Patent Application
20140287961
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SEQLIST.TXT, which is 5.51 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
1. Field of the Invention
The present embodiments relate to an array system for detecting and identifying biomolecules and organisms. More specifically, the present embodiments relate to an array system comprising a microarray configured to simultaneously detect a plurality of organisms in a sample at a high confidence level.
2. Description of the Related Art
In the fields of molecular biology and biochemistry, biopolymers such as nucleic acids and proteins from organisms are identified and/or fractionated in order to search for useful genes, diagnose diseases or identify organisms. A hybridization reaction is frequently used as a pretreatment for such process, where a target molecule in a sample is hybridized with a nucleic acid or a protein having a known sequence. For this purpose, microarrays, or DNA chips, are used on which probes such as DNAs, RNAs or proteins with known sequences are immobilized at predetermined positions.
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip forming an array. The affixed DNA segments are known as probes (although some sources will use different nomenclature), thousands of which can be used in a single DNA microarray. Measuring gene expression using microarrays is relevant to many areas of biology and medicine, such as studying treatments, disease, and developmental stages. For example, microarrays can be used to identify disease genes by comparing gene expression in diseased and normal cells.
Molecular approaches designed to describe organism diversity routinely rely upon classifying heterogeneous nucleic acids amplified by universal 16S RNA gene PCR (polymerase chain reaction). The resulting mixed amplicons can be quickly, but coarsely, typed into anonymous groups using T-/RFLP (Terminal Restriction Fragment Length Polymorphism), SSCP (single-strand conformation polymorphism) or T/DGGE (temperature/denaturing gradient gel. electrophoresis). These groups may be classified through sequencing, but this requires additional labor to physically isolate each 16S RNA type, does not scale well for large comparative studies such as environmental monitoring, and is only suitable for low complexity environments. Also, the number of clones that would be required to adequately catalogue the majority of taxa in a sample is too large to be efficiently or economically handled. As such, an improved array and method is needed to efficiently analyze a plurality of organisms without the disadvantages of the above technologies.
Some embodiments relate to an array system including a microarray configured to simultaneously detect a plurality of organisms in a sample, wherein the microarray comprises fragments of 16s RNA unique to each organism and variants of said fragments comprising at least 1 nucleotide mismatch, wherein the level of confidence of species-specific detection derived from fragment matches is about 90% or higher.
In one aspect, the plurality of organisms comprise bacteria or archaea.
In another aspect, the fragments of 16s RNA are clustered and aligned into groups of similar sequence such that detection of an organism based on at least 11 fragment matches is possible.
In yet another aspect, the level of confidence of species-specific detection derived from fragment matches is about 95% or higher.
In still another aspect, the level of confidence of species-specific detection derived from fragment matches is about 98% or higher.
In some embodiments, the majority of fragments of 16s RNA unique to each organism have a corresponding variant fragment comprising at least 1 nucleotide mismatch.
In some aspects, every fragment of 16s RNA unique to each organism has a corresponding variant fragment comprising at least 1 nucleotide mismatch.
In other aspects, the fragments are about 25 nucleotides long.
In some aspects, the sample is an environmental sample.
In other aspects, the environmental sample comprises at least one of soil, water or atmosphere.
In yet other aspects, the sample is a clinical sample.
In still other aspects, the clinical sample comprises at least one of tissue, skin, bodily fluid or blood.
Some embodiments relate to a method of detecting an organism including applying a sample comprising a plurality of organisms to the array system which includes a microarray that comprises fragments of 16s RNA unique to each organism and variants of said fragments comprising at least 1 nucleotide mismatch, wherein the level of confidence of species-specific detection derived from fragment matches is about 90% or higher; and identifying organisms in the sample.
In some aspects, the plurality of organisms comprise bacteria or archaea.
In other aspects, the majority of fragments of 16s RNA unique to each organism have a corresponding variant fragment comprising at least 1 nucleotide mismatch.
In still other aspects, every fragment of 16s RNA unique to each organism has a corresponding variant fragment comprising at least 1 nucleotide mismatch.
