes highly conserved and highly variable regions is used to characterize the presence of microbial community members. Species-specific or OTU-specific primers that anneal to internal variable regions can then be used to test for the presence of individual species. For both eukaryotic and prokaryotic rDNA, the variable rDNA regions show sufficient variability to develop amplification primers and probes that may be sensitive and specific to the organism to be tested. 
     For increased specificity and sensitivity in microarray experiments, variable sequence regions within the SSU rDNA may be utilized in a two-tiered strategy: (1) species-specific or OTU-specific PCR primers (e.g., Tables 2 and 3) can be multiplexed in individual PCR reactions to selectively amplify individual species or OTUs; (2) PCR products may then be labeled and hybridized to corresponding species-specific or OTU-specific oligonucleotide probes (e.g., Tables 2 and 3) immobilized on a microarray, where the probes immobilized on the array have sequences that are included in the amplified DNA products. The process may provide improved specificity and sensitivity because each of the species-specific or OTU-specific primer pairs and probes are designed using the same rDNA sequence. 
     To determine nucleic acid sequences that are specific to a single OTU, and thus can be used to detect the presence of a specific OTU in an ecosystem of interest, water samples were used to isolate DNA sequences derived from microbial ribosomal DNA. The individual ribosomal DNAs were cloned and the nucleic acid sequence for each clone was determined. Then, sequences from each rDNA clone were simulataneously aligned with either a prokaryotic rDNA sequence or a eukaryotic rDNA sequence to determine regions of variability for the cloned rDNAs. In this way, primers and probes (e.g., Tables 2 and 3) specific a particular OTU (or microbe population) were identified. 
     To ascertain the relative location and degree of variability among variable regions, rDNA sequences can be initially aligned using the multiple alignment computer program CLUSTAL W (Thompson et al., 1994 , Nucleic Acids Res.,  22:4673–4680). Multiple alignment parameters including a gap initiation penalty of three, a gap extension penalty of one, a base match score of one, and a base mismatch penalty of one were used. Transitions were not weighted and terminal gaps were not penalized. By aligning the cloned sequences with either the prokaryotic or eukaryotic rDNA, the SSU rDNA was demarcated into conserved and variable sequence regions, which were further aligned by hand to optimize the multiple alignment result where necessary. For prokaryotic primer sequences and probes (Table 3), variable sequence regions were used within nucleotides 50–880 (V1, V2, V3, and V4), based on alignments with  E. coli  rDNA (SEQ ID NO: 317) ( FIG. 10A ) (Brosius et al., 1981, J. Mol. Biol., 148:107–127; GenBank Accession No. V00348). Specifically, nucleotides 50–150 (V1 region) (SEQ ID NO: 319) or 160–250 (V2 region) (SEQ ID NO: 320) in the forward direction and nucleotides 430–510 (V3 region) (SEQ ID NO: 321) or 820–880 (V4 region) (SEQ ID NO: 322) ( FIG. 10C ) in the reverse direction allow for OTU-specific amplification by PCR that includes a region that complements microarray probes, which include nucleotides 160–250 (V2) (SEQ ID NO: 320) or 430–510(V3) (SEQ ID NO: 321). Primer sequences within these regions were selected to maximize specificity for each individual organism. Based on alignments with  S. cerevisiae  (Rubstov et al., 1980, Nucl. Acids Res., 8:5779–5794; GenBank Accession No. V01335) (SEQ ID NO: 318) ( FIG. 10B ) using the same alignment conditions, eukaryotic primer sequences and probes (Table 2) included variable sequence regions within nucleotides 50–1100 (V1, V2, V3, and V4). Specifically, positions 50–550 (V1 plus flanking sequence region) (SEQ ID NO: 324) in the forward direction and positions 800–870 (V3) (SEQ ID NO: 326) or 1000–1100 (V4) (SEQ ID NO: 327) in the reverse direction allow for OTU-specific amplification of a region that includes a region that complements probes within nucleotides 600–800 (V2) (SEQ ID NO: 325) or 1000–1100 (V4). When the sequence regions specified for primer and probe design do not include OTU-specific sequences, such that primers designed for two microbes would be the same or substantially similar, other variable regions are used. For example, prokaryotic primer sequences have also included nucleotides 1100–1160 (V6) (SEQ ID NO: 323) in the reverse direction and eukaryotic primer sequences have also included nucleotides 1350–1450 (V5) (SEQ ID NO: 328) in the reverse direction, where necessary. 
     Thus, in one embodiment of the present invention, amplification of a genomic sample DNA may be performed by multiplex PCR using primers chosen to provide products that can hybridize to taxon-specific ribosomal DNAs. Using this protocol can dramatically reduce non-specific labeling, and eliminate the need for intermediate PCR reactions, which reduce sensitivity. Oligonucleotide probes that may be spotted to provide a taxon-specific array (e.g., SEQ ID NOS: 5–113), and the primers used to detect (i.e., amplify) such sequences in water samples (e.g., SEQ ID NOS. 114–316), are shown in Tables 2 and 3. For example, primers having the sequences SEQ ID NOS. 114 and 115 (1F and 1R, respectively) may be used to amplify DNA from a water sample such that the amplification product contains sequences that will hybridize with an oligonucleotide probe (1P) immobilized on an array, where probe 1P has the sequence described by SEQ ID NO: 5. In one embodiment, multiple primer pairs (e.g., SEQ ID NOS: 114–214) are used to amplify DNA sequences that include SEQ ID NOS: 5–60). In addition, as is known in the art, the reverse complementary sequence of each probe sequence in Tables 2 and 3 may also be spotted as an oligonucleotide on the array. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Eukaryotic probes and primer pairs used for multiplex PCR 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Eukaryotic 
                 SEQ 
                   
                   
                 GenBank 
                   
                 SEQ 
                   
               
               
                 primer sequence 
                 ID 
                 PCR 
                   
                 Accession 
                 Probe Sequence 
                 ID 
                 Oligo 
               
               
                 (5′–3′) 
                 NO: 
                 No. 
                 Template 
                 No. 
                 (5′–3′) 
                 NO: 
                 No. 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 ATACAGGCGC 
                 114 
                 1F 
                 
                   Acanthamoeba 
                 
                 AY35164 
                 ACTAACTCAATAGC 
                 5 
                 1P 
               
               
                 TCGATAAGAG 
                   
                   
                 
                   mauritaniensis 
                 
                 7 
                 AGGAACGGGAATC 
               
               
                 AGCTGCTAGG 
                 115 
                 1R 
                   
                   
                 CAGAAGGAGGGGA 
               
               
                 GGAGTCATTC 
                   
                   
                   
                   
                 CGGGCGGGCC 
               
               
                 AACTCGACTTT 
                 116 
                 2F 
                 
                   Cryptosporidi 
                 
                 AF22299 
                 GATTTCTCATAAGG 
                 6 
                 2P 
               
               
                 ATGGAAGGG 
                   
                   
                 
                   um parvum 
                 
                 8 
                 TGCTGAAGGAG- 
               
               
                 CAAAGTCCCTC 
                 117 
                 2R 
                   
                   
                 TAAGGAACAACCT 
               
               
                 TAAGAAGAC 
                   
                   
                   
                   
                 CCAATCTCTAGT 
               
               
                 TTGGCTTTAGC 
                 118 
                 3F 
                 
                   Cyclospora 
                 
                 AF11118 
                 AGTTCCGGAACACC 
                 7 
                 3P 
               
               
                 CGGCGATAG 
                   
                   
                 
                   cayetanensis 
                 
                 3 
                 AACGCACGCAGCG 
               
               
                 AAGCCAAGGT 
                 119 
                 3R 
                   
                   
                 AAGCGCGGAAGGC 
               
               
                 AGGCGTTTCC 
                   
                   
                   
                   
                 TACCGGAAGA 
               
               
                 GACGACACAT 
                 120 
                 4F 
                 
                   Entamoeba 
                 
                 X65163 
                 GAAATGTCTTATTG 
                 8 
                 4P 
               
               
                 AACTCTAGAG 
                   
                   
                 
                   histolytica 
                 
                   
                 ACATCCCCTCAGCA 
               
               
                 TCATCCAATCC 
                 121 
                 4R 
                   
                   
                 TTGTCCCATGCTTG 
               
               
                 TTGGTTGAC 
                   
                   
                   
                   
                 AATATTCA 
               
               
                 AACTTGCCCAA 
                 122 
                 5F 
                 
                   Giardia 
                 
                 AF19944 
                 CCCACGCGGCGGG 
                 9 
                 5P 
               
               
                 TGCGCGG 
                   
                   
                 
                   intestinalis 
                 
                 9 
                 TCCAACGGGCCTGC 
               
               
                 GGGAATACGG 
                 123 
                 5R 
                 isolate Dog19 
                   
                 CTGGAGCGCTCCCG 
               
               
                 TGGTGTCTG 
                   
                   
                   
                   
                 TTTCCTCGT 
               
               
                 GATTGGAATG 
                 124 
                 6F 
                 
                   Isospora belli 
                 
                 AF10693 
                 GAATTTCACCACGT 
                 10 
                 6P 
               
               
                 ATGGGAATCC 
                   
                   
                   
                 5 
                 ACACACCCCTAAG 
               
               
                 AGGAGAAGTC 
                 125 
                 6R 
                   
                   
                 GGCGGACTGGCTG 
               
               
                 AAGTATGACG 
                   
                   
                   
                   
                 CTTCCAGCAG 
               
               
                 GCGGTAGTAA 
                 126 
                 7F 
                 
                   Microsporidiu 
                 
                 AY14064 
                 CTTTATCATCGGAC 
                 11 
                 7P 
               
               
                 GGAGACGTG 
                   
                   
                   m  sp. STF 
                 7 
                 TCGCCCCTGGCCAG 
               
               
                 GCATCGGCATC 
                 127 
                 7R 
                   
                   
                 CGCTTTCGCCTCTG 
               
               
                 GTTTACTGC 
                   
                   
                   
                   
                 TCGCTCCT 
               
               
                 TTCGGTGGTGA 
                 128 
                 8F 
                 
                   Naegleria 
                 
                 AF33842 
                 CCTCCAACCATCTC 
                 12 
                 8P 
               
               
                 GGTATTATC 
                   
                   
                 
                   fowleri 
                 
                 3 
                 CTGATGGAACTAGT 
               
               
                 AAGATCGCTG 
                 129 
                 8R 
                   
                   
                 TACCCCGTAAACAC 
               
               
                 GGATAGTGTC 
                   
                   
                   
                   
                 TCTTAGGT 
               
               
                 ATCGAGTATCA 
                 130 
                 9F 
                 OTU LT3A27, 
                 N/A 
                 ACGGAGACAAACA 
                 13 
                 9P 
               
               
                 ATTGGAGGG 
                   
                   
                 multi-copy, 
                   
                 AGCACCAACACAA 
               
               
                 GACGGGGTCA 
                 131 
                 9R 
                 identified as 
                   
                 GTGAAGGGCACGT 
               
               
                 ATACAACGAC 
                   
                   
                 
                   Asterionella 
                 
                   
                 TGCTCCAACCA 
               
               
                   
                   
                   
                 
                   formosa 
                 
               
               
                 GCCAATGGTCT 
                 132 
                 10F 
                 OTU LT1A42, 
                 N/A 
                 CAAGCAGAAAGGC 
                 14 
                 10P 
               
               
                 TCTTATTGG 
                   
                   
                 multi-copy, 
                   
                 ACGCGCGCACCGTC 
               
               
                 GAGGTCGTAA 
                 133 
                 10R 
                 identified as 
                   
                 CAACCAGAGGCTG 
               
               
                 ATTGACACTC 
                   
                   
                 
                   Cryptomonas 
                 
                   
                 ACAGTTCACA 
               
               
                   
                   
                   
                 sp., strain 
               
               
                   
                   
                   
                 M420 
               
               
                 TTCAAACCGGC 
                 134 
                 11F 
                 OTU LT1A4, 
                 N/A 
                 GCACGCGCATGCC 
                 15 
                 11P 
               
               
                 CTCGTTCTG 
                   
                   
                 multi-copy, 
                   
                 GTCCGACCAGAGG 
               
               
                 CCCATAACCA 
                 135 
                 11R 
                 identified as 
                   
                 CCGACAGCCCACA 
               
               
                 ACGAAATAGC 
                   
                   
                 
                   Cryptomonas 
                 
                   
                 CGCGCCCAAAA 
               
               
                   
                   
                   
                   ovata , strain 
               
               
                   
                   
                   
                 CCAP 979/61 
               
               
                 TTAGCGAATCG 
                 136 
                 12F 
                 OTU LT2A7, 
                 N/A 
                 TAACTGTCCCTGAT 
                 16 
                 12P 
               
               
                 TGGCACGTC 
                   
                   
                 multi-copy, 
                   
                 GGGACTAGTAGGG 
               
               
                 AATGTATTCCT 
                 137 
                 12R 
                 identified as 
                   
                 ATTGGTTTAAAGCC 
               
               
                 GCAAACGCC 
                   
                   
                   Dileptus  sp. 
                   
                 TCTCCCTAG 
               
               
                 GGGTTCTTACG 
                 138 
                 13F 
                 OTU LT2A19, 
                 N/A 
                 TCTCAGACGGATGA 
                 17 
                 13P 
               
               
                 AACTTTGGG 
                   
                   
                 single-copy, 
                   
                 ACGCCTATACCTCG 
               
               
                 CTGATCGGGCT 
                 139 
                 13R 
                 identified as 
                   
                 ACCGGAGCCGCTGT 
               
               
                 TGAAAGACC 
                   
                   
                   Coleps  sp. 
                   
                 ACAAACGC 
               
               
                 TATCGAGGAC 
                 140 
                 14F 
                 OTU LT3A2, 
                 N/A 
                 ACCTAATGCCACAC 
                 18 
                 14P 
               
               
                 CAATTGGAGG 
                   
                   
                 single-copy, 
                   
                 AGATTCCACCCAAG 
               
               
                 GACGGAGTCA 
                 141 
                 14R 
                 unidentified 
                   
                 GATGGACGAGCTG 
               
               
                 ATACAACGAC 
                   
                   
                   
                   
                 CCCAAGTAC 
               
               
                 TGGACTCTTTT 
                 142 
                 15F 
                 OTU LT1A3, 
                 N/A 
                 CCATCTGCGCCTCA 
                 19 
                 15P 
               
               
                 GAGTCCGGC 
                   
                   
                 single-copy, 
                   
                 ACATGCAGGTAAA 
               
               
                 ATCAATACTAA 
                 143 
                 15R 
                 unidentified 
                   
                 TCGTAAAGAAAAG 
               
               
                 CACCCACCG 
                   
                   
                   
                   
                 GCCAAATAGC 
               
               
                 TAACGATAGC 
                 144 
                 16F 
                 OTU LT1A10, 
                 N/A 
                 GTATCACACCAGG 
                 20 
                 16P 
               
               
                 GGGCTCGTTC 
                   
                   
                 multi-copy, 
                   
                 GAGGTTATTGAACG 
               
               
                 CATAGGGTGCT 
                 145 
                 16R 
                 unidentified 
                   
                 CAGACCACCTAGGT 
               
               
                 GATAGAGTC 
                   
                   
                   
                   
                 AACACCTAA 
               
               
                 CCGAGATTTCT 
                 146 
                 17F 
                 OTU LT2A12, 
                 N/A 
                 AAGGATGCTTTCAG 
                 21 
                 17P 
               
               
                 CGGAAATTG 
                   
                   
                 multi-copy, 
                   
                 GCACTGATCGCGCA 
               
               
                 TTTCTCACGAG 
                 147 
                 17R 
                 unidentified 
                   
                 CACTGAGGTGGGA 
               
               
                 GTGCTGAGG 
                   
                   
                   
                   
                 AGTGCCGTT 
               
               
                 ATGGTGGAGG 
                 148 
                 18F 
                 OTU TL1A16, 
                 N/A 
                 TAAGTGCAACGGG 
                 22 
                 18P 
               
               
                 TGATTCATTC 
                   
                   
                 multi-copy, 
                   
                 ATCCTCATGCAGAA 
               
               
                 AATTGACATCC 
                 149 
                 18R 
                 unidentified 
                   
                 AGACCCGAGCCTG 
               
               
                 ACTGATCCC 
                   
                   
                   
                   
                 CCGTCCGACC 
               
               
                 GATACAGGAC 
                 150 
                 19F 
                 OTU TL1A1, 
                 N/A 
                 AAAGTAAACCTGC 
                 23 
                 19P 
               
               
                 TCATCCGAGG 
                   
                   
                 multi-copy, 
                   
                 CAGCACAGACGGA 
               
               
                 AAACGCCTGC 
                 151 
                 19R 
                 unidentified 
                   
                 CACTCGGCGAAGA 
               
               
                 AGATCGCTAG 
                   
                   
                   
                   
                 GCACCCGCCTG 
               
               
                 ACAATGCCGG 
                 152 
                 20F 
                 OTU TL1A2, 
                 N/A 
                 TTAATGCCAGATAT 
                 24 
                 20P 
               