In yet other aspects, the fragments are about 25 nucleotides long.
In some aspects, the organism to be detected is the most metabolically active organism in the sample.
Some embodiments relate to a method of fabricating an array system including identifying 16s RNA sequences corresponding to a plurality of organisms of interest; selecting fragments of 16s RNA unique to each organism; creating variant RNA fragments corresponding to the fragments of 16s RNA unique to each organism which comprise at least 1 nucleotide mismatch; and fabricating said array system.
In some aspects, the plurality of organisms comprise bacteria or archaea.
In other aspects, the majority of fragments of 16s RNA unique to each organism have a corresponding variant fragment comprising at least 1 nucleotide mismatch.
In still other aspects, every fragment of 16s RNA unique to each organism has a corresponding variant fragment comprising at least 1 nucleotide mismatch.
In yet other aspects, the fragments are about 25 nucleotides long.
The present embodiments are related to an array system for detecting and identifying biomolecules and organisms. More specifically, the present embodiments relate to an array system comprising a microarray configured to simultaneously detect a plurality of organisms in a sample at a high confidence level.
In some embodiments, the array system uses multiple probes for increasing confidence of identification of a particular organism using a 16S rRNA gene targeted high density microarray. The use of multiple probes can greatly increase the confidence level of a match to a particular organism. Also, in some embodiments, mismatch control probes corresponding to each perfect match probe can be used to further increase confidence of sequence-specific hybridization of a target to a probe. Probes with one or more mismatch can be used to indicate non-specific binding and a possible non-match. This has the advantage of reducing false positive results due to non-specific hybridization, which is a significant problem with many current microarrays.
Some embodiments of the invention relate to a method of using an array to simultaneously identify multiple prokaryotic taxa with a relatively high confidence. A taxa is an individual microbial species or group of highly related species that share an average of about 97% 16S rRNA gene sequence identity. The array system of the current embodiments may use multiple confirmatory probes, each with from about 1 to about 20 corresponding mismatch control probes to target the most unique regions within a 16S rRNA gene for about 9000 taxa. Preferably, each confirmatory probe has from about 1 to about 10 corresponding mismatch probes. More preferably, each confirmatory probe has from about 1 to about 5 corresponding mismatch probes. The aforementioned about 9000 taxa represent a majority of the taxa that are currently known through 16S rRNA clone sequence libraries. In some embodiments, multiple targets can be assayed through a high-density oligonucleotide array. The sum of all target hybridizations is used to identify specific prokaryotic taxa. The result is a much more efficient and less time consuming way of identifying unknown organisms that in addition to providing results that could not previously be achieved, can also provide results in hours that other methods would require days to achieve.
In some embodiments, the array system of the present embodiments can be fabricated using 16s rRNA sequences as follows. From about 1 to about 500 short probes can be designed for each taxonomic group. In some embodiments, the probes can be proteins, antibodies, tissue samples or oligonucleotide fragments. In certain examples, oligonucleotide fragments are used as probes. In some embodiments, from about 1 to about 500 short oligonucleotide probes, preferably from about 2 to about 200 short oligonucleotide probes, more preferably from about 5 to about 150 short oligonucleotide probes, even more preferably from about 8 to about 100 short oligonucleotide probes can be designed for each taxonomic grouping, allowing for the failure of one or more probes. In one example, at least about 11 short oligonucleotide probes are used for each taxonomic group. The oligonucleotide probes can each be from about 5 bp to about 100 bp, preferably from about 10 bp to about 50 bp, more preferably from about 15 bp to about 35 bp, even more preferably from about 20 bp to about 30 bp. In some embodiments, the probes may be 5-mers, 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, 25-mers, 26-mers, 27-mers, 28-mers, 29-mers, 30-mers, 31-mers, 32-mers, 33-mers, 34-mers, 35-mers, 36-mers, 37-mers, 38-mers, 39-mers, 40-mers, 41-mers, 42-mers, 43-mers, 44-mers, 45-mers, 46-mers, 47-mers, 48-mers, 49-mers, 50-mers, 51-mers, 52-mers, 53-mers, 54-mers, 55-mers, 56-mers, 57-mers, 58-mers, 59-mers, 60-mers, 61-mers, 62-mers, 63-mers, 64-mers, 65-mers, 66-mers, 67-mers, 68-mers, 69-mers, 70-mers, 71-mers, 72-mers, 73-mers, 74-mers, 75-mers, 76-mers, 77-mers, 78-mers, 79-mers, 80-mers, 81-mers, 82-mers, 83-mers, 84-mers, 85-mers, 86-mers, 87-mers, 88-mers, 89-mers, 90-mers, 91-mers, 92-mers, 93-mers, 94-mers, 95-mers, 96-mers, 97-mers, 98-mers, 99-mers, 100-mers or combinations thereof.