               
                 GCCTTTCAAG 
                   
                   
                 multi-copy, 
                   
                 GCTCTCCCCGAGGA 
               
               
                 TGGAGTCGTTA 
                 153 
                 20R 
                 unidentified 
                   
                 TGGCTGCAGACAC 
               
               
                 CAAACTTCC 
                   
                   
                   
                   
                 ATAGTACAG 
               
               
                 TCGGCGACGA 
                 154 
                 21F 
                 OTU TL1A9, 
                 N/A 
                 AGTCGACCAGTTCT 
                 25 
                 21P 
               
               
                 TGATTCATTC 
                   
                   
                 multi-copy, 
                   
                 GACCCATGAGGCC 
               
               
                 TGAACAAACC 
                 155 
                 21R 
                 unidentified 
                   
                 GACCGGCTGAGCTC 
               
               
                 ACGCCCAATC 
                   
                   
                   
                   
                 ACTCTGAAC 
               
               
                 CGGTTTACCGG 
                 156 
                 22F 
                 OTU TL1A12, 
                 N/A 
                 TCAAACCTGATTCA 
                 26 
                 22P 
               
               
                 CGATAGATC 
                   
                   
                 multi-copy, 
                   
                 AACCCGTATGGGTC 
               
               
                 TTCTCTCGAGG 
                 157 
                 22R 
                 unidentified 
                   
                 GATCGGTCGTCCTC 
               
               
                 TGCTGAAGG 
                   
                   
                   
                   
                 AGCAGAAA 
               
               
                 AATCGGATCG 
                 158 
                 23F 
                 OTU TL1A21, 
                 N/A 
                 TGGTAGGCTACCAC 
                 27 
                 23P 
               
               
                 CATGGGCTAG 
                   
                   
                 multi-copy, 
                   
                 TGCGCATCCACAAG 
               
               
                 GAACGGGATA 
                 159 
                 23R 
                 unidentified 
                   
                 GAGGCAGAAACTA 
               
               
                 ATTCTCGCCC 
                   
                   
                   
                   
                 GCCAACCAG 
               
               
                 CCCACTTATGT 
                 160 
                 24F 
                 OTU CL1A3, 
                 N/A 
                 GCTTCATGCAGGAG 
                 28 
                 24P 
               
               
                 GGGTTTGAC 
                   
                   
                 single-copy, 
                   
                 CATCTCAGCATCCA 
               
               
                 GAAGTAGAGG 
                 161 
                 24R 
                 unidentified 
                   
                 GTGTTGGGACCAG 
               
               
                 ATCTTGCCTC 
                   
                   
                   
                   
                 GACATACTG 
               
               
                 GACAGCTTCTT 
                 162 
                 25F 
                 OTU CL1A4, 
                 N/A 
                 GTTATGATTCTATC 
                 29 
                 25P 
               
               
                 TAATGGAGG 
                   
                   
                 single-copy, 
                   
                 TCAAGGAGGAGCG 
               
               
                 ATCTGTTGGTC 
                 163 
                 25R 
                 unidentified 
                   
                 TCCTGTGCTCTCCC 
               
               
                 CTCCAAATC 
                   
                   
                   
                   
                 ACTTCACTC 
               
               
                 AATACAGGGC 
                 164 
                 26F 
                 OTU CL1A5, 
                 N/A 
                 TCCAGAAGGTGAG 
                 30 
                 26P 
               
               
                 TCTTTGAGTC 
                   
                   
                 single-copy, 
                   
                 GCCGACGCAAAGA 
               
               
                 AAGACGTACC 
                 165 
                 26R 
                 unidentified 
                   
                 GTACTCACCGCTAG 
               
               
                 ACCGATCCTG 
                   
                   
                   
                   
                 GTGGACCCTC 
               
               
                 TAACAATGCG 
                 166 
                 27F 
                 OTU CL1A6, 
                 N/A 
                 AGAGTAAAGGACG 
                 31 
                 27P 
               
               
                 GAGCCTTCGG 
                   
                   
                 single-copy, 
                   
                 CAGGTCCGGACGC 
               
               
                 AAGAACGTCC 
                 167 
                 27R 
                 unidentified 
                   
                 CGACAAGTGAATG 
               
               
                 GCCAATCCTG 
                   
                   
                   
                   
                 CCGACGCCTTC 
               
               
                 TATCTGGCGCT 
                 168 
                 28F 
                 OTU CL1A8, 
                 N/A 
                 TCTCTAGAAGGATG 
                 32 
                 28P 
               
               
                 TTTGCGTCG 
                   
                   
                 multi-copy, 
                   
                 CCCAACCCGCACCG 
               
               
                 CAACGTCTACC 
                 169 
                 28R 
                 unidentified 
                   
                 GCACTCACAGGCC 
               
               
                 CATCCCAAG 
                   
                   
                   
                   
                 AAAAAGGCC 
               
               
                 ACTCGGGAAC 
                 170 
                 29F 
                 OTU LT2A20, 
                 N/A 
                 CGAAGACGGATGA 
                 33 
                 29P 
               
               
                 CTAGTTCTAC 
                   
                   
                 single-copy, 
                   
                 CTAACTATATACTG 
               
               
                 TCTCTTACGGC 
                 171 
                 29R 
                 unidentified 
                   
                 ACGTAAGCCAGCA 
               
               
                 GCCGAAAAG 
                   
                   
                   
                   
                 TATAAATAGC 
               
               
                 AGGGCCAACG 
                 172 
                 30F 
                 OTU LT1A5, 
                 N/A 
                 CACAATTAAGTGCA 
                 34 
                 30P 
               
               
                 GTCTTGTTAT 
                   
                   
                 multi-copy, 
                   
                 ACGGGATCCTCATG 
               
               
                 TCGGAAATTGA 
                 173 
                 30R 
                 unidentified 
                   
                 CAGAAAGACCCGA 
               
               
                 CATCCACTG 
                   
                   
                   
                   
                 GCCTGCCGT 
               
               
                 CTCTCTCCGAG 
                 174 
                 31F 
                 OTU LT1A8, 
                 N/A 
                 TATTAACGCACTAC 
                 35 
                 31P 
               
               
                 TATCAATTG 
                   
                   
                 multi-copy, 
                   
                 GCCCTGGAAGGAT 
               
               
                 ACTTCCCTCAA 
                 175 
                 31R 
                 unidentified 
                   
                 GCTTTCAGGCACTG 
               
               
                 TCGCTAGTC 
                   
                   
                   
                   
                 ATCGCGCAC 
               
               
                 GCAGAGCTTC 
                 176 
                 32F 
                 OTU LT1A9, 
                 N/A 
                 ACAGCTACCACCAC 
                 36 
                 32P 
               
               
                 ACAGTTTTGC 
                   
                   
                 single-copy, 
                   
                 CCTAAGGTGGGGA 
               
               
                 AGACGTCTCCT 
                 177 
                 32R 
                 unidentified 
                   
                 GGTCATCCCGATCA 
               
               
                 GATCGCAAG 
                   
                   
                   
                   
                 GAGATTCAA 
               
               
                 N/A 
                   
                   
                 OTU LT1A11, 
                 N/A 
                 TTCCAAGAGGATGC 
                 37 
                 33P 
               
               
                   
                   
                   
                 single-copy, 
                   
                 CTCGGTCTAACCAG 
               
               
                   
                   
                   
                 unidentified 
                   
                 ACACAAACCCGTAT 
               
               
                   
                   
                   
                   
                   
                 GGGTCGGT 
               
               
                 N/A 
                   
                   
                 OTU LT1A13, 
                 N/A 
                 AAGTGTTTTCCGGA 
                 38 
                 34P 
               
               
                   
                   
                   
                 single-copy, 
                   
                 AGATGGACGCAAA 
               
               
                   
                   
                   
                 unidentified 
                   
                 CACCCGGTACACA 
               
               
                   
                   
                   
                   
                   
                 GACCGCGAGT 
               
               
                 ATACGTCCCGG 
                 178 
                 35F 
                 OTU LT3A5, 
                 N/A 
                 TAACAGAAGGATG 
                 39 
                 35P 
               
               
                 GACTGCAAT 
                   
                   
                 multi-copy, 
                   
                 GTAGGGCGGCTCA 
               
               
                 CGAAGGCGGA 
                 179 
                 35R 
                 unidentified 
                   
                 GCGCACTCAACTTG 
               
               
                 TAATTCTCGC 
                   
                   
                   
                   
                 AGGGCAAAGT 
               
               
                 ACAATGCAGG 
                 180 
                 36F 
                 OTU LT3A6, 
                 N/A 
                 ACAGTACAAGTCTT 
                 40 
                 36P 
               
               
                 GCCTTTACGG 
                   
                   
                 multi-copy, 
                   
                 GCGACTAGACCGTC 
               
               
                 GAATAACACT 
                 181 
                 36R 
                 unidentified 
                   
                 CGGCCCAAAACCT 
               
               
                 CACTGATCCC 
                   
                   
                   
                   
                 GAAATCCAA 
               
               
                 ATACAGGACT 
                 182 
                 37F 
                 OTU LT3A7, 
                 N/A 
                 AAACAAGCCAGTA 
                 41 
                 37P 
               
               
                 CATCCGAGGC 
                   
                   
                 multi-copy, 
                   
                 CCGAAAGCATTCG 
               
               
                 AAACGCCTGC 
                 183 
                 37R 
                 unidentified 
                   
                 GACCGACTTCTGTC 
               
               
                 AGATCGCTAG 
                   
                   
                   
                   
                 CGCCGAGATC 
               
               
                 N/A 
                   
                   
                 OTU LT3A11, 
                 N/A 
                 GCAAGCGGATGAC 
                 42 
                 38P 
               
               
                   
                   
                   
                 single-copy, 
                   
                 TGTCAGAATCCCCG 
               
               
                   
                   
                   
                 unidentified 
                   
                 TCTAATGACTGAAG 
               
               
                   
                   
                   
                   
                   
                 ACCTGAACA 
               
               
                 CTTTACAGGTC 
                 184 
                 39F 
                 OTU LT3A13, 
                 N/A 
                 ACCTAATGCCACAC 
                 43 
                 39P 
               
               
                 TGGCAATTG 
                   
                   
                 single-copy, 
                   
                 AGATTCCACCCAAG 
               
               
                 CATACAGTGCT 
                 185 
                 39R 
                 unidentified 
                   
                 GATGGACGAGCTG 
               
               
                 GACAGGGTC 
                   
                   
                   
                   
                 CCCAAGTAC 
               
               
                 CAGGGCCTTTT 
                 186 
                 40F 
                 OTU LT1A1, 
                 N/A 
                 TTCAGAAAAGAAG 
                 44 
                 40P 
               
               
                 CAGGTCTTG 
                   
                   
                 multi-copy, 
                   
                 TGTCGTCCCGATCG 
               
               
                 CACAAGGTGC 
                 187 
                 40R 
                 unidentified 
                   
                 CACTACCGTAAGGC 
               
               
                 CAACAGAGTC 
                   
                   
                   
                   
                 GGCAAGCGT 
               
               
                 AACAATGTCTG 
                 188 
                 41F 
                 OTU LT1A38, 
                 N/A 
                 AATGCCGCTGGTCA 
                 45 
                 41P 
               
               
                 GCCCTACGG 
                   
                   
                 multi-copy, 
                   
                 CACGGAAGAAAGA 
               
               
                 GTAAACAACG 
                 189 
                 41R 
                 unidentified 
                   
                 AGCCGACCAAACA 
               
               
                 CCCACCGATC 
                   
                   
                   
                   
                 GTGCGACTTG 
               
               
                 GAGGGCAAGT 
                 190 
                 42F 
                 All LT1A &amp; 
                 N/A 
                 N/A 
               
               
                 CTGGTGCCAG 
                   
                   
                 LT3A OTUs 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Prokaryotic probes and primer pairs used for multiplex PCR 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Prokaryotic 
                 SEQ 
                   
                   
                 GenBank 
                   
                 SEQ 
                   
               
               
                 primer Sequence 
                 ID 
                 PCR 
                   
                 Accession 
                 Prokaryotic probe 
                 ID 
                 Oligo 
               
               
                 (5′–3′) 
                 NO: 
                 No. 
                 Template 
                 No. 
                 Sequence (5′–3′) 
                 NO: 
                 No. 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CGAATGGATT 
                 191 
                 43F 
                 
                   Bacillus 
                 
                 AB116124 
                 GTGACAGCCGAA 
                 46 
                 43P 
               
               
                 AAGAGCTTGC 
                   
                   
                   anthracis  strain: 
                   
                 GCCGCCTTTCAAT 
               
               
                 TGCCAGCTTAT 
                 192 
                 43R 
                 S51 
                   
                 TTTCGAACCATGC 
               
               
                 TCAACTAGC 
                   
                   
                   
                   
                 GGTTCAAAATGT 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 GAACGTACCA 
                 193 
                 44F 
                 
                   Brucella 
                 
                 AF220149 
                 CCAACGCGGGCC 
                 47 
                 44P 
               
               
                 TTTGCTACGG 
                   
                   
                 
                   melitensis 
                 
                   
                 GATCATTTGCCG 
               
               
                 ACCGTCATTAT 
                 194 
                 44R 
                   
                   
                 ATAAATCTTTCCC 
               
               
                 CTTCACCGG 
                   
                   
                   
                   
                 CCGAAGGGCACA 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 ACGGGCTTCG 
                 195 
                 45F 
                 
                   Burkholderia 
                 
                 AY305760 
                 AGGCCCGAAGGT 
                 48 
                 45P 
               
               
                 GCCTGGTG 
                   
                   
                   mallei  strain 
                   
                 CCCCCGCTTTCAT 
               
               
                 TCCGGGTATTA 
                 196 
                 45R 
                 2000031063 
                   
                 CCTCAGATCGTAT 
               
               
                 GCCAGAATG 
                   
                   
                   
                   
                 GCGGTATTAATC 
               
               
                 GCTTGCTAGAA 
                 197 
                 46F 
                 
                   Campylobacter 
                 
                 AF550630 
                 TCCTACACCGAA 
                 49 
                 46P 
               
               
                 GTGGATTAG 
                   
                   
                   jejuni  strain 
                   
                 AAACTTTCCCTAC 
               
               
                 CGTCAGAATTC 
                 198 
                 46R 
                 B99/206 
                   
                 TCAACTTGTGTTA 
               
               
                 TTCCCTAAG 
                   
                   
                   
                   
                 AGCAGGAGTATA 
               
               
                 TTTAGTGGCGG 
                 199 
                 47F 
                 
                   Chlamydophila 
                 
                 AY334530 
                 GGTCCGAAGATC 
                 50 
                 47P 
               
               
                 AAGGGTTAG 
                   
                   
                   psittaci  clone 
                   
                 CCCTTCTTTAATA 
               
               
                 ATCTCTCTTAT 
                 200 
                 47R 
                 cvCps2 
                   
                 TGTTTTAGATGCC 
               
               
                 TCCCAAGCG 
                   
                   
                   
                   
                 TAAACATACCAC 
               
               
                 AAGCTTCCTTC 
                 201 
                 48F 
                 
                   Clostridium 
                 
                 AY303799 
                 CGCCGCGGGTCC 
                 51 
                 48P 
               
               
                 GGGAAGTGG 
                   
                   
                   botulinum  strain 
                   
                 ATCTCAAAGCAA 
               
               
                 GGTACCGTCAT 
                 202 
                 48R 
                 AIP 355.02 
                   
                 TAAATCTTTGATA 
               
               
                 TATCGTCC 
                   
                   
                   
                   
                 AGAAAATCATGC 
               
               
                   
                   
                   
                   
                   
                 G 
               
               
                 ATAACCTGGG 
                 203 
                 49F 
                 
                   Coxiella burnetii 
                 
                 Y11502 
                 TCATCTTATAGCA 
                 52 
                 49P 
               
               
                 GAAACTCGGG 
                   
                   
                 strain Nine Mile 
                   
                 CGAGGTCCGAAG 
               
               
                 CCAAGGATATT 
                 204 
                 49R 
                   
                   
                 ATCCCCCGCTTTG 
               
               
                 ACCCTTGAG 
                   
                   
                   
                   
                 CTCCAAAGAGAT 
               
               
                 AGAAGCTTGCT 
                 205 
                 50F 
                 
                   Escherichia coli 
                 
                 AB035920 
                 ACATCCGATGGC 
                 53 
                 50P 
               
               
                 TCTTTGCTG 
                   
                   
                 O157:H7 
                   
                 AAGAGGCCCGAA 
               
               
                 TTCCTCCCCGC 
                 206 
                 50R 
                   
                   
                 GGTCCCCCTCTTT 
               
               
                 TGAAAGTAC 
                   
                   
                   
                   
                 GGTCTTGCGACG 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 CAATTCTGGGA 
                 207 
                 51F 
                 
                   Escherichia coli 
                 
                 NC 002655 
                 CAATTCTGGGAA 
                 54 
                 51P 
               
               
                 AGCGTGG 
                   
                   
                 O157:H7 
                   
                 GCGTGGCATTAA 
               
               
                 CGATGCATGAT 
                 208 
                 51R 
                 EDL933 
                   
                 TACTGAATTGTCA 
               
               
                 GATGACA 
                   
                   
                   
                   
                 TCATCATGCATCG 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 002655 
                 GGTTGATGAAAA 
                 55 
                 52P 
               
               
                   
                   
                   
                 O157:H7 
                   
                 AGCATTTGGAGC 
               
               
                   
                   
                   
                 EDL933 
                   
                 CGCGAAATTTAC 
               
               
                   
                   
                   
                   
                   
                 CAGTGTCTTAAA 
               
               
                   
                   
                   
                   
                   
                 AC 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 002655 
                 TGTCCGATTCAGC 
                 56 
                 53P 
               
               
                   
                   
                   
                 O157:H7 
                   
                 ACGGGTAAATAG 
               
               
                   
                   
                   
                 EDL933 
                   
                 TCGTATTGTTAGT 
               
               
                   
                   
                   
                   
                   
                 GGCCGAATTTAA 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 002655 
                 TTGCTGGAGAGT 
                 57 
                 54P 
               
               
                   
                   
                   
                 O157:H7 
                   
                 CCTTCTCGGGTAT 
               
               
                   
                   
                   
                 EDL933 
                   
                 CGATTGTCGAAG 
               
               
                   
                   
                   
                   
                   
                 ATAAACATATTT 
               
               
                   
                   
                   
                   
                   
                 A 
               
               
                 GTGGTGGATTA 
                 209 
                 55F 
                 
                   Escherichia coli 
                 
                 NC 002655 
                 GTGGTGGATTAC 
                 58 
                 55P 
               
               
                 CGCCATG 
                   
                   
                 O157:H7 
                   
                 GCCATGACATGG 
               
               
                 GCTATTACTCC 
                 210 
                 55R 
                 EDL933 
                   
                 GAGGATTAACGG 
               
               
                 CCCCCGT 
                   
                   
                   
                   
                 GGGGGAGTAATA 
               
               
                   
                   
                   
                   
                   
                 GC 
               
               
                 TCTGGAGTATC 
                 211 
                 56F 
                 
                   Escherichia coli 
                 
                 NC 002655 
                 TCTGGAGTATCA 
                 59 
                 56P 
               
               
                 AAGCACT 
                   
                   
                 O157:H7 
                   
                 AGCACTTATAAC 
               
               
                 CCAGGAAGAG 
                 212 
                 56R 
                 EDL933 
                   
                 CTAATAACACAA 
               
               
                 GGTTTTGT 
                   
                   
                   
                   
                 AACCCTCTTCCTG 
               
               
                   
                   
                   
                   
                   
                 G 
               
               
                 GCCCTGACGTA 
                 213 
                 57F 
                 
                   Escherichia coli 
                 
                 NC 002655 
                 GCCCTGACGTAT 
                 60 
                 57P 
               
               
                 TGGCGGG 
                   
                   
                 O157:H7 
                   
                 GGCGGGTACGAA 
               
               
                 GTAATGGTCAC 
                 214 
                 57R 
                 EDL933 
                   
                 ATGAAGCCAGTG 
               
               
                 CGTCACT 
                   
                   
                   
                   
                 ACGGTGACCATT 
               
               
                   
                   
                   
                   
                   
                 AC 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 002655 
                 ACTGGCGGGAAC 
                 61 
                 58P 
               
               
                   
                   
                   
                 O157:H7 
                   
                 ACATGAAAACGT 
               
               
                   
                   
                   
                 EDL933 
                   
                 AACCACGCTACC 
               
               
                   
                   
                   
                   
                   
                 AGTAGCCAGAAG 
               
               
                   
                   
                   
                   
                   
                 AA 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 002655 
                 CCATTAAAACTA 
                 62 
                 59P 
               
               
                   
                   
                   
                 O157:H7 
                   
                 ATGCCTGTCATA 
               
               
                   
                   
                   
                 EDL933 
                   
                 ATGGAGGGGGAT 
               
               
                   
                   
                   
                   
                   
                 TCAGCGAAGTTA 
               
               
                   
                   
                   
                   
                   
                 TT 
               
               
                 AAGACATCTTC 
                 215 
                 60F 
                 
                   Escherichia coli 
                 
                 NC 000913 
                 AAGACATCTTCA 
                 63 
                 60P 
               
               
                 ACCGTTC 
                   
                   
                 K12 
                   
                 CCGTTCACGATAT 
               
               
                 TCAGATTTCCC 
                 216 
                 60R 
                   
                   
                 TTTGAAAGCACG 
               
               
                 CTCGTGC 
                   
                   
                   
                   
                 AGGGGAAATCTG 
               
               
                   
                   
                   
                   
                   
                 A 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 000913 
                 CACCGTCGCTTTA 
                 64 
                 61P 
               
               
                   
                   
                   
                 K12 
                   
                 AAACGCGCCCGG 
               
               
                   
                   
                   
                   
                   
                 TGGGAGAATCGT 
               
               
                   
                   
                   
                   
                   
                 CGTTGTACATTTA 
               
               
                 N/A 
                   
                   
                 
                   Escherichia coli 
                 
                 NC 000913 
                 TTTCTGATCGCGT 
                 65 
                 62P 
               
               
                   
                   
                   
                 K12 
                   
                 TGCTGCGCTGATC 
               
               
                   
                   
                   
                   
                   
                 AAAGAAGTAAAC 
               
               
                   
                   
                   
                   
                   
                 AAAGCAGCTTAA 
               
               
                 ATGGCATCCGT 
                 217 
                 63F 
                 
                   Escherichia coli 
                 
                 NC 000913 
                 ATGGCATCCGTG 
                 66 
                 63P 
               
               
                 GGTATCC 
                   
                   
                 K12 
                   
                 GTATCCCGACTCT 
               
               
                 CACTTCACCGT 
                 218 
                 63R 
                   
                   
                 GCTGCTGTTCAA 
               
               
                 TTTTGAA 
                   
                   
                   
                   
                 AAACGGTGAAGT 
               
               
                   
                   
                   
                   
                   
                 G 
               
               
                 AACAGCTTGCT 
                 219 
                 64F 
                 
                   Shigella sonnei 
                 
                 X96964 
                 N/A 
               
               
                 GTTTCGCTG 
                   
               
               
                 TTCCTCCCCGC 
                 220 
                 64R 
               
               
                 TGAAAGTAC 
                   
               
               
                 CAGGTCTTAGG 
                 221 
                 65F 
                 
                   Francisella 
                 
                 AY243028 
                 AGGCTCATCCAT 
                 67 
                 65P 
               
               
                 ATGCTGACG 
                   
                   
                   tularensis  strain 
                   
                 CTGCGACACGCC 
               
               
                 AAGGCTATTA 
                 222 
                 65R 
                 3523 
                   
                 GAAAGCCACCTT 
               
               
                 ACCTTGAGGC 
                   
                   
                   
                   
                 TAATCCACAGAT 
               
               
                   
                   
                   
                   
                   
                 AT 
               
               
                 AGACTATCTAC 
                 223 
                 66F 
                 
                   Legionella 
                 
                 AJ496383 
                 AATCCTTAAAAG 
                 68 
                 66P 
               
               
                 TTCTGGTGC 
                   
                   
                 
                   pneumophila 
                 
                   
                 TCGGTCGTAGTCC 
               
               
                 ATACAGGTGCT 
                 224 
                 66R 
                 serogroup 6 
                   
                 GGATTGGAGTCT 
               
               
                 GCATGGCTG 
                   
                   
                   
                   
                 GCAACTCGACTC 
               
               
                   
                   
                   
                   
                   
                 C 
               
               
                 GAGTAGCAAT 
                 225 
                 67F 
                 
                   Leptospira 
                 
                 Z12817 
                 ATCTCCGAGCAA 
                 69 
                 67P 
               
               
                 ACTCAGCGGC 
                   
                   
                 
                   interrogans 
                 
                   
                 TAAATCTTTACCC 
               
               
                 TACCATCATCA 
                 226 
                 67R 
                   
                   
                 GAAAAATCTTAT 
               
               
                 CATTGCTGC 
                   
                   
                   
                   
                 GATCTCTCGGGA 
               
               
                   
                   
                   
                   
                   
                 C 
               
               
                 GAGCTTGCTCC 
                 227 
                 68F 
                 
                   Pseudomonas 
                 
                 AB117953 
                 TCATCTGATAGC 
                 70 
                 68P 
               
               
                 TGGATTCAG 
                   
                   
                 
                   aeruginosa 
                 
                   
                 GTGAGGTCCGAA 
               
               
                 GTAACGTCAA 
                 228 
                 68R 
                 strain WatG 
                   
                 GATCCCCCACTTT 
               
               
                 AACAGCAAGG 
                   
                   
                   
                   
                 CTCCCTCAGGAC 
               
               
                   
                   
                   
                   
                   
                 G 
               
               
                 AGTTAATTAGT 
                 229 
                 69F 
                 
                   Rickettsia 
                 
                 M21789 
                 ATCTGACGCGGG 
                 71 
                 69P 
               
               
                 GGCAGACGG 
                   
                   
                 
                   prowazekii 
                 
                   
                 CCCATCCATCAG 
               
               
                 ACTAAACCGC 
                 230 
                 69R 
                   
                   
                 CGATAAATCTTTC 
               
               
                 CTACGCACTC 
                   
                   
                   
                   
                 CTCCGTAGAGAA 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 AGCTTGCTGCT 
                 231 
                 70F 
                 
                   Salmonella 
                 
                 Z49264 
                 CTTGGTGAGCCG 
                 72 
                 70P 
               
               
                 TTGCTGACG 
                   
                   
                 
                   typhimurium 
                 
                   
                 TTACCTCACCAAC 
               
               
                 TAACCACAAC 
                 232 
                 70R 
                   
                   
                 AAGCTAATCCCA 
               
               
                 ACCTTCCTCC 
                   
                   
                   
                   
                 TCTGGGCACATCT 
               
               
                 GAACTTGTTCC 
                 233 
                 71F 
                 
                   Vibrio cholerae 
                 
                 X76337 
                 ATCCCACCTGGG 
                 73 
                 71P 
               
               
                 TTGGGTGGC 
                   
                   
                 CECT 514 T 
                   
                 CATATCCGGTAG 
               
               
                 TTAACCACCTT 
                 234 
                 71R 
                   
                   
                 CGCAAGGCCCGA 
               
               
                 CCTCCCTAC 
                   
                   
                   
                   
                 AGGTCCCCTGCTT 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 GTAGTTTACTA 
                 235 
                 72F 
                 
                   Yersinia pestis 
                 
                 AF366383 
                 TCTGGGTTCATCC 
                 74 
                 72P 
               
               
                 CTTTGCCGG 
                   
                   
                   
                   
                 GATGGCGTGAGG 
               
               
                 GAGCGTATTA 
                 236 
                 72R 
                   
                   
                 CCCTAAGGTCCC 
               
               
                 AACTCAACCC 
                   
                   
                   
                   
                 CCACTTTGCTCTT 
               
               
                 GGAAAGTGGC 
                 237 
                 73F 
                 Arsenite- 
                 AF406554 
                 TCAAGACCCACG 
                 75 
                 73P 
               
               
                 CCTCTGATTC 
                   
                   
                 oxidizing 
                   
                 GCTATTAACCGT 
               
               
                 CCATAAATGA 
                 238 
                 73R 
                 bacterium 
                   
                 AAGCTTTTCCTCC 
               
               
                 ACCCAACGGC 
                   
                   
                 MLHE-1 
                   
                 CTGCTGAAAGTG 
               
               
                   
                   
                   
                   
                   
                 C 
               
               
                 ACCGGATACA 
                 239 
                 74F 
                 
                   Thiomicrospira 
                 
                 U46506 
                 GCCGGTGCTTATT 
                 76 
                 74P 
               
               
                 CCTTCATACC 
                   
                   
                 sp. CVO 
                   
                 CATATGCTACCGT 
               
               
                 CCGCAATGAC 
                 240 
                 74R 
                   
                   
                 CATTTTCTTGACA 
               
               
                 AAGCATCACG 
                   
                   
                   
                   
                 TATAAAAGGAG 
               
               
                 ACGCTCCGATT 
                 241 
                 75F 
                 
                   Desulfovibrio 
                 
                 Z24450 
                 GTACCGTCAGAC 
                 77 
                 75P 
               
               
                 TCACAGTTC 
                   
                   
                 
                   longreachii 
                 
                   
                 CATGGCTGATTA 
               
               
                 AAGTCCAGCA 
                 242 
                 75R 
                   
                   
                 GCACCATGGCGG 
               
               
                 GTATCAAGGG 
                   
                   
                   
                   
                 TTCTTCCCTCCTG 
               
               
                   
                   
                   
                   
                   
                 A 
               
               
                 TGGGTTTACCT 
                 243 
                 76F 
                 
                   Bacillus 
                 
                 AF064705 
                 CAAGGTACCGCC 
                 78 
                 76P 
               
               
                 AACACTACG 
                   
                   
                 
                   arsenicoselenatis 
                 
                   
                 CTATTTGAACGGT 
               
               
                 TAGAGTCGAG 
                 244 
                 76R 
                   
                   
                 ACTTGTTCTTCCC 
               
               
                 TTACAGACCG 
                   
                   
                   
                   
                 TAGCAACAGAGC 
               
               
                 ATCATGAGTTC 
                 245 
                 77F 
                 Uncultured 
                 AF233412 
                 TATTCATAAGGT 
                 79 
                 77P 
               
               
                 ACATGTCCG 
                   
                   
                 human fecal 
                   
                 ACATACAAAACA 
               
               
                 CAATCGGAGTT 
                 246 
                 77R 
                 bacterium HF74 
                   
                 CCACACGTGGCG 
               
               
                 CTTCGTG 
                   
                   
                   
                   
                 AACTTTATTCCCT 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 ATCATGAGTTC 
                 247 
                 78F 
                 Uncultured 
                 AF233408 
                 TATTCATAAAGT 
                 80 
                 78P 
               
               
                 ACATGTCCG 
                   
                   
                 human fecal 
                   
                 ACATGCAAACGG 
               
               
                 CAATCGGAGTT 
                 248 
                 78R 
                 bacterium HF8 
                   
                 GTATGCATACCC 
               
               
                 CTTCGTG 
                   
                   
                   
                   
                 GACTTTATTCCTT 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 GCCGTCTACTC 
                 249 
                 79F 
                 Uncultured 
                 AF233413 
                 TATTCATACGGTA 
                 81 
                 79P 
               
               
                 TTGGCC 
                   
                   
                 human fecal 
                   
                 CATACAAAAAGG 
               
               
                 CCTGCCTCTAC 
                 250 
                 79R 
                 bacterium HF10 
                   
                 CACACGTGCCTC 
               
               
                 TGTACTC 
                   
                   
                   
                   
                 ACTTTATTCCCGT 
               
               
                 ACGGGTGCTTG 
                 251 
                 80F 
                 
                   Burkholderia 
                 
                 AB091761 
                 AGGCCCGAAGGT 
                 82 
                 80P 
               
               
                 CACCTGG 
                   
                   
                 
                   cepacia 
                 
                   
                 CCCCCGCTTTCAT 
               
               
                 CGACTGTATTA 
                 252 
                 80R 
                   
                   
                 CCGTAGATCGTA 
               
               
                 GAGCCAAGG 
                   
                   
                   
                   
                 TGCGGTATTAATC 
               
               
                 GTTGGCCGATG 
                 253 
                 81F 
                 
                   Burkholderia 
                 
                 AF148556 
                 CGGTACCGTCAT 
                 83 
                 81P 
               
               
                 GCTGATTAG 
                   
                   
                 
                   cepacia 
                 
                   
                 CCCCCGACTGTAT 
               
               
                 TCTGCCATACT 
                 254 
                 81R 
                 genomovar III 
                   
                 TAGAGCCAAGGA 
               
               
                 CTAGCCTGC 
                   
                   
                   
                   
                 TTTCTTTCCGGAC 
               
               
                 ACATGCAAGT 
                 255 
                 82F 
                 OTU LT3A11, 
                 N/A 
                 AGCCGCAAGCTT 
                 84 
                 82P 
               
               
                 CGTACGAGAG 
                   
                   
                 multi-copy, 
                   
                 CTCTTTAGGCGG 
               
               
                   
                   
                   
                 identified as 
                   
                 AAATCCATTTCAC 
               
               
                 ACACGTCATTT 
                 256 
                 82R 
                 Unidentified 
                   
                 TCGAAAGCATAT 
               
               
                 ATTCCTCCC 
                   
                   
                 cyanobacterium 
                   
                 G 
               
               
                   
                   
                   
                 clone LD27 
               
               
                 ACGAACCTTCG 
                 257 
                 83F 
                 OTU LT1A53- 
                 N/A 
                 AGACGCGAGCTC 
                 85 
                 83P 
               
               
                 GGTTAGTGG 
                   
                   
                 3A9, multi-copy, 
                   
                 ATCCTCAGGCGA 
               
               
                 TCAAGTACCGT 
                 258 
                 83R 
                 identified as 
                   
                 AATTCATTTCACC 
               
               
                 CAGATCTTC 
                   
                   
                 
                   Synechococcus 
                 
                   
                 TCTCGGCATATG 
               
               
                   
                   
                   
                 sp. 
                   