Non-specific cross hybridization can be an issue when an abundant 16S rRNA gene shares sufficient sequence similarity to non-targeted probes, such that a weak but detectable signal is obtained. The use of sets of perfect match and mismatch probes (PM-MM) effectively minimizes the influence of cross-hybridization. In certain embodiments, each perfect match probe (PM) has one corresponding mismatch probe (MM) to form a pair that is useful for analyzing a particular 16S rRNA sequence. In other embodiments, each PM has more than one corresponding MM. Additionally, different PMs can have different numbers of corresponding MM probes. In some embodiments, each PM has from about 1 to about 20 MM, preferably, each PM has from about 1 to about 10 MM and more preferably, each PM has from about 1 to about 5 MM.
Any of the nucleotide bases can be replaced in the MM probe to result in a probe having a mismatch. In one example, the central nucleotide base sequence can be replaced with any of the three non-matching bases. In other examples, more than one nucleotide base in the MM is replaced with a non-matching base. In some examples, 10 nucleotides are replaced in the MM, in other examples, 5 nucleotides are replaced in the MM, in yet other examples 3 nucleotides are replaced in the MM, and in still other examples, 2 nucleotides are replaced in the MM. This is done so that the increased hybridization intensity signal of the PM over the one or more MM indicates a sequence-specific, positive hybridization. By requiring multiple PM-MM probes to have a confirmation interaction, the chance that the hybridization signal is due to a predicted target sequence is substantially increased.
In other embodiments, the 16S rRNA gene sequences can be grouped into distinct taxa such that a set of the short oligonucleotide probes that are specific to the taxon can be chosen. In some examples, the 16s rRNA gene sequences grouped into distinct taxa are from about 100 bp to about 1000 bp, preferably the gene sequences are from about 400 bp to about 900 bp, more preferably from about 500 bp to about 800 bp. The resulting about 9000 taxa represented on the array, each containing from about 1% to about 5% sequence divergence, preferably about 3% sequence divergence, can represent substantially all demarcated bacterial and archaeal orders.
In some embodiments, for a majority of the taxa represented on the array, probes can be designed from regions of gene sequences that have only been identified within a given taxon. In other embodiments, some taxa have no probe-level sequence that can be identified that is not shared with other groups of 16S rRNA gene sequences. For these taxonomic groupings, a set of from about 1 to about 500 short oligonucleotide probes, preferably from about 2 to about 200 short oligonucleotide probes, more preferably from about 5 to about 150 short oligonucleotide probes, even more preferably from about 8 to about 100 short oligonucleotide probes can be designed to a combination of regions on the 16S rRNA gene that taken together as a whole do not exist in any other taxa. For the remaining taxa, a set of probes can be selected to minimize the number of putative cross-reactive taxa. For all three probe set groupings, the advantage of the hybridization approach is that multiple taxa can be identified simultaneously by targeting unique regions or combinations of sequence.
In some embodiments, oligonucleotide probes can then be selected to obtain an effective set of probes capable of correctly identifying the sample of interest. In certain embodiments, the probes are chosen based on various taxonomic organizations useful in the identification of particular sets of organisms.