                 G 
               
               
                 AAAGGCCTAC 
                 259 
                 84F 
                 OTU LT1A53, 
                 N/A 
                 CCATCGCAGTAA 
                 86 
                 84P 
               
               
                 CAAGGCTTCG 
                   
                   
                 multi-copy, 
                   
                 TGGAGTTAAGGT 
               
               
                 GGCACTCTCTC 
                 260 
                 84R 
                 identified as 
                   
                 CCACGCTTTGAC 
               
               
                 GTTTCCAAG 
                   
                   
                 
                   Synechococcus 
                 
                   
                 GACAGACTTAAA 
               
               
                   
                   
                   
                 sp. LBG2 
                   
                 AG 
               
               
                 AAAGGCTTAC 
                 261 
                 85F 
                 OTU LT3A9, 
                 N/A 
                 CCATCGCTGAAA 
                 87 
                 85P 
               
               
                 CAAGGCATTG 
                   
                   
                 multi-copy, 
                   
                 TGGAGTTGAGCT 
               
               
                 CCTCCGGTTTC 
                 262 
                 85R 
                 identified as 
                   
                 CCACGCTTTAAC 
               
               
                 CCAGAG 
                   
                   
                 
                   Synechococcus 
                 
                   
                 GACAGACTTGTA 
               
               
                   
                   
                   
                 sp. LBP1 
                   
                 AA 
               
               
                 GTAACAGGTCT 
                 263 
                 86F 
                 OTU TL1A7, 
                 N/A 
                 CGCTCTAGTAGC 
                 88 
                 86P 
               
               
                 TTCGGGATG 
                   
                   
                 multi-copy, 
                   
                 ACAAGGCCCGAA 
               
               
                 CAAGACTTTTC 
                 264 
                 86R 
                 identified as 
                   
                 GGTCCCCTGCTTT 
               
               
                 GTTCCGTAC 
                   
                   
                 Uncultured beta 
                   
                 CATCCATAGATCT 
               
               
                   
                   
                   
                 proteobacterium 
               
               
                   
                   
                   
                 clone OSlL-16 
               
               
                 TCTTTCACCGG 
                 265 
                 87F 
                 OTU TL1A9, 
                 N/A 
                 TCAGTGACGCAA 
                 89 
                 87P 
               
               
                 AGCTTGCTC 
                   
                   
                 multi-copy, 
                   
                 AAGCGCCTTTCA 
               
               
                 CTCTCATCCTT 
                 266 
                 87R 
                 identified as 
                   
                 ACTTTCTTCCATG 
               
               
                 GTTCTTCTC 
                   
                   
                 
                   Enterococcus 
                 
                   
                 CGGAAAATAGTG 
               
               
                   
                   
                   
                   gallinarum  strain 
                   
                 T 
               
               
                   
                   
                   
                 LMG 13129 
               
               
                 ACGGTCGCGT 
                 267 
                 88F 
                 OTU LT1A31, 
                 N/A 
                 TCCTGAAGCGAT 
                 90 
                 88P 
               
               
                 AACACGTAAG 
                   
                   
                 multi-copy, 
                   
                 AAATCTTTAGAC 
               
               
                 CGTCAAATTTC 
                 268 
                 88R 
                 identified as 
                   
                 ACAAGTCGATGC 
               
               
                 TTCCCACTC 
                   
                   
                 Uncultured 
                   
                 CGACTCGTGACC 
               
               
                   
                   
                   
                 Crater Lake 
                   
                 AC 
               
               
                   
                   
                   
                 bacterium 
               
               
                   
                   
                   
                 CL500-18 
               
               
                 ATGAAGCTACT 
                 269 
                 89F 
                 OTU CL1A15, 
                 N/A 
                 AGGTCATCTTCA 
                 91 
                 89P 
               
               
                 TCGGTAGTG 
                   
                   
                 single-copy, 
                   
                 ACCGAAAAACTT 
               
               
                 TGTAGGTACCG 
                 270 
                 89R 
                 identified as 
                   
                 TCCAGCCCCGAT 
               
               
                 TCACTTTCG 
                   
                   
                 Uncultured 
                   
                 CATGCGATCAGA 
               
               
                   
                   
                   
                 Crater Lake 
                   
                 GC 
               
               
                   
                   
                   
                 bacterium CL0- 
               
               
                   
                   
                   
                 27 
               
               
                 ATGAAGCACC 
                 271 
                 90F 
                 OTU LT1A54, 
                 N/A 
                 TCATCTTCAACCG 
                 92 
                 90P 
               
               
                 TTCGGGTGTG 
                   
                   
                 multi-copy, 
                   
                 AAAAACTTTCCA 
               
               
                 TGCAGGTACC 
                 272 
                 90R 
                 identified as 
                   
                 AACCCGCGGATG 
               
               
                 GTCACTTTCG 
                   
                   
                 Uncultured 
                   
                 CCCGCAGGTTTC 
               
               
                   
                   
                   
                 Crater Lake 
                   
                 A 
               
               
                   
                   
                   
                 bacterium 
               
               
                   
                   
                   
                 CL0-64 
               
               
                 GATCTTTGATC 
                 273 
                 91F 
                 OTU LT1A55, 
                 N/A 
                 CAGACGCGAGCT 
                 93 
                 91P 
               
               
                 TTAGTGGCG 
                   
                   
                 multi-copy, 
                   
                 CTTCCTAAGGTG 
               
               
                 TCAAGTACCGT 
                 274 
                 91R 
                 identified as 
                   
                 GATAAATCCTTTT 
               
               
                 CAGAACTTC 
                   
                   
                 Uncultured 
                   
                 ACCTCTCGGCGT 
               
               
                   
                   
                   
                 freshwater 
                   
                 A 
               
               
                   
                   
                   
                 bacterium 
               
               
                   
                   
                   
                 LCK-26 
               
               
                 AACGTACCCA 
                 275 
                 92F 
                 OTU CL1A9, 
                 N/A 
                 GGCCGCTCCAGG 
                 94 
                 92P 
               
               
                 AGAGTGGG 
                   
                   
                 multi-copy, 
                   
                 AGCACGAGGTCT 
               
               
                 AAGGATATTA 
                 276 
                 92R 
                 identified as 
                   
                 TGCGATCCCCCG 
               
               
                 GCCTCTACCG 
                   
                   
                 
                   Zoogloea 
                 
                   
                 CTTTCATCCTTAG 
               
               
                   
                   
                   
                 
                   ramigera 
                 
                   
                 A 
               
               
                 TGAAGTTCCTT 
                 277 
                 93F 
                 OTU LT1A27, 
                 N/A 
                 ATCTTTCATCAAA 
                 95 
                 93P 
               
               
                 CGGGAATGG 
                   
                   
                 multi-copy, 
                   
                 ATTTTTTCCCGGC 
               
               
                 TTCTTCCCTAC 
                 278 
                 93R 
                 identified as 
                   
                 TCGGCGATGCCG 
               
               
                 TGAAAGAGG 
                   
                   
                 Uncultured 
                   
                 CCAAGACGGAGT 
               
               
                   
                   
                   
                 actinomycete 
               
               
                   
                   
                   
                 clone SFD1-39 
               
               
                 CTCATCAGCAA 
                 279 
                 94F 
                 OTU LT1A46, 
                 N/A 
                 TCATGTAAGCCG 
                 96 
                 94P 
               
               
                 TGGTGGGAG 
                   
                   
                 multi-copy, 
                   
                 CTCCTCCGGCGG 
               
               
                 TCAACTCCGGA 
                 280 
                 94R 
                 identified as 
                   
                 AATCACACCTTTG 
               
               
                 GGAGAACC 
                   
                   
                 Uncultured 
                   
                 CTCCGCAGAGTT 
               
               
                   
                   
                   
                 planctomycete 
                   
                 C 
               
               
                   
                   
                   
                 clone CY0ARA- 
               
               
                   
                   
                   
                 031E04 
               
               
                 GGCAGCACGG 
                 281 
                 95F 
                 OTU TL1A1, 
                 N/A 
                 TATTCTTAAAGCG 
                 97 
                 95P 
               
               
                 TCTAGTTTAC 
                   
                   
                 multi-copy, 
                   
                 CCAGGCCTTGCG 
               
               
                 TCAAATCCTCC 
                 282 
                 95R 
                 unidentified 
                   
                 GTCCCCAGCTTTT 
               
               
                 TCCCCACTG 
                   
                   
                   
                   
                 CTCCTCAGAGAT 
               
               
                 GTCAGACTTCG 
                 283 
                 96F 
                 OTU TL1A2, 
                 N/A 
                 CTCCATCAGCGC 
                 98 
                 96P 
               
               
                 GTCTGATTG 
                   
                   
                 multi-copy, 
                   
                 CCTTGCGAGCTTT 
               
               
                 GGTACTTCTTC 
                 284 
                 96R 
                 unidentified 
                   
                 CATCCCTTCTGCG 
               
               
                 CCGAGCAAC 
                   
                   
                   
                   
                 ACGAAGGGATCG 
               
               
                 ATGTAGCAAT 
                 285 
                 97F 
                 OTU TL1A6, 
                 N/A 
                 GGGGCACGGGCT 
                 99 
                 97P 
               
               
                 ACAGGACAGC 
                   
                   
                 single-copy, 
                   
                 CATCTTGGGGCG 
               
               
                 CGTACATTTGA 
                 286 
                 97R 
                 unidentified 
                   
                 GAATCACACCTTT 
               
               
                 TTCCCTACG 
                   
                   
                   
                   
                 GGTCCGCAAACA 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 ATGAAGCTGG 
                 287 
                 98F 
                 OTU CL1A2, 
                 N/A 
                 TCCTTGACCAAA 
                 100 
                 98P 
               
               
                 AGCTTGCTCC 
                   
                   
                 multi-copy, 
                   
                 ATTCTTTCCACGC 
               
               
                 GCGAGCTCATC 
                 288 
                 98R 
                 unidentified 
                   
                 CCGTGGGATGCC 
               
               
                 CTTGACC 
                   
                   
                   
                   
                 CCAAGGCGTCGT 
               
               
                   
                   
                   
                   
                   
                 A 
               
               
                 ACGGGAGCAA 
                 289 
                 99F 
                 OTU CL1A10, 
                 N/A 
                 TGATATCGGCCG 
                 101 
                 99P 
               
               
                 TCCTGGTG 
                   
                   
                 multi-copy, 
                   
                 CTCCAATCGCGC 
               
               
                 CCACTGTATTA 
                 290 
                 99R 
                 unidentified 
                   
                 GAGGTCTTGCGA 
               
               
                 GAGCAGACC 
                   
                   
                   
                   
                 TCCCCCGCTTTCA 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 ACGGCTTCGGC 
                 291 
                 100F 
                 OTU LT2A3, 
                 N/A 
                 GCTCTTGCGAGCT 
                 102 
                 100P 
               
               
                 CTAGTAAAG 
                   
                   
                 multi-copy, 
                   
                 CCCTTTCCCGAAA 
               
               
                 AGGGCTGTTCA 
                 292 
                 100R 
                 unidentified 
                   
                 AACTCCTTACGA 
               
               
                 CCCTAATGG 
                   
                   
                   
                   
                 GTTCCGTCGCTC 
               
               
                 TTAACTTAAGT 
                 293 
                 101F 
                 OTU LT2A12, 
                 N/A 
                 AGACGCGAGCTT 
                 103 
                 101P 
               
               
                 GGCGGACGG 
                   
                   
                 multi-copy, 
                   
                 CTCTTTAGGCGG 
               
               
                 GGTACACGTC 
                 294 
                 101R 
                 unidentified 
                   
                 ATTACTCCATTTC 
               
               
                 GTTTTATTCC 
                   
                   
                   
                   
                 ACTCGGAAGCAT 
               
               
                   
                   
                   
                   
                 A 
               
               
                 TAACGCGGGG 
                 295 
                 102F 
                 OTU LT2A16, 
                 N/A 
                 CGCTCCAATAGC 
                 104 
                 102P 
               
               
                 CAACCTGG 
                   
                   
                 multi-copy, 
                   
                 GAGAGGTCTTGC 
               
               
                 GGGTATTAGCC 
                 296 
                 102R 
                 unidentified 
                   
                 GATCCCCCCCTTT 
               
               
                 CAGAGCG 
                   
                   
                   
                   
                 CACCCGAAGGTC 
               
               
                   
                   
                   
                   
                   
                 G 
               
               
                 AGAGTTTGATC 
                 297 
                 103F 1   
                 OTU LT1A15, 
                 N/A 
                 CGGTCCCAGCGTT 
                 105 
                 103P 
               
               
                 CTGGCTCAG 
                 298 
                 103F 2   
                 single-copy, 
                   
                 TCCAGTAATCTCT 
               
               
                 ACGGAGGTAG 
                   
                   
                 unidentified 
                   
                 CTCTAGACTACTG 
               
               
                 CAATACCTTA 
                   
                   
                   
                   
                 CTTACGACGTA 
               
               
                 GTGCTTCTTCT 
                 299 
                 103R 
               
               
                 TCCGGTACC 
                   
               
               
                 TTCGGTTATGT 
                 300 
                 104F 
                 OTU LT1A16, 
                 N/A 
                 TAATCCTAAAGC 
                 106 
                 104P 
               
               
                 TGATGGCGA 
                   
                   
                 multi-copy, 
                   
                 GCCAGGCCTTGC 
               
               
                 TCGGGTAACGT 
                 301 
                 104R 
                 unidentified 
                   
                 GGTCCCCAGCTTT 
               
               
                 CAATAAACC 
                   
                   
                   
                   
                 CCTCCTAAGAGA 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 AACCCCGGTG 
                 302 
                 105F 1   
                 OTU LTIA18, 
                 N/A 
                 GTCCCCCGCTTTC 
                 107 
                 105P 
               
               
                 GCGAGTGG 
                 303 
                 105F 2   
                 multi-copy, 
                   
                 ATCCATAGATCG 
               
               
                 AACCCTGGTG 
                   
                   
                 unidentified 
                   
                 TATGCGGTATTA 
               
               
                 GCGAGTGG 
                   
                   
                   
                   
                 GCGTAACTTTCGC 
               
               
                 TTCTTACGGTA 
                 304 
                 105R 
               
               
                 CCGTCATG 
                   
                   
               
               
                 GAGCGATGAA 
                 305 
                 106F 
                 OTU LT3A1, 
                 N/A 
                 CAATATTCGGTAT 
                 108 
                 106P 
               
               
                 GTTTCTTCGG 
                   
                   
                 single-copy, 
                   
                 TAGCACCGGTTTC 
               
               
                 AGCCGGTGCTT 
                 306 
                 106R 
                 unidentified 
                   
                 CCGGTGTTATCCC 
               
               
                 CTTTTGTAG 
                   
                   
                   
                   
                 AAAGTGGAGGG 
               
               
                 GGTAACAGGT 
                 307 
                 107F 
                 OTU LT3A2, 
                 N/A 
                 AGGTCTTGCGAT 
                 109 
                 107P 
               
               
                 TAAGCTGACG 
                   
                   
                 multi-copy, 
                   
                 CCCCCCCTTTCAC 
               
               
                 CAGAGTATTA 
                 308 
                 107R 
                 unidentified 
                   
                 CCGTAGGTCGTA 
               
               
                 ATCCGAAGCG 
                   
                   
                   
                   
                 TGCGGTATTAATC 
               
               
                 GGTCTAGTTTA 
                 309 
                 108F 
                 OTU LT3A7, 
                 N/A 
                 CAGCTTTTCTCCT 
                 110 
                 108P 
               
               
                 CTAGATGGG 
                   
                   
                 multi-copy, 
                   
                 CAGAGATTACGC 
               
               
                 TTCTTCTGTGG 
                 310 
                 108R 
                 unidentified 
                   
                 GGTATTAGCCTG 
               
               
                 GTAACGTCC 
                   
                   
                   
                   
                 AGTTTCCCCAGGT 
               
               
                 CATCGGAACG 
                 311 
                 109F 
                 OTU LT1A35, 
                 N/A 
                 CTTTCCCCCTCAG 
                 111 
                 109P 
               
               
                 TACCTTATCG 
                   
                   
                 multi-copy, 
                   
                 GGCGTATGCGGT 
               
               
                 CGCAGTCTGTG 
                 312 
                 109R 
                 unidentified 
                   
                 ATTAGCGCAACT 
               
               
                 TTAGAGCTG 
                   
                   
                   
                   
                 TTCGCTGCGTTAT 
               
               
                 CGTGAGAATCT 
                 313 
                 110F 
                 OTU LT1A55, 
                 N/A 
                 CAGACGCGAGCT 
                 112 
                 110P 
               
               
                 ACCCTTAGG 
                   
                   
                 multi-copy, 
                   
                 CTTCCTAAGGTG 
               
               
                 GCTTGCATCCT 
                 314 
                 110R 
                 unidentified 
                   
                 GATAGATCCTTTT 
               
               
                 CTGTATTAC 
                   
                   
                   
                   
                 ACCTCTCGGCAT 
               
               
                   
                   
                   
                   
                   
                 A 
               
               
                 TGTCGTCAGCT 
                 315 
                 111F 
                 Control 1372 
                 N/A 
                 TGACGGGCGGTG 
                 113 
                 111P 
               
               
                 CGTGTCG 
                   
                   
                   
                   
                 TGTACAAGGCCC 
               
               
                 AAGGAGGTGA 
                 316 
                 111R 
                   
                   
                 G 
               
               
                 TCCAGCCG 
               
               
                   
               
            
           
         
       
     
     The PCR products in each reaction mixture (e.g., 16S rDNA and 18S rDNA) may be generated from dNTPs which contain a mixture of dATP, dGTP, dCTP, dTTP, and amino allyl-dUTP. The labeling step may employ dye incorporation resulting from a coupling reaction between a cyanine (Cy) and the PCR product. The pools of labeled PCR products may be hybridized with the array, whose immobilized oligonucleotides specify 50-mer sequences that are complementary to at least some of the individual rDNA sequences amplified from each sample. In some cases, the experiment may be replicated by performing a second “dye swap” experiment to minimize any false signals due to differential incorporation of the dye in the amplification products. 
     Also, probes that target taxa at different hierarchical levels may be included in the array in order to optimize detection of desired bioindicator organisms. For example, in addition to developing PCR primers that are specific to a single species, primers that are capable of detecting several species in a particular genus may be developed (e.g., Oldach, D. W., et al., 2000 , Proc. Natl Acad. Sci. U.S.A.  97:4303–4308). The more generic primers may be used both as a confirmation that a particular genus is present in any sample that tested positive with the more specific primers, and as an indicator that potentially unknown or undetected members of the genus are also present. 
     The array may provide a qualitative result and/or a quantitative result. For example, as shown in  FIG. 11 , an array of prokaryotic rDNA sequences and/or an array of eukaryotic rDNA sequences may be probed using PCR amplified rDNA sequences amplified from genomic DNA from a water sample to provide qualitatively different patterns of hybridization. For example, DNA from a water sample may be amplified using prokaryotic specific primers and then labeled with a red dye (Cy5) to provide a “red” prokaryotic probe ( FIG. 11A-1 ) that recognizes prokaryotic sequences immobilized at designated positions on an array ( FIG. 11B ). Alternatively or additionally, DNA from a water sample may be amplified using eukaryotic specific primers and then labeled with a green dye (Cy3) to provide a “green” eukaryotic probe ( FIG. 11A-2 ) that recognizes eukaryotic sequences immobilized at designated positions on an array ( FIG. 11B ). 
     The array may also be used as a quantitative assessment tool, to monitor the change in various microbes over time, or to monitor the relative amounts of a microbe between two samples that vary in location (e.g., for two different bodies of water, or two locations in one body of water) or time of sampling (e.g., a single body of water sampled at two different seasons). Referring now to  FIG. 12 , an array may be probed with amplified DNA that corresponds to both 16S (prokaryotic) rDNA sequences and/or 18S (eukaryotic) rDNA sequences. To distinguish the two samples, amplification from the first sample may be labeled with a first dye (e.g., Cy3, green), whereas the amplification from the second sample may be labeled with a second dye (e.g., Cy5, red). As shown in  FIG. 12A , the nucleic acids immobilized at a particular position (e.g., location or address) on the array, will bind to a complementary nucleic acid probe that is labeled either with the green dye (from the first sample), or the red dye (from the second sample). Upon hybridization, if hybridization is specific to probe from sample  1 , a green spot will result; if hybridization is specific to probe from sample  2 , a red spot will result; if hybridization occurs for probe molecules that are found in both samples, the color will vary dependent upon relative abundance of the amplified probe, and also the target, in each sample ( FIGS. 12A and 12B ).  FIG. 12C  shows the results for this type of experiment for two lake samples amplified by PCR to generate either 16S probes (top panel) or 18S probes (bottom panel). In an embodiment, there may be at least one position per array for a known sequence that serves as a control to allow for orientation of the array (e.g., green spots in lower right hand position of each grid in  FIG. 12C ). 
     As described herein, the array may also be used to determine how chemical additions to water (e.g. cadmium, atrazine, perchlorate) may change the microbial community, and/or to detect known water pathogens. For example, the microarray may include 16S rDNA sequences from several known pathogens.  FIG. 13  shows results for such an experiment, where multiplex PCR amplification and direct labeling of cyanobacteria present in a water sample are detected using cyanobacterial sequences on the array (Panel  13 D). 
     In one embodiment, an array of oligonucleotides that are isolated from known and unknown OTUs may be prepared. As used herein, known OTUs comprise sequences that are ≧97.5% identical to sequences that have been reported in public databases, whereas unknown OTUs comprise sequences that do not meet this criterion and are believed to represent as yet unknown organisms.  FIG. 14  shows an array comprising 105 olignonucleotides isolated from predetermined known and unknown freshwater lake OTUs probed with amplified DNAs from two different lake samples, where multiplex PCR reactions of each sample were identical and products each reaction were either labeled with a red dye (Cy5) or a green dye (Cy3), allowing comparison of samples. Shown are results from a test of two lake samples used to hybridize first singly (top panels) (left panel: green probe, sample  1 ; right panel: red probe, sample  2 ) and then together (bottom panel) to the same array of 105 oligonucleotide probes. 
     Such taxon-specific arrays may be used to detect microbes that are known to be characteristic of a particular type of water system. For example, in one embodiment, the array may be formulated to detect microbes common to fresh water systems. Or, the array may be formulated to detect microbes common to marshlands or small tidal pools. Or, arrays having nucleic acids derived from estuary water samples may be used to analyze water from various estuaries. The development of an array that utilizes freshwater nucleotide sequences is described in the Examples, below. 
     EXAMPLES 
     Example 1 
     Materials and Methods 
     Sample collection and DNA extraction. To develop probes for an array, three lakes were sampled: Lake Townsend (LT) (Greensboro, N.C.); City Lake (CL) (High Point, N.C.); and Toolik Lake (T L) (Alaska). Lake Townsend and City Lake are temperate mesotrophic and eutrophic municipal drinking water reservoirs, respectively. Toolik Lake is a highly oligotrophic, glacial lake located in the Arctic Long Term Ecological Research Site above the Arctic Circle in Alaska (Moon-van der Staay, S. Y., et al., 2001 , Nature,  409:607–610). 
     Five samples of surface water were collected: (1) LT-1J—at an open water location in Lake Townsend (station 1), depth=8.3 m, on Jun. 14, 2000; (2) LT-2J—at a shallow, near shore location in Lake Townsend (station 2), depth=1.3 m, on Jun. 14, 2000; (3) LT-1M—at station 1 of Lake Townsend, on Mar. 28, 2001; (4) CL—at an artificially aerated location in City Lake near a subsurface water treatment system intake on May 22, 2001; and (5) TL—at a location near the main LTER sampling station in Toolik Lake on Aug. 11, 2000. Each sample (˜100 ml) was drawn through GF/C and GF/F glass fiber filters that were placed in cetyltrimethylammonium bromide (CTAB) buffer for storage at room temperature until later DNA extraction. Other lake samples, such as water samples from lakes that may be diagnostic of deformities found in members of the ecosystem may be used. For example, tests have been conducted using a group of North Dakota lakes that were paired according to geographical proximity. Each pair of lakes includes one lake that has been associated with a high level of frog deformities and one lake that has not been associated with any known deformities in frogs or any other organism. Samples from the lakes were used to isolate DNA for array analysis to ascertain microbial bioindicators associated with conditions leading to such deformities. 
     Genomic DNA was extracted from each water sample using a CTAB (cetyltrimethylammonium bromide) buffer DNA isolation technique (Rublee, P. A., et al., 1999 , Va. J Sci.,  50:325–335). Briefly, the glass fiber filter was macerated in 2 ml CTAB in a 15 ml polypropylene conical tube using a sterile wooden applicator stick. After heating for 1 hr at 65° C., the mixture was extracted with 2 ml 24:1 (v/v) chloroform-isoamyl alcohol, and the DNA isolated from the aqueous portion by precipitation with 0.7 volumes 100% 2-propanol. The precipitate was pelleted, air-dried, and the DNA rehydrated in 25 μl TE buffer (pH 7.4) and stored at −20° C. 
     PCR amplification of genomic DNA from water samples for cloning rDNAs. SSU rDNA was amplified by PCR using prokaryotic-specific and eukaryotic-specific forward and reverse primers in 50 μl reactions (Table 1). Using the appropriate primer pairs, separate reactions were prepared for 16S rDNA and 18S rDNA. The amplification reactions consisted of: 5 μl 10× PCR Buffer; 5 μl Promega 25 mM MgCl 2 ; 5 μl 100 mM BSA; 2.5 μl 16 mM dNTP stock (4 mM each of dATP, dCTP, dGTP and dTTP); 1 μl each of 10 μM forward and reverse primers; 1 U Taq DNA Polymerase; 30.2 μl sterile deionized H 2 O; and 1 μl genomic DNA. An MJ Research PTC-100 Programmable Thermal Controller was used to amplify samples under the following conditions: 2 min initial denaturation at 94° C.; 30 cycles of 1 min denaturation at 94° C.; 1 min annealing at 56° C. for 16S primers or 58° C. for 18S primers; and 2 min extension at 72° C.; 5 min final extension at 72° C. PCR products were verified by gel electrophoresis. 
     Cloning and sequencing of amplified PCR products. Purified PCR products were subcloned in plasmid vectors using the Invitrogen TOPO TA Cloning Kit according to the manufacturer&#39;s protocol. Ligation reaction mixtures were then used to transform TOP10 Chemically Competent  E. coli  cells and recombinant plasmids were identified by growth of bacterial colonies on LB agar plates containing 50 μg/ml ampicillin. Individual colonies from each plating were inoculated into LB liquid medium containing 50 μg/ml ampicillin and grown overnight with antibiotic selection. Inserts were purified from cultures using a QIAGEN QIAprep Spin Miniprep Kit according to the manufacturer&#39;s protocol. After removal of the insert, gel electrophoresis was used to verify the presence of a DNA fragment corresponding in size to 16S rDNA or 18S rDNA. 
     Fifty verified clones from each library were sequenced using a LI-COR Global Edition IR 2  System for automated DNA sequencing. Sequencing reactions were prepared using an Epicentre Technologies SequiTherm EXCEL™ II DNA Sequencing Kit-LC (for 25–41 cm gels) according to the Cycle Sequencing Protocol provided. Sequencing reactions included: 3.5× SequiTherm EXCEL™ II Sequencing Buffer; fluorescent (IR)-labeled M13–20 forward and reverse primers; purified cloned 16S rDNA or 18S rDNA; sterile deionized H 2 O; SequiTherm EXCEL II DNA Polymerase; and SequiTherm EXCEL II-LC Termination Mixes A, C, G and T (each containing dATP, dCTP, dTTP, 7-deaza-dGTP, and one ddNTP). Using an MJ Research PTC-100 Programmable Thermal Controller, reactions were amplified by PCR using the following conditions: 4.5 min initial denaturation at 95° C., followed by 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 51° C., and 1 min extension at 70° C. After amplification, Stop/Loading Buffer was added to reaction products, which were denatured for 5 min at 95° C. and loaded into a 41 cm polyacrylamide gel. During electrophoresis of reaction products, dual 700 and 800 nm detection channels were used for simultaneous bidirectional sequencing of each clone and sequence read lengths were at least 550 bp in each direction. 
     Sequence Alignments and Community Analyses. It is generally accepted in the art that 1000 nucleotides of sequence information is sufficient for even a detailed phylogenetic analysis (Oldach, D. W., et al., 2000 , Proc. Natl Acad. Sci., U.S.A.  97:4303–4308). Thus, partial sequences were obtained for each clone and the first 500 nucleotides from both 5′ and 3′ ends. The use of 1000 nucleotides for phylogenetic ordering of the sequences was subsequently verified by generating complete sequences for 18S rDNA clones, with no improvement in the data (results not shown). After checking each sequence for the presence of chimeric properties using the program CHIMERA_CHECK (version 2.7; Ribosomal Database Project II, Center for Microbial Ecology, Michigan State University, available on-line), one sequence was excluded. The remaining sequences were submitted to the National Center for Biotechnology Information website for BLAST analysis (Altschul, S. F., et al., 1990 , J. Mol. Biol.,  215:403–410). 
     Sequence alignments and analyses were performed using the BioEdit Sequence Alignment Editor and Analysis software (version 5.0.9; Department of Microbiology, North Carolina State University, available on-line), which includes CLUSTAL W as an accessory application for multiple alignments. Libraries were compared in pairwise fashion by combining and aligning two libraries at a time (100 sequences of 16S rDNA or 18S rDNA). Libraries were also merged into one group for alignment and comparison (249 16S rDNA sequences or 250 18S rDNA sequences). 
     Taxonomic classifications were based on identity matrices generated using an analysis tool incorporated into BioEdit. Sequences having identity scores of 0.975 or greater were considered to be sufficiently similar to group them into the same operational taxonomic unit (OTU). After grouping clones into OTUs, several diversity indices were calculated for each library. These included: (1) species richness, or total number of OTUs; (2) Simpson&#39;s dominance index, used to describe the distribution of clones among OTUs, or evenness; and (3) the Shannon-Wiener index, which serves as a statistical measure of the probability of correctly guessing the OTU identity of a randomly selected clone (Colinvaux, P. 1993. Ecology 2. John Wiley &amp; Sons, Inc., New York, U.S.A). In addition, Sorensen coefficients (Lemke, M. J., et al., 1997 , Microb. Ecol.,  34:224–231) were calculated to measure the similarity in species composition between two communities, or the proportion of OTUs shared between two libraries. 
     Also estimated was total species richness (S) using four methods based on the distribution of OTUs within a library. These included: Scov, an estimate based on “coverage” (Finlay, B. J., 2002 , Science,  296:1061–1063; Giovannoni, S. J., et al., 1990 , Nature,  345:60–63); Smax, an estimate based on rarefaction analysis (Haldeman, D. L., et al, 1994 , Appl. Environ. Microbiol.,  60:2697–2703; Methe, B. A., and J. P. Zehr, 1999 , Hydrobiologia,  401 :77–96), which was performed using the program Analytic Rarefaction (version 1.3; Stratigraphy Lab, University of Georgia, available on-line); and SACE and SChao 1, two estimates that were evaluated using a form processor and spreadsheet available through a web-based interface (ASLO, Limnology and Oceanography Methods, available on-line). 
     Phylogenetic analyses were conducted to assess molecular evolutionary relationships using MEGA software (version 2.1; Molecular Evolutionary Genetics Analysis, available on-line). All phylogenetic analyses are sensitive to alignment methods, assumptions regarding mutational rates, and the types and amount of sequence data used (Troesch, A., et al., 1999 , J. Clin. Microbiol.,  37:49–55.). Trees were constructed using the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) and distances were estimated according to the Kimura 2-parameter model for nucleotide exchange with a transition/transversion ratio of 2.0 (Hurlbert, S. H., 1971 , Ecology  52:577–586). 
     Real-time Quantitative PCR. Real-time Quantitative PCR (Q-PCR) experiments were designed to analyze the relative abundance of two 16S rDNA OTUs and one 18S rDNA OTU in three different samples using a Cepheid Smart Cycler system. The presence and accumulation of fluorescence bound to each target OTU was measured directly and compared among LT-1J, LT-1M and CL. Individual 25 μl reactions included: 2.5 μl Takara 10× Ex Taq Buffer; 1.25 μl Takara dNTP Mixture (2.5 mM each); 1.25 μl SYBR® Green I nucleic acid gel stain (10×); 0.25 μl Takara Ex Taq™; 1 μl OTU-specific forward and reverse primers (10 μM each); 16.75 μl sterile deionized H 2 O; and 1 μl experimental template (genomic DNA sample —0.025 μg/μl), positive control (mixed clone standard—25 pg/μl each), or negative control (sterile deionized H 2 O). The mixed clone standard was prepared by combining three clones representing three experimental samples used in the experiment (i.e., genomic DNA samples from three different OTUs). Relative abundance estimates were calculated using a 1:10 dilution series of the mixed clone standard to determine cycle number differences between 25 pg, 2.5 pg, 0.25 pg, and 0.025 pg template concentrations. 
     Multiplex PCR of probes for array. To generate the target sequences used for hybridization to the array, a multiplex PCR amplification using at least 45 primer pairs developed from an analysis of individual OTUs, may be performed. The sequences of the eukaryotic primers and associated 50-mer probes are shown in Table 2. The sequences of the prokaryotic primers and associated 50-mer probes are shown in Table 3. 
     The conditions for multiplex PCR are as follows. Multiplexed PCR reactions containing ˜250 ng genomic DNA are prepared in 0.5 ml thin-walled microcentrifuge tubes. The final reaction volume is 50 μl and contains a 200 nM final concentration of each primer, 5 μl 10× buffer, 5 μl BSA, 5 μl dNTPs, 1.25 U Taq, and sd H 2 O (remaining volume). Reactions are placed in a programmable thermal controller and DNA amplification by PCR is carried out under the following conditions: initial denaturation at 94° C. for 2 minutes; 30 cycles of denaturation at 94° C. for 1 minute, annealing at 1 to 3 degrees below lowest primer T m , extension at 72° C. for 1 minute; final extension at 72° C. for 1 minute; and storage at 4° C. of reaction product until use. The dNTP mixture is prepared by adding 16.6 μl sd H 2 O to 2 μl 50× dNTP stock solution for an 8.33-fold dilution, where 50× dNTP stock solution is 10 μl each 100 mM dATP, dGTP, dCTP; 8 μl 100 mM amino allyl-dUTP; and 2 μl 100 mM dTTP. 
     The PCR products may, in some cases, be labeled using fluorescent dyes. The PCR product is transferred to a 1.5 ml microcentrifuge tube and 5 μl 3 M sodium acetate and 500 μl 100% ethanol are added. The contents are thoroughly mixed and stored overnight at −70° C. The samples are removed from −70° C. storage and spun in a microcentrifuge at 14,000 rpm for 20 minutes. After pouring off the supernatant, 500 μl 70% ethanol is added to wash the DNA pellet and this mixture is microcentrifuged at 14,000 rpm for 10 minutes. The supernatant is again poured off, the tube is allowed to air dry to remove all ethanol, and the pellet is then resuspended in 15 μl nuclease-free H 2 O. The Cy dyes are removed from −20° C. storage and resuspended in 15 μl 0.1 M sodium bicarbonate, pH 9.0. The Cy dye and DNA resuspensions are combined, mixed well, and allowed to incubate for 1 hr at room temperature in the dark. To quench any unbound Cy dye after the dye coupling reaction, 15 μl 4 M hydroxylamine are added to each sample and these are incubated for 15 minutes at room temperature in the dark. Labeled samples are then purified using a PCR purification kit according to the manufacturer&#39;s protocol. 
     Spotting of oligonucleotides on the array. To immobilize nucleic acids on the array the following protocol was used. Each 50-mer oligonucleotide to be used as a probe sequence (i.e., sequences corresponding to SEQ ID NOS: 5–113 or the complement of these sequences) on the array is diluted 1:10 with 3×SSC/0.1% sarkosyl in a 96-well microplate for a final oligonucleotide concentration of ˜250 ng/μl. Epoxy-coated slides are secured in slide positions of arrayer for printing. After the oligonucleotides are printed, the slides are UV cross-linked at 60 mJoules, baked at 80° C. in an oven for 2 hours, and stored at room temperature. The 3×SSC is prepared using a 20×SSC concentrate containing 3 M NaCl and 0.3 M sodium citrate, pH 7.0. 
     Hybridization of DNA sample to array. After purification, the Cy3 and Cy5-labeled sample eluates may be combined and lyophilized until almost dry, leaving approximately 5–10 μl behind. The sample may be resuspended using 80 μl Roche DIG Easy Hyb hybridization buffer and a clean lifterslip is placed on the microarray being used. The sample is heated at 95° C. for 2 minutes, cooled on ice for 1 minute, spun down to collect any condensation, and pipetted under the lifterslip. Next, the microarray is sealed within a hybridization cassette using the cassette lid and placed into a 45–55° C. water bath overnight. The next morning, three wash solutions are heated to 37° C., the hybridization cassette is removed from the water bath, and the microarray is removed from the cassette. The microarray with lifterslip is gently dipped into a staining dish containing wash buffer #1 (1×SSC, 0.1% SDS), to release the lifterslip. The microarray is then placed in a staining dish cassette and gently washed in wash buffer #1 for 5 minutes. Using the same technique, the microarray is next washed in wash buffer #2 (1×SSC), for 5 minutes. Finally, the microarray is washed in wash buffer #3 (0.05×SSC), by gently dipping it 5–10 times. The microarray is removed from the staining dish cassette and tapped on edge against the benchtop to remove all solution droplets. Once dry, the microarray is scanned. 
     Example 2 
     Grouping rDNAs into Operational Taxonomic Units 
     Analysis of Clones. Approximately 50 prokaryotic rDNA and 50 eukaryotic rDNA clones for each of the five lake samples (500 rDNA sequences) were sequenced through a 1700 bp segment of the eukaryotic 18S rDNA, or a 1540 bp segment of the prokaryotic 16S rDNA, to provide for phylogenetic classification of known and novel species (Pace et al., 1986 , Adv. Microb. Ecol.,  9:1–55; Sogin and Gunderson, 1987 , Annals. NY Acad. Sci.,  503:125–139). Based on the rDNA sequencing alignments, a level of 97.5% sequence identity was the criterion by which rDNAs were placed in the same operational taxonomic unit (OTU). Because multiple small subunit rDNA copies may reside within a species genome (Farrelly et al., 1995 , Appl. Environ. Microbiol.,  61:2798–2801), a 97.5% level of sequence identity allows for the possibility that a different sequence in the same species was recovered. A software program (CHIMERA-CHECK; Kopzcysnski et al., 1994 , Appl. Environ. Microbiol.,  60:746–748; Wang and Wang, 1995 , Appl. Environ. Microbiol.,  63:4645–4650; Qui et al, 2001 , App. Environ. Microbiol.,  58:2717–2722) was also employed to reduce the possibility of misidentifying a chimeric rDNA as an unique clone. 
     For each sample, libraries of SSU rDNA clones were produced, individual clones were sampled, sequences for each clone were generated, and standard diversity statistics were computed (Table 4). Based on a comparative analysis of all prokaryotic (16S) rDNA sequences, it was determined that 49 OTUs contained multiple sequences and that 62 OTUs were unique, each containing a single sequence. An analysis of all eukaryotic (18S) sequences resulted in 42 OTUs containing multiple sequences and 67 unique OTUs. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Comparison of prokaryotic and eukaryotic diversity 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 LT-1J 
                 LT-2J 
                 LT-1M 
                 CL 
                 TL 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 rDNA library 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Number of clones 
                 50 
                 50 
                 50 
                 50 
                 50 
                 50 
                 49 
                 50 
                 50 
                 50 
               