In some embodiments, the chosen oligonucleotide probes can then be synthesized by any available method in the art. Some examples of suitable methods include printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing or electrochemistry. In one example, a photolithographic method can be used to directly synthesize the chosen oligonucleotide probes onto a surface. Suitable examples for the surface include glass, plastic, silicon and any other surface available in the art. In certain examples, the oligonucleotide probes can be synthesized on a glass surface at an approximate density of from about 1,000 probes per μm2 to about 100,000 probes per μm2, preferably from about 2000 probes per μm2 to about 50,000 probes per μm2, more preferably from about 5000 probes per μm2 to about 20,000 probes per μm2. In one example, the density of the probes is about 10,000 probes per μm2. The array can then be arranged in any configuration, such as, for example, a square grid of rows and columns. Some areas of the array can be oligonucleotide 16S rDNA PM or MM probes, and others can be used for image orientation, normalization controls or other analyses. In some embodiments, materials for fabricating the array can be obtained from Affymetrix, GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) or Agilent Technologies (Palo Alto, Calif.)
In some embodiments, the array system is configured to have controls. Some examples of such controls include 1) probes that target amplicons of prokaryotic metabolic genes spiked into the 16S rDNA amplicon mix in defined quantities just prior to fragmentation and 2) probes complimentary to a pre-labeled oligonucleotide added into the hybridization mix. The first control collectively tests the fragmentation, biotinylation, hybridization, staining and scanning efficiency of the array system. It also allows the overall fluorescent intensity to be normalized across all the arrays in an experiment. The second control directly assays the hybridization, staining and scanning of the array system. However, the array system of the present embodiments is not limited to these particular examples of possible controls.
The accuracy of the array of some embodiments has been validated by comparing the results of some arrays with 16S rRNA gene sequences from approximately 700 clones in each of 3 samples. A specific taxa is identified as being present in a sample if a majority (from about 70% to about 100%, preferably from about 80% to about 100% and more preferably from about 90% to about 100%) of the probes on the array have a hybridization signal about 100 times, 200 times, 300 times, 400 times or 500 times greater than that of the background and the perfect match probe has a significantly greater hybridization signal than its one or more partner mismatch control probe or probes. This ensures a higher probability of a sequence specific hybridization to the probe. In some embodiments, the use of multiple probes, each independently indicating that the target sequence of the taxonomic group being identified is present, increases the probability of a correct identification of the organism of interest.
Biomolecules, such proteins, DNA, RNA, DNA from amplified products and native rRNA from the 16S rRNA gene, for example can be probed by the array of the present embodiments. In some embodiments, probes are designed to be antisense to the native rRNA so that directly labeled rRNA from samples can be placed directly on the array to identify a majority of the actively metabolizing organisms in a sample with no bias from PCR amplification. Actively metabolizing organisms have significantly higher numbers of ribosomes used for the production of proteins, therefore, in some embodiments, the capacity to make proteins at a particular point in time of a certain organism can be measured. This is not possible in systems where only the 16S rRNA gene DNA is measured which encodes only the potential to make proteins and is the same whether an organism is actively metabolizing or quiescent or dead. In this way, the array system of the present embodiments can directly identify the metabolizing organisms within diverse communities.
In some embodiments, the array system is able to measure the microbial diversity of complex communities without PCR amplification, and consequently, without all of the inherent biases associated with PCR amplification. Actively metabolizing cells typically have about 20,000 or more ribosomal copies within their cell for protein assembly compared to quiescent or dead cells that have few. In some embodiments, rRNA can be purified directly from environmental samples and processed with no amplification step, thereby avoiding any of the biases caused by the preferential amplification of some sequences over others. Thus, in some embodiments the signal from the array system can reflect the true number of rRNA molecules that are present in the samples, which can be expressed as the number of cells multiplied by the number of rRNA copies within each cell. The number of cells in a sample can then be inferred by several different methods, such as, for example, quantitative real-time PCR, or FISH (fluorescence in situ hybridization.) Then the average number of ribosomes within each cell may be calculated.