               
                 Number of OTUs 
                 33 
                 31 
                 31 
                 32 
                 26 
                 34 
                 28 
                 28 
                 23 
                 12 
               
               
                 Evenness 
                 0.039 
                 0.049 
                 0.050 
                 0.053 
                 0.089 
                 0.043 
                 0.079 
                 0.102 
                 0.078 
                 0.358 
               
               
                 Shannon-Wiener 
                 3.38 
                 3.24 
                 3.24 
                 3.24 
                 2.84 
                 3.35 
                 3.00 
                 2.90 
                 2.86 
                 1.62 
               
               
                 index 
               
               
                   
               
               
                 LT-1J: Lake Townsend, station 1, June; 
               
               
                 LT-2J: Lake Townsend, station 2, June; 
               
               
                 LT-1M: Lake Townsend, station 1, March; 
               
               
                 CL: City Lake; 
               
               
                 TL: Toolik Lake. 
               
            
           
         
       
     
     Species Diversity Measures. Prokaryotic and eukaryotic species richness, evenness, and the Shannon-Wiener index differed among lakes. In general, Lake Townsend, NC, contained more species and showed the most even distribution of species; consequently, its Shannon-Wiener index values were the highest. In contrast, Toolik Lake, AK, had the lowest richness estimates, especially for eukaryotes, indicating that species diversity was much lower here than in the temperate lakes and that a few successful competitors dominated the arctic community. 
     As a first indication of diversity, various statistical measurements of the recovered sequences were made to determine the species coverage, species evenness (Simpson&#39;s Index of Dominance; Colinvaux, 1993 , Ecology  2, John Wiley &amp; Sons, Inc. New York, N.Y.), species richness (the number of recovered species in a sample), species diversity (Shannon-Wiener index; Nubel et al., 1999 , Appl. Environ. Microbiol.,  65:422–430) and the estimated proportion of shared OTUs between samples (Sorenson similarity coefficient; Lincoln et al, 1998 , A Dictionary of Ecology, Evolution, and Systematics , Cambridge University Press, New York, N.Y.; McCaig et al., 1999 , Appl. Environ. Microbiol.,  65:1721–1730). Coverage estimates of the percentage of OTUs recovered from a source were based on the relative abundance of the clones already obtained, and indicated that 48 to 76% of the prokaryotic rDNAs, and between 48 and 90% of the eukaryotic rDNAs, had been recovered from the samples. The highest coverage (i.e., 90%) was for Toolik Lake; apparently caused by the high level of recovery of a single rDNA. Not unexpectedly, the species evenness and richness was about the same for all the North Carolina lake samples, but the distribution of recovered species was much more skewed and lower in Toolik Lake, Alaska. To generate a nucleic acid array for monitoring water, however, the purpose of the census is not necessarily to identify all the microbial species that exist in these samples, but rather to survey water sources for relatively common microbes whose appearance and abundance can be monitored on a microarray platform. 
     Rank-abundance curves ( FIGS. 2 and 3 ) for most samples showed that a few taxa were abundant and that many taxa were represented by a single clone. It is highly likely that the samples also contain several other clones at low frequencies. In Toolik Lake, fewer species were detected and these were more abundant relative to the other lakes tested, implying that Toolik Lale contained a smaller number of species. This was supported by the estimates of total taxonomic diversity using the four methods described above (Table 5). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Estimates of species richness (S) for each prokaryotic and eukaryotic community 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 LT-1J 
                 LT-2J 
                 LT-1M 
                 CL 
                 TL 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 rDNA library 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Number of OTUs 
                 33 
                 31 
                 31 
                 32 
                 26 
                 34 
                 28 
                 28 
                 23 
                 12 
               
               
                 Scov 
                 57 
                 56 
                 54 
                 62 
                 44 
                 71 
                 46 
                 47 
                 29 
                 14 
               
               
                   S max 
                 93 
                 72 
                 73 
                 74 
                 40 
                 93 
                 53 
                 47 
                 40 
                 12 
               
               
                 SACE 
                 61 
                 80 
                 77 
                 110 
                 110 
                 106 
                 67 
                 65 
                 38 
                 17 
               
               
                 SChaol 
                 54 
                 77 
                 63 
                 88 
                 91 
                 99 
                 54 
                 60 
                 28 
                 14 
               
               
                   
               
               
                 LT-1J: Lake Townsend, station 1, June; 
               
               
                 LT-2J: Lake Townsend, station 2, June; 
               
               
                 LT-1M: Lake Townsend, station 1, March; 
               
               
                 CL: City Lake; 
               
               
                 TL: Toolik Lake. 
               
            
           
         
       
     
     Sequence Identifications and Library Comparisons. Clone sequences were submitted for BLAST analysis to assess phylogenetic affiliations with reported sequences in the GenBank database. Sequence alignments having ≧97.5% identity were used to propose OTU identifications and determine the proportion of known OTUs for each library (Table 6). The Lake Townsend March sample contained the lowest average proportion of known OTUs (24.8%), while Toolik Lake had the highest (43.0%). Identifications for clones within the same OTU were consistent, suggesting that a 97.5% identity threshold grouped individuals at the species level. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Summary of prokaryotic and eukaryotic OTUs 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 LT-1J 
                 LT-2J 
                 LT-IM 
                 CL 
                 TL 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 rDNA library 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 No. known clones 
                 21 
                 17 
                 16 
                 21 
                 21 
                 19 
                 31 
                 20 
                 25 
                 32 
               
               
                 Proportion known 
                 12/33 
                 8/31 
                 10/31 
                 8/32 
                 6/26 
                 9/34 
                 13/28 
                 6/28 
                 14/23 
                 3/12 
               
               
                 OTUs (%) 
                 (36.4) 
                 (25.8) 
                 (32.3) 
                 (25.0) 
                 (23.1) 
                 (26.5) 
                 (46.4) 
                 (21.4) 
                 (60.9) 
                 (25.0) 
               
               
                   
               
               
                 LT-1J: Lake Townsend, station 1, June; 
               
               
                 LT-2J: Lake Townsend, station 2, June; 
               
               
                 LT-1M: Lake Townsend, station 1, March; 
               
               
                 CL: City Lake; 
               
               
                 TL: Toolik Lake. 
               
               
                 Number of known OTUs = number of OTUs with clones having ≧97.5% sequence similarity to GenBank entries. Known clones refer to clones whose sequences correspond to known microbial species or unidentified species in the GenBank database. 
               
            
           
         
       
     
     Samples having similar estimates of species diversity were distinguished by differences in community composition. For instance, 8 eukaryotic OTUs were identified in each Lake Townsend sample, but the species composition of this group varied between samples. Comparing only the OTUs with multiple sequences, the following observations were made: LT-1J species included  Chilomonas paramecium, Cryptomonas ovata , and  Geminigera cryophila ; LT-2J species included  Brachionus plicatilis, Cryptomonas ovata, Didinium nasutum , and  Dileptus  sp., and the LT-1M species included  Cryptomonas ovata  and  Tabularia tabulata . A comparison of the unique OTUs distinguished these samples even more. 
     Based on sequence alignments and comparisons that included all OTUs, phylogenetic trees were generated according to the UPGMA method to show the overall distribution of OTUs among the five samples. The prokaryote tree in  FIG. 4  was constructed using 111 different 16S rDNA OTUs, including 40 that are known rDNAs (about 36%). The eukaryote tree in  FIG. 5  represents 109 18S rDNA OTUs and includes 22 known rDNA sequences (about 20%). 
     To determine which OTUs, if any, appeared in more than one library, the sequences from each library were compared to those in every other library in pairwise library comparisons and a similarity coefficient was calculated for each pairing (Table 7). Although no prokaryotic sequences were shared between LT-2J and TL, there was some overlap between every pair of samples and the degree of overlap varied considerably. In some comparisons, roughly equal proportions of prokaryotic and eukaryotic OTUs were shared, but other comparisons revealed different patterns. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Sorensen coefficients for prokaryotic and eukaryotic sample comparisons 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 LT-1J 
                 LT-2J 
                 LT-1M 
                 CL 
                 TL 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 rDNA library 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
                 16S 
                 18S 
               
               
                   
               
               
                 LT-1J 
                 1 
                 1 
                 0.344 
                 0.159 
                 0.169 
                 0.215 
                 0.230 
                 0.068 
                 0.071 
                 0.093 
               
               
                   
                   
                   
                 (11) 
                 (5) 
                 (5) 
                 (7) 
                 (7) 
                 (2) 
                 (2) 
                 (2) 
               
               
                 LT-2J 
                   
                   
                 1 
                 1 
                 0.070 
                 0.061 
                 0.169 
                 0.133 
                 0 
                 0.091 
               
               
                   
                   
                   
                   
                   
                 (2) 
                 (2) 
                 (5) 
                 (4) 
                 (2) 
               
               
                 LT-1M 
                   
                   
                   
                   
                 1 
                 1 
                 0.074 
                 0.161 
                 0.122 
                 0.174 
               
               
                   
                   
                   
                   
                   
                   
                   
                 (2) 
                 (5) 
                 (3) 
                 (4) 
               
               
                 CL 
                   
                   
                   
                   
                   
                   
                 1 
                 1 
                 0.039 
                 0.200 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 (1) 
                 (4) 
               
               
                 TL 
                   
                   
                   
                   
                   
                   
                   
                   
                 1 
                 1 
               
               
                   
               
               
                 Coefficients were calculated as follows: S = 2 × C/(A + B), where A and B represent the numbers of OTUs in libraries A and B, respectively, and C represents the number of OTUs shared by A and B (34, 40). ( ) indicate the number of OTUs that were shared by the paired libraries. LT-1J: Lake Townsend, station 1, June; LT-2J: Lake Townsend, station 2, June; LT-1M: Lake Townsend, station 1, March; CL: City Lake; TL: Toolik Lake. 
               
            
           
         
       
     
     Venn diagrams depict sample comparisons at different spatial and temporal scales ( FIG. 6 ). In the fine-scale spatial comparison of LT-1J and LT-2J, each shared OTU contained nearly equal numbers of sequences from both samples, except for two sequences that occurred four times as often in LT-2J. One of these, a 16S rDNA OTU, was not identified, while the other 18S rDNA OTU aligned with  Geminigera cryophila , and was also found in Toolik Lake (three copies). Such unequal occurrences of an OTU between samples may signify a detectable difference in the relative abundance of this particular microbial population between samples. OTUs that consistently vary in frequency among samples are potential bioindicators. For instance, one new bacterial species and the  Geminigera cryophila  OTU appear to be more abundant in temperate lakes, especially pelagic waters. 
     Thus, it was found that for the five lake samples analyzed, 26 different eukaryotic OTUs were represented by multiple copies, including 11 that are associated with known species. Another 79 eukaryotic OTUs were obtained as single copy clones and almost all of these represent unidentified species (Marshall, 2002, Masters Thesis, University of North Carolina at Greensboro). Similarly, 45 different prokaryotic OTUs were found in multiple copies among the collection of samples, of which 10 are associated with a known species, and another 19 resemble reported sequences for as yet unidentified species. Another 92 single copy rDNA sequences, most from unidentified prokaryotes, were recovered from the samples (Amos, 2002, Masters Thesis, University of North Carolina at Greensboro). Both known and unidentified rDNA sequences may be used as probes printed on the assay matrix of the present invention. 
     The ability to assess environmental parameters of water quality may require a sufficient number of potential bioindicator species, but may also require sequences having sufficient similarity across samples to allow for some general application. As noted in Table 8, a diversity of microbial species may be readily retrievable from a single body of water for even an oligotrophic source such as Toolik Lake, Alaska. While every sample produced several unique OTUs, all of the lakes produced multiply represented OTUs, and the most common OTU in each sample accounted for 10% or more of all the rDNAs analyzed in each of the samples. 
                     TABLE 8                  Relative abundance of eukaryotic and prokaryotic OTUs                                     Most abundant                       OTU   2X OTUs   1X OTUs   Total OTUs                                                     Eukary.   Prokary.   Eukary.   Prokary.   Eukary.   Prokary.   Eukary.   Prokary.                                                             LT-1J   10%   8%   9   10   23   26   32   36       LT-2J   14%   10%   9   8   22   26   31   34       LT-1M   10%   14%   8   7   26   23   34   30       CL   14%   22%   10   9   21   22   31   31       TL   58%   20%   7   12   5   12   12   24               LT-1J; Townsend, NC; June, Station 1;       LT-2J; Townsend, NC; June, Station 2;       LT-1M, Townsend, NC; March, Station 1;       CL, City Lake, NC May;       TL, Toolik Lake, AK;            
August. 2× represents OTUs with at least 2 members; 1× represents OTUs with only one member.
 
     Table 9 shows the pattern of shared and unshared OTUs among the five freshwater lake samples. Unshared OTUs refer to rDNA sequences that were recovered and characterized only in the designated sample either as a single copy, referred to as a unique OTU, or in multiple copies. It can be seen that the distribution of some OTUs is broad, while for others it is more localized (Table 9), suggesting that many microbial species exist across a range of watersheds and that a general purpose DNA microarray, that may be used for multiple watersheds, may be developed. An example analysis of specific OTUs is shown in Table 10. 
     A preliminary analysis of rDNA sequences from paired North Dakota lake samples (where one lake sample that exhibits substantial deformities in the frog population and one lake sample does not) acquired from the Fargo, N. Dak. USDA station indicates that some of the OTUs for unknown species are shared with sequences found in North Carolina lakes. Moreover, preliminary real-time PCR experiments suggest that at least some microbes exist an endemic level in almost all freshwater lakes, suggesting that that relative abundance for these endemic microbes is primarily dictated by local water conditions. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Shared and unshared OTUs among the five freshwater lake samples 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 A 
                 B 
                 C 
                 D 
                 E 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Euk. 
                 Prok. 
                 Euk. 
                 Prok. 
                 Euk. 
                 Prok. 
                 Euk. 
                 Prok. 
                 Euky. 
                 Prok. 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Unshared 
                 18 
                 22 
                 19 
                 19 
                 19 
                 19 
                 18 
                 20 
                 5 
                 12 
               
               
                 OTUs 
               
               
                 OTUs in 2 
                 2 
                 5 
                 1 
                 4 
                 3 
                 8 
                 2 
                 4 
                 2 
                 19 
               
               
                 samples only 
               
               
                 Toolik only 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 — 
                 — 
               
               
                 Townsend 
                 5 
                 6 
                 3 
                 6 
                 5 
                 2 
                 — 
                 — 
                 — 
                 — 
               
               
                 only 
               
               
                 Townsend or 
                 2 
                 2 
                 1 
                 3 
                 2 
                 1 
                 5 
                 6 
                 — 
                 — 
               
               
                 City 
               
               
                 All lakes* 
                 4 
                 0 
                 3 
                 0 
                 5 
                 0 
                 5 
                 0 
                 5 
                 0 
               
               
                   
               
               
                 *All lakes does not necessarily mean all samples. 
               
               
                 A: Townsend, NC, June, Station 1; 
               
               
                 B: Townsend, NC, June, Station 2; 
               
               
                 C: Townsend, NC, March, Station 1; 
               
               
                 D: City Lake, NC, May; 
               
               
                 E, Toolik Lake, AK, August 
               
               
                 Euk = Eukaryotic; 
               
               
                 Prok = Prokaryotic 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Relative abundance of five eukaryotic rDNA species found in all three 
               
               
                 sampled lakes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 A 
                 B 
                 C 
                 D 
                 E 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 
                   Cryptomonas 
                 
                 30% 
                 12% 
                 20% 
                 30% 
                 8% 
               
               
                   
                 
                   ovata 
                 
               
               
                   
                 Unidentified 
                 0% 
                 0% 
                 10% 
                 6% 
                 58% 
               
               
                   
                 18s rDNA-1 
               
               
                   
                 
                   Ochromonas 
                 
                 12% 
                 6% 
                 6% 
                 8% 
                 4% 
               
               
                   
                 
                   tuberculata 
                 
               
               
                   
                 
                   Oxytricha 
                 
                 2% 
                 10% 
                 2% 
                 8% 
                 10% 
               
               
                   
                 
                   longa 
                 
               
               
                   
                 Unidentified 
                 10% 
                 0% 
                 4% 
                 2% 
                 10% 
               
               
                   
                 18s rDNA-2 
               
               
                   
                   
               
               
                   
                 A: Townsend, NC, June, Station 1; 
               
               
                   
                 B: Townsend, NC, June, Station 2; 
               
               
                   
                 C: Townsend, NC, March, Station 1; 
               
               
                   
                 D: City Lake, NC, May, 
               
               
                   
                 E: Toolik Lake, AK, August 
               
            
           
         
       
     
     Library Screening vs. Real-time Q-PCR. Several OTUs may be found at quantitatively different levels in different ecosystems. Three OTUs that appeared to be recovered differentially from one water sample were used in fluorescence detection real-time PCR experiments to determine if they were also present in other water samples ( FIG. 7 ). It was found that each of the test OTUs was in fact present in all of the samples investigated. Panels ( 7 A) and ( 7 C) show the detection of two OTUs that appeared in LT-1M clone libraries at frequencies of 10 and four copies (out of 50), respectively. The OTU in panel ( 7 B) was originally recovered from a City Lake library (11 copies of 50). 
     The relative abundance of each OTU was compared and estimated across the same three samples based on a dilution series of a known standard (data not shown). According to cycle threshold differences between growth curves in  FIG. 7A , the amount of product in the LT-1M sample was estimated to be 34 times greater than the amount of this product in LT-1J and 13 times greater than the amount in CL. For the experiment shown in  FIG. 7B , it was found that the amount of product in CL was about 219 times greater than that found in LT-1J and 41× greater than that found in LT-1M. For the experiment shown in  FIG. 7C , it was found that the amount of product in LT-1M was about 29 times more abundant than in LT-1J and 55 times more abundant than in CL. 
     Real-time PCR assays also allowed for an estimation of the sensitivity of rDNA amplification for detecting community members. Based on four completed  E. coli  genomes from GenBank entries AE014075 (Venter, J. C., et al., 2004 , Science,  304:66–74), U00096 (Blattner, F. R., et al., 1997 , Science,  277:1453–1474), BA000007 (Lincoln, R., G. Boxshall, and P. Clark, 1998, A Dictionary of Ecology, Evolution and Systematics. Cambridge University Press, New York, N.Y.), and AE005174 (Pace, N. R., et al., 1986 , Adv. Microb. Ecol.,  9:1–55), it was estimated that 16S rDNA makes up about 0.206% of the  E. coli  genome, and therefore, about 52 pg of 16S rDNA should be present in 25 ng of genomic DNA material. Experiments indicated that Q-PCR detected a specific rDNA signal from as little as 3.6×10 −3  pg of genomic DNA, based on a comparison with the Q-PCR signal evoked by individual rDNA clones, indicating that clones occurring at a frequency as low as 6.9×10 −5  should be detectable by Q-PCR, and that a sampling of 14.5×10 3  clones would be required for a mean recovery of one target clone. Assuming a Poisson distribution of clones in a library, it was estimated that to ensure the recovery of any one clone at a probability of greater than 99%, a library screening method would require 66.7×10 3  clones. 
     Generally, the results indicated that the Toolik Lake microbial community differs more from the North Carolina lakes than the North Carolina lake communities differ from each other. The three Lake Townsend samples also showed variation as the samples differed with respect to the location and season of sampling. While the March and June collections at the same Lake Townsend location shared several OTUs with the other Lake Townsend and City lake samples, several multiple-copy OTUs were only found in the March collection, indicating that some microbial species thrive in the relatively cold waters of early Spring. Also, the two Lake Townsend samples collected on the same day showed the highest level of overall similarity. The library sampling method was able to detect similarities between samples, indicating that the microbial community is not too heterogeneous to analyze with molecular methods. In addition, the existence of unshared OTUs and the ability to detect quantitative differences between shared OTUs indicated differences between microbial communities that may be diagnostic of specific environmental conditions. 
     Example 3 
     Qualitative and Quantitative Analysis of Water Samples Using Microarrays 
     Based on the sequence information provided by the clones and information available in GenBank, two microarrays for testing water samples were made. The protocol for testing the microarray was as follows: appropriate prokaryotic and eukaryotic primers were used to amplify either 16S rDNA or 18S rDNA, respectively, from DNA that was extracted from the water samples as described above. The PCR products were then labeled by chemical attachment to either Cy3 (green) or Cy5 (red) dye, and upon denaturation, the labeled PCR products were hybridized to the array. 
     To make the arrays, 50-mer oligonucleotide sequences were spotted onto the surface of epoxy-coated glass slides. In Table 11, sequence identifications from Tables 2 and 3 are provided for each probe that was spotted to make the array shown in  FIGS. 11 and 12 . As indicated, there were some locations at which no probes were spotted. 
     
       
         
           
               
             
               
                 TABLE 11 
               
               
                   
               
             
            
               
                 Prokaryotic (16S rDNA) probes 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 103P 
                   
                   
                 103P 
                   
                   
               
               
                   
                 103P 
                 103P 
                 104P 
                 105P 
                 106P 
                 107P 
               
               
                   
                 108P 
                 109P 
                 110P 
                 103F 1   
                 103F 1 * 
                 103F 1 * 
               
               
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
            
               
                 Eukaryotic (18S rDNA) probes 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 15P 
                 15P 
                 30P 
                 31P 
                 32P 
                 16P 
               
               
                   
                 33P 
                 34P 
                 14P 
                 35P 
                 36P 
                 38P 
               
               
                   
                 39P 
                 40P 
                 41P 
                 37P 
                 42F 
                 42F** 
               
               
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                 9mer 
               
               
                   
                   
               
               
                   
                 *reverse complementary sequence of 103F 1;   
               
               
                   
                 **reverse complementary sequence of 42F 
               
            
           
         
       
     
     The experimental design is illustrated in  FIG. 11A , showing labeling of a subset of sequences (e.g., prokaryotic) with red dye ( FIG. 11A-1 ) and labeling of a second subset of sequences (e.g., eukaryotic) with green dye ( FIG. 11A-2 ). Aligned sequences were used to design taxon-specific PCR primers (20–26 mer) and oligonucleotide probes (50-mers) to complementary variable regions. As a printing control and orientation marker, a Cy3-labeled random 9-mer probe was also printed with each grouping of probes. 
     In the experiment shown in  FIG. 11B , two aliquots drawn from a single genomic DNA sample (Lake Townsend, Station  1 , June) were subjected to PCR with either a universal prokaryotic rDNA primer pair or a universal eukaryotic rDNA primer pair (Table 1) with dUTP added to the reaction mixture. The conditions for amplification were as described above for generation of the libraries. As described herein, the PCR reactions were then labeled with either Cy5 (red, prokaryotic), or Cy3 (green, eukaryotic) by chemically attaching the incorporated dUTP. The reactions were mixed and hybridized to a glass slide spotted with the oligonucleotide probes. As shown in  FIG. 11B , the eukaryotic and prokaryotic PCR products are clearly detected on the prototype microarray. The scanned array image showed complete specificity of 12 of 12 prokaryotic probes and 19 of 21 eukaryotic probes. The green spot in the lower right of each sub-array was a Cy3-labeled 9-mer marker for orientation. Also, locations at which no spots appear were printed with probes for sequences that were not experimental targets, thus acting as negative controls and, indicating that there was minimal DNA carryover during the array printing process. The actual array carried three sets of each grid, and scanned images of the replicate grids produced substantially identical images. 
     The experiment illustrated in  FIG. 12  illustrated that the microarray may be used as a semi-quantitative assessment to compare two samples (for instance, the same location within a lake at two different times), or a sample and a standard. In this approach, two multiplex PCR amplifications were run—one for each lake sample. Both 16S and 18S rDNA were amplified together in a single reaction, and the amplified products from each sample were then labeled with either the red (Sample  1 ) or green (Sample  2 ) dye. When the amplified products were hybridized to a microarray, any resulting signal varied in color from red (target found in only sample  1 ) to yellow (target in both samples) to green (target found only in sample  2 ). The continuum from red to green is indicative of the relative abundance of the target in the samples. 
     Example 4 
     Testing for Specific Pathogens 
     The microarray is also capable of detecting known water pathogens and contaminants which affect water quality, thus raising the possibility of an “all in one” testing system. To test the feasibility of this additional feature, a microarray including 16S rDNA sequences from several cyanobacteria species was made. This microarray was tested with primers designed to amplify the rDNA of these species specifically, and the labeled products were hybridized to the microarray. As  FIG. 13  shows, the multiplex/direct labeling methodology resulted in the appearance of signals specific for the cyanobacterial sequences from water samples known to contain cyanobacteria. For the experiment shown in  FIG. 13 , oligonucleotide probes to 18S rDNA are spotted in Grid A (16 probes) and B (15 probes). Twelve probes to 16S rDNA of  Escherichia coli  (strains K12 and O157:H7; Prena et al, 2001 , Nature,  409: 465–466) are spotted in Grid C, and twenty one 16S rDNA probes are spotted in Grid D, which contains cyanobacteria sequences, sequences associated with arsenic-responsive microbes (Oremland and Stolz, 2003 , Science  300:939–943), and sequences identified in collected samples. Grids A, B, C, and D from  FIG. 13  are shown in Table 12, and the identity of each probe (Tables 2 and 3) is provided at its location on the array 
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
             
            
               
                 Oligonucleotide probes on array of FIG. 13 
               
               
                 A 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 15P 
                 30P 
                 31P 
                 32P 
                 16P 
                 33P 
               
               
                   
                 34P 
                 14P 
                 35P 
                 36P 
                 38P 
                 39P 
               
               
                   
                 40P 
                 41P 
                   
                   
                   
                   
               
               
                   
                 17P 
                 29P 
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
            
               
                 B 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 10P 
                 11P 
                 12P 
                 13P 
                   
                   
               
               
                   
                 19P 
                 20P 
                 21P 
                 22P 
                 18P 
                 23P 
               
               
                   
                 24P 
                 25P 
                 26P 
                 27P 
                 28P 
               
               
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
            
               
                 C 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 51P 
                 52P 
                 53P 
                 54P 
                 55P 
                 56P 
               
               
                   
                 57P 
                 58P 
                 60P 
                 61P 
                 62P 
                 63P 
               
               
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
            
               
                 D 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 83P 
                 81P 
                 73P 
                 74P 
                 75P 
                 76P 
               
               
                   
                 84P 
                 85P 
                 82P 
                 80P 
               
               
                   
                 103P 
                 104P 
                 105P 
                 106P 
                 107P 
                 108P 
               
               
                   
                 109P 
                 110P 
                 77P 
                 78P 
                 79P 
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                 9mer 
               
               
                   
                   
               
            
           
         
       
     
     To generate the labeled probe for this experiment, Toolik Lake, AK and City Lake, NC genomic samples were amplified using cyanobacteria specific PCR primers in a multiplex format. Toolik Lake products were labeled with Cy3 (green) and City Lake products were labeled with Cy5 (red). The primer pairs were:  Synechococcus . sp. LBP1,  Synechococcus  sp. LBG2, an unknown cyanobacteria clone LD27, and a degenerate  Synechococcus  primer set (Table 3). The four probes to cyanobacteria hybridized with amplicons from both lake samples as evidenced by the yellow signal in grid D ( FIG. 13 ). The additional signal in Grid D resulted from a labeled product that recognized a probe corresponding to the bacterium  Burkholderia cepacia  genomovar III. Subsequent analysis revealed that at least one of the  Synechococus  primers resembled the  B. cepacia  sequence, and that it likely amplified  B. cepacia  rDNA in the sample, indicating the importance of designing primer pairs to prevent cross-reactivity. 
     Example 5 
     Taxon-Specific Arrays 
     Amplification of genomic sample DNA may be performed by multiplex PCR using primers chosen to provide products that can hybridize to taxon-specific DNAs. Using this protocol can dramatically reduce non-specific labeling, and eliminates the need for intermediate PCR reactions, which reduce sensitivity. 
       FIG. 14  shows a taxon-specific array comprising 105 oligonucleotides generated from known and unknown OTUs isolated from freshwater lakes and from GenBank pathogen sequences. DNA samples from two different lakes were amplified in identical multiplex PCR reactions and reaction products were either labeled with a red dye (Cy5) or a green dye (Cy3), allowing for comparison of samples. Shown are results from a test of two lake samples used to hybridize first singly (top panels) (left panel: Cy3-labeled sample, sample  1 ; right panel: Cy5-labeled sample) and then together (bottom panel) to the same array of 105 oligonucleotide probes. 
     The arrays included nucleic acid sequences from variable regions of individual eukaryotic and prokaryotic rDNAs. Sequences spotted on the arrays included 32 probes derived from the literature that target known pathogens or contaminant microbes and 73 probes to sequences derived from freshwater environmental samples, including both known and novel sequences, and four cyanobacteria. The actual probe sequences spotted on the array shown in  FIG. 14  are provided in Table 13, and the location of each sequence on the array is provided in Table 14. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Primer/Probe combinations spotted on microarrays 
               
            
           
           
               
               
               
               
            
               
                   
                 GenBank 
                   
                   
               
               
                   
                 accession 
                   
                 GenBank 
               
               
                 16S rDNA sequence 
                 no. 
                 18S rDNA sequence 
                 accession no. 
               