In some embodiments, the samples used can be environmental samples from any environmental source, for example, naturally occurring or artificial atmosphere, water systems, soil or any other sample of interest. In some embodiments, the environmental samples may be obtained from, for example, atmospheric pathogen collection systems, sub-surface sediments, groundwater, ancient water deep within the ground, plant root-soil interface of grassland, coastal water and sewage treatment plants. Because of the ability of the array system to simultaneously test for such a broad range of organisms based on almost all known 16s rRNA gene sequences, the array system of the present embodiments can be used in any environment, which also distinguishes it from other array systems which generally must be targeted to specific environments.
In other embodiments, the sample used with the array system can be any kind of clinical or medical sample. For example, samples from blood, the lungs or the gut of mammals may be assayed using the array system. Also, the array system of the present embodiments can be used to identify an infection in the blood of an animal. The array system of the present embodiments can also be used to assay medical samples that are directly or indirectly exposed to the outside of the body, such as the lungs, ear, nose, throat, the entirety of the digestive system or the skin of an animal. Hospitals currently lack the resources to identify the complex microbial communities that reside in these areas.
Another advantage of the present embodiments is that simultaneous detection of a majority of currently known organisms is possible with one sample. This allows for much more efficient study and determination of particular organisms within a particular sample. Current microarrays do not have this capability. Also, with the array system of the present embodiments, simultaneous detection of the top metabolizing organisms within a sample can be determined without bias from PCR amplification, greatly increasing the efficiency and accuracy of the detection process.
Some embodiments relate to methods of detecting an organism in a sample using the described array system. These methods include contacting a sample with one organism or a plurality of organisms to the array system of the present embodiments and detecting the organism or organisms. In some embodiments, the organism or organisms to be detected are bacteria or archaea. In some embodiments, the organism or organisms to be detected are the most metabolically active organism or organisms in the sample.
Some embodiments relate to a method of fabricating an array system including identifying 16s RNA sequences corresponding to a plurality of organisms of interest, selecting fragments of 16s RNA unique to each organism and creating variant RNA fragments corresponding to the fragments of 16s RNA unique to each organism which comprise at least 1 nucleotide mismatch and then fabricating the array system.
The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.
An array system was fabricated using 16s rRNA sequences taken from a plurality of bacterial species. A minimum of 11 different, short oligonucleotide probes were designed for each taxonomic grouping, allowing one or more probes to not bind, but still give a positive signal in the assay. Non-specific cross hybridization is an issue when an abundant 16S rRNA gene shares sufficient sequence similarity to non-targeted probes, such that a weak but detectable signal is obtained. The use of a perfect match-mismatch (PM-MM) probe pair effectively minimized the influence of cross-hybridization. In this technique, the central nucleotide is replaced with any of the three non-matching bases so that the increased hybridization intensity signal of the PM over the paired MM indicates a sequence-specific, positive hybridization. By requiring multiple PM-MM probe-pairs to have a positive interaction, the chance that the hybridization signal is due to a predicted target sequence is substantially increased.
The known 16S rRNA gene sequences larger than 600 bp were grouped into distinct taxa such that a set of at least 11 probes that were specific to each taxon could be selected. The resulting 8,935 taxa (8,741 of which are represented on the array), each containing approximately 3% sequence divergence, represented all 121 demarcated bacterial and archaeal orders. For a majority of the taxa represented on the array (5,737, 65%), probes were designed from regions of 16S rRNA gene sequences that have only been identified within a given taxon. For 1,198 taxa (14%) no probe-level sequence could be identified that was not shared with other groups of 16S rRNA gene sequences, although the gene sequence as a whole was distinctive. For these taxonomic groupings, a set of at least 11 probes was designed to a combination of regions on the 16S rRNA gene that taken together as a whole did not exist in any other taxa. For the remaining 1,806 taxa (21%), a set of probes were selected to minimize the number of putative cross-reactive taxa. Although more than half of the probes in this group have a hybridization potential to one outside sequence, this sequence was typically from a phylogenetically similar taxon. For all three probe set groupings, the advantage of the hybridization approach is that multiple taxa can be identified simultaneously by targeting unique regions or combinations of sequence.