               
                   
               
               
                   Enterococcus gallinarum,  strain LMG 13129 
                 AJ301833 
                 
                   Cryptosporidium parvum 
                 
                 AF222998 
               
               
                 
                   Burkholderia cepacia 
                 
                 AB091761 
                 
                   Acanthamoeba mauritaniensis 
                 
                 AY351647 
               
               
                   Burkholderia cepacia  genomovar III 
                 AF148556 
                 
                   Cyclospora cayetanensis 
                 
                 AF111183 
               
               
                 Uncultured human fecal bacterium HF74 
                 AF233412 
                 
                   Entamoeba histolytica 
                 
                 X65163 
               
               
                 Uncultured human fecal bacterium HF8 
                 AF233408 
                   Giardia intestinalis  isolate 
                 AF199449 
               
               
                 Uncultured human fecal bacterium HF10 
                 AF233413 
                 
                   Isospora belli 
                 
                 AF106935 
               
               
                   Bacillus anthracis  strain S51 
                 AB116124 
                   Microsporidium  sp. STF 
                 AY140647 
               
               
                   Clostridium botulinum  strain AIP 355.02 
                 AY303799 
                 
                   Naegleria fowleri 
                 
                 AF338423 
               
               
                   Francisella tularensis  strain 3523 
                 AY243028 
                 OTU TL1A1, multi-copy, unidentified 
               
               
                 
                   Yersinia pestis 
                 
                 AF366383 
                 OTU TL1A2, multi-copy, unidentified 
               
               
                 
                   Brucella melitensis 
                 
                 AF220149 
                 OTU TL1A9, multi-copy, unidentified 
               
               
                   Burkholderia mallei  strain 2000031063 
                 AY305760 
                 OTU TL1A12, multi-copy, unidentified 
               
               
                   Chlamydophila psittaci  clone cvCps2 
                 AY334530 
                 OTU TL1A16, multi-copy, unidentified 
               
               
                   Coxiella burnetii,  strain Nine Mile 
                 Y11502 
                 OTU TL1A21, multi-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 
                 AB035920 
                 OTU CL1A3, single-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU CL1A4, single-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU CL1A5, single-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU CL1A6, single-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU CL1A8, multi-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU LT2A12, multi-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU LT2A20, single-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU LT1A3, single-copy, unidentified 
               
               
                   Escherichia coli  O157:H7 EDL933 
                 NC 002655 
                 OTU LT1A42, multi-copy, identified 
               
               
                   
                   
                 as  Cryptomonas  sp., strain M420 
               
               
                   Escherichia coli  K12 
                 NC 000913 
                 OTU LT1A5, multi-copy, unidentified 
               
               
                   Escherichia coli  K12 
                 NC 000913 
                 OTU LT1A4, multi-copy, identified 
               
               
                   
                   
                 as  Cryptomonas ovata , 
               
               
                   
                   
                 strain CCAP 979/61 
               
               
                   Escherichia coli  K12 
                 NC 000913 
                 OTU LT1A8, multi-copy, unidentified 
               
               
                   Escherichia coli  K12 
                 NC 000913 
                 OTU LT2A7, multi-copy, identified 
               
               
                   
                   
                 as  Dileptus  sp. 
               
               
                 
                   Rickettsia prowazekii 
                 
                 M21789 
                 OTU LT1A9, single-copy, unidentified 
               
               
                 
                   Salmonella typhimurium 
                 
                 Z49264 
                 OTU LT2A19, single-copy, identified 
               
               
                   
                   
                 as  Coleps  sp. 
               
               
                   Vibrio cholerae  (CECT 514 T) 
                 X76337 
                 OTU LT1A10, multi-copy, unidentified 
               
               
                   Campylobacter jejuni  strain B99/206 
                 AF550630 
                 OTU LT1A11, single-copy, unidentified 
               
               
                   Legionella pneumophila  serogroup 6 
                 AJ496383 
                 OTU LT1A13, single-copy, unidentified 
               
               
                 
                   Leptospira interrogans 
                 
                 Z12817 
                 OTU LT3A2, single-copy, unidentified 
               
               
                   Pseudomonas aeruginosa,  strain WatG 
                 AB117953 
                 OTU LT3A5, multi-copy, unidentified 
               
               
                 OTU TL1A1, multi-copy, unidentified 
                   
                 OTU LT3A6, multi-copy, unidentified 
               
               
                 OTU TL1A2, multi-copy, unidentified 
                   
                 OTU LT3A11, single-copy, unidentified 
               
               
                 OTU TL1A6, single-copy, unidentified 
                   
                 OTU LT3A13, single-copy, unidentified 
               
               
                 OTU TL1A7, multi-copy, identified as 
                   
                 OTU LT1A1, multi-copy, unidentified 
               
               
                 Uncultured beta proteobacterium clone OS1L-16 
               
               
                 OTU LT1A31, multi-copy, identified as 
                   
                 OTU LT1A38, multi-copy, unidentified 
               
               
                 Uncultured Crater Lake bacterium CL500-18 
               
               
                 OTU LT1A55, multi-copy, identified as 
               
               
                 Uncultured freshwater bacterium LCK-26 
               
               
                 OTU CL1A2, multi-copy, unidentified 
               
               
                 OTU CL1A9, multi-copy, identified as 
               
               
                 
                   Zoogloea ramigera 
                 
               
               
                 OTU CL1A10, multi-copy, unidentified 
               
               
                 OTU CL1A15, single-copy, identified as 
               
               
                 Uncultured Crater Lake bacterium CL0-27 
               
               
                 OTU LT1A54, multi-copy, identified as 
               
               
                 Uncultured Crater Lake bacterium CL0-64 
               
               
                 OTU LT1A27, multi-copy, identified as 
               
               
                 Uncultured actinomycete clone SFD1-39 
               
               
                 OTU LT2A3, multi-copy, unidentified 
               
               
                 OTU LT1A46, multi-copy, identified as 
               
               
                 Uncultured planctomycete clone 
               
               
                 CY0ARA031E04 
               
               
                 OTU LT2A12, multi-copy, unidentified 
               
               
                 OTU LT2A16, multi-copy, unidentified 
               
               
                 OTU LT1A53-3A9, multi-copy, identified as 
               
               
                   Synechococcus  sp. 
               
               
                 OTU LT1A53, multi-copy, identified as 
               
               
                   Synechococcus  sp. LBG2 
               
               
                 OTU LT3A9, multi-copy, identified as 
               
               
                   Synechococcus  sp. LBP1 
               
               
                 OTU LT3A11, multi-copy, identified as 
               
               
                 Unidentified cyanobacterium clone LD27 
               
               
                 Arsenite-oxidizing bacterium MLHE-1 
               
               
                   Thiomicrospira  sp. CVO 
               
               
                 
                   Desulfovibrio longreachii 
                 
               
               
                 
                   Bacillus arsenicoselenatis 
                 
               
               
                 OTU LT3A1, single-copy, unidentified 
               
               
                 OTU LT3A2, multi-copy, unidentified 
               
               
                 OTU LT3A7, multi-copy, unidentified 
               
               
                 OTU LT1A15, single-copy, unidentified 
               
               
                 OTU LT1A16, multi-copy, unidentified 
               
               
                 OTU LT1A18, multi-copy, unidentified 
               
               
                 OTU LT1A35, multi-copy, unidentified 
               
               
                 OTU LT1A55, multi-copy, unidentified 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
               
                   
               
             
            
               
                 Oligonucleotide probes on array of FIG. 14 
               
            
           
           
               
               
            
               
                 A 
                 B 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 19P 
                 20P 
                 21P 
                 22P 
                 18P 
                 23P 
               
               
                   
                   
                   
                   
                   
                   
                 24P 
                 25P 
                 26P 
                 27P 
                 28P 
               
               
                   
                   
                   
                   
                   
                   
                 17P 
                 29P 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
               
            
               
                 C 
                 D 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 43P 
                 44P 
                 45P 
                 46P 
                 47P 
                 48P 
                 95P 
                 96P 
                 97P 
                 86P 
                 87P 
                 88P 
               
               
                 49P 
                 50P 
                 65P 
                 66P 
                 67P 
                 68P 
                 91P 
                 98P 
                 92P 
                 99P 
                 89P 
                 90P 
               
               
                 69P 
                 70P 
                 71P 
                 72P 
                   
                   
                 93P 
                 100P 
                 94P 
                 101P 
                 102P 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
               
            
               
                 E 
                 F 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 15P 
                 30P 
                 31P 
                 32P 
                 16P 
                 33P 
                 10P 
                 11P 
                 12P 
                 13P 
                 1P 
                 2P 
               
               
                 34P 
                 14P 
                 35P 
                 36P 
                 38P 
                 39P 
                 3P 
                 4P 
                 5P 
                 6P 
                 7P 
                 8P 
               
               
                 40P 
                 41P 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 9mer 
               
            
           
           
               
               
            
               
                 G 
                 H 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 51P 
                 52P 
                 53P 
                 54P 
                 55P 
                 56P 
                 83P 
                 81P 
                 73P 
                 74P 
                 75P 
                 76P 
               
               
                 57P 
                 58P 
                 60P 
                 61P 
                 62P 
                 63P 
                 84P 
                 85P 
                 82P 
                 80P 
                 77P 
                 78P 
               
               
                   
                   
                   
                   
                   
                   
                 79P 
                 106P 
                 107P 
                 108P 
               
               
                   
                   
                   
                   
                   
                   
                 103P 
                 104P 
                 105P 
                 109P 
                 110P 
                 Cy3 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 9mer 
               
               
                   
               
            
           
         
       
     
     Such taxon-specific arrays may be developed for specific bodies of water. For example, arrays may be developed for lakes, marshes, tidal pools, or estuaries. Such arrays may include probes developed for freshwater systems, as these may be diagnostic for known pathogens (e.g., coliform bacteria) or environmental conditions (e.g., eutrophication). Also, sequences specific to microbes known to be common in the body of water of interest may be used. For example, for estuaries, phytoplankton populations have been described (Williams, R. B. and M. B. Murdoch 1966 , Limnology and Oceanography  11:73–82., Thayer, G. W., 1971  Estuaries  12:240–253; Mallin 1994, Mallin, et al. 2000). A review of the literature indicates that many of the sequences that may be used are known (Table 15). Using this information, primers and probes to these common organisms, may be designed. Finally, probes for the array may be derived by sequencing clonal libraries derived from field samples as described herein for the lake samples to develop additional OTUs. For example, to develop an estuary array, water samples may be collected every few months in estuarine tidal creeks at various sites representing a range of estuarine conditions. Specific sampling locations may be water quality monitoring stations, and samples may be taken late on the flooding tide and six hours later on the ebbing tide, to provide a representative sample of both the community that enters the estuarine site as well as the community that leaves the estuarine site with the falling tide. 
     It should be understood that various changes and modifications to the embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant scope and/or advantages. 
     
       
         
           
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 Examples of organisms of interest for North Carolina 
               
               
                 estuarine microarray targets based on literature reports 4   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Phytoplankton 
                   
               
               
                   Cyclotella  spp. 
                   Melosira  spp. 
               
               
                   Nitzschia  spp. 
                   Navicula  spp. 
               
               
                   Psuedo - nitzschia australis   3   
                   Skeletonema  costatum 1   
               
               
                   Thalassiosira  spp. 
               
               
                   Amphidinium  spp. 
                   Ceratium  spp. 
               
               
                   Chattonella  spp. 3  ( C. antiqua,   
                 
                   Gymnodinium sanguineum 
                 
               
               
                   C. verruculosa ) 
               
               
                 
                   Heterocapsa triquetra 
                   1 
                 
                 
                   Heterosgima akashiwo 
                   3 
                 
               
               
                 
                   Hematodinium perezi 
                   3 
                 
                 
                   Karenia brevis 
                   3 
                 
               
               
                 
                   Karlodinium micrum 
                   1 
                 
                 
                   Katodinium rotundatum 
                   1 
                 
               
               
                   Pfiesteria piscicida   3 ,  P. shumwayae   3   
                 
                   Prorocentrum minimum 
                   1 
                 
               
               
                 
                   Calicomonas ovalis 
                 
                   Chlamydomonas  spp. 
               
               
                   Chroomonas  spp. ( C. minuta,   
                 
                   Cyrptomonas testaceae 
                 
               
               
                   C. amphioxiae ) 
               
               
                 
                   Hemiselmis virescens 
                 
                 
                   Phaeocystis globosa 
                 
               
               
                 
                   Pyramimonas 
                 
                 
                   Eutreptia 
                   1 
                 
               
               
                 Bacteria 
               
               
                   Acinetobcter  spp. 
                   Alcaligenes  spp. 
               
               
                   Bacteroides  spp. 2   
                   Enterococcus  spp. 2   
               
               
                   
                 ( E. faecalis, E. faecium ) 
               
               
                   Escherichia  spp. 2,3  ( E. coli,   
                   Flavobacterium  spp. 
               
               
                   E. coli  O157H7) 
               
               
                   Oceanospirillum  spp. 
                   Salmonella  spp. 2,3  ( S. typhi,   
               
               
                   
                   S. non - typhi ) 
               
               
                 
                   Psuedomonas aeruginosa 
                 
                   Shigella  sp. 2,3   
               
               
                   Clostridium  sp. 2,3  ( C. perfringens,   
               
               
                   C. botulinum  Type E) 
               
               
                   Vibrio  sp. 3  ( V. anguillarum,   
               
               
                   V. cholerae  01,  V. cholerae  non 01, 
               
               
                 
                   V. parahaemolyticus V. vulnificus 
                 
               
               
                 Virus 
               
               
                 Hepatitis A 3   
                 Norwalk virus 3   
               
               
                 Adenovirus 3   
                 rotavirus 3   
               
               
                 Protozoa 
               
               
                   Kudoa  spp. 3   
                   Cryptosporidium  spp. 2,3   
               
               
                 ( K. clupeidae, K. fundulae ) 
               
               
                 
                   Giardia 
                   2,3 
                 
                 
                   Perkinsus marinus 
                   3 
                 
               
               
                 
                   Haplosporidium 
                   3 
                 
               
               
                 Fungi 
               
               
                 
                   Aphanomyces invadens 
                   3 
                 
               
               
                   
               
               
                   1 Genera or species referenced in the literature as commonly found in southeastern estuarine systems, and likely to be indicators of good ecosystem health (e.g. Campbell, 1973, Univ. of N.C. Sea Grant Publication, UNC-SG-73-07; Mallin, et al. 2000, American Scientist, 88:26–37; Shubert, 1984, In Algae as Ecological Markers, Academic Press, NY, p. 434; Stoermer and Smol, 1999, In The Diatoms: Applications For the Environmental and Earth Sciences, Cambridge Univ. Press, Cambridge, UK, page 469; Thayer, 1971, Estuaries, 12:240–253, Williams and Murdoch, 1966, Limnology and Oceanography, 11:73–82;). 
               
               
                   2 Microbes linked to specific contamination sources (e.g. human sewage) which indicate point or non-point source pollution (e.g. Bernhard and Field, 2000, Applied and Environmental Microbiology, 66:4641–4648; Lipp, et al. 2001, Marine Pollution Bull., 42:286–293; Mallin, et al., 2000b, Ecological Applications, 10:1047–1056&#39; USEPA, 1985, Test methods for  Escherichia coli  and  Enterococci  in water by the membrane filter procedure, EPA600/4-85/076) 
               
               
                   3 Known human, fish, or shellfish pathogens or parasites (e.g. DeLeon, et al. 1990, In Proceeding of the Water Quality Conference, San Diego, CA, American Water Works Association, 18:833–853; Grimes 1991, Estuaries, 14:345–360; Kane, et al., 1998, Maryland Medical Journal, 37:106–112; Lipp, 1999, , Reed and Francis-Floyd, 1996, Vibrio Infections of Fish, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida web-site; Shields, 1997, An investigation into the epidemiology of  Hematodinium perezi , a parasitic dinoflagellate in the blue crab,  Callinectes sapidus , available on-line) 
               
               
                   4 Many of the taxa listed have GenBank sequence entries for one or more species.