An array system was fabricated according to the following protocol. 16S rDNA sequences (Escherichia coli base pair positions 47 to 1473) were obtained from over 30,000 16S rDNA sequences that were at least 600 nucleotides in length in the 15 Mar. 2002 release of the 16S rDNA database, “Greengenes.” This region was selected because it is bounded on both ends by universally conserved segments that can be used as PCR priming sites to amplify bacterial or archaeal genomic material using only 2 to 4 primers. Putative chimeric sequences were filtered from the data set using computer software preventing them from being misconstrued as novel organisms. The filtered sequences are considered to be the set of putative 16S rDNA amplicons. Sequences were clustered to enable each sequence of a cluster to be complementary to a set of perfectly matching (PM) probes. Putative amplicons were placed in the same cluster as a result of common 17-mers found in the sequence.
The resulting 8,988 clusters, each containing approximately 3% sequence divergence, were considered operational taxonomic units (OTUs) representing all 121 demarcated prokaryotic orders. The taxonomic family of each OTU was assigned according to the placement of its member organisms in Bergey's Taxonomic Outline. The taxonomic outline as maintained by Philip Hugenholtz was consulted for phylogenetic classes containing uncultured environmental organisms or unclassified families belonging to named higher taxa. The OTUs comprising each family were clustered into sub-families by transitive sequence identity. Altogether, 842 sub-families were found. The taxonomic position of each OTU as well as the accompanying NCBI accession numbers of the sequences composing each OTU are recorded and publicly available.
The objective of the probe selection strategy was to obtain an effective set of probes capable of correctly categorizing mixed amplicons into their proper OTU. For each OTU, a set of 11 or more specific 25-mers (probes) were sought that were prevalent in members of a given OTU but were dissimilar from sequences outside the given OTU. In the first step of probe selection for a particular OTU, each of the sequences in the OTU was separated into overlapping 25-mers, the potential targets. Then each potential target was matched to as many sequences of the OTU as possible. First, a text pattern was used for a search to match potential targets and sequences, however, since partial gene sequences were included in the reference set additional methods were performed. Therefore, the multiple sequence alignment provided by Greengenes was used to provide a discrete measurement of group size at each potential probe site. For example, if an OTU containing seven sequences possessed a probe site where one member was missing data, then the site-specific OTU size was only six.
In ranking the possible targets, those having data for all members of that OTU were preferred over those found only in a fraction of the OTU members. In the second step, a subset of the prevalent targets was selected and reverse complimented into probe orientation, avoiding those capable of mis-hybridization to an unintended amplicon. Probes presumed to have the capacity to mis-hybridize were those 25-mers that contained a central 17-mer matching sequences in more than one OTU. Thus, probes that were unique to an OTU solely due to a distinctive base in one of the outer four bases were avoided. Also, probes with mis-hybridization potential to sequences having a common tree node near the root were favored over those with a common node near the terminal branch.
Probes complementary to target sequences that were selected for fabrication were termed perfectly matching (PM) probes. As each PM probe was chosen, it was paired with a control 25-mer (mismatching probe, MM), identical in all positions except the thirteenth base. The MM probe did not contain a central 17-mer complimentary to sequences in any OTU. The probe complementing the target (PM) and MM probes constitute a probe pair analyzed together.
The chosen oligonucleotides were synthesized by a photolithographic method at Affymetrix Inc. (Santa Clara, Calif., USA) directly onto a 1.28 cm by 1.28 cm glass surface at an approximate density of 10,000 probes per μm2. Each unique probe sequence on the array had a copy number of roughly 3.2×106 (personal communication, Affymetrix). The entire array of 506,944 features was arranged as a square grid of 712 rows and columns. Of these features, 297,851 were oligonucleotide 16S rDNA PM or MM probes, and the remaining were used for image orientation, normalization controls or other unrelated analyses. Each DNA chip had two kinds of controls on it: 1) probes that target amplicons of prokaryotic metabolic genes spiked into the 16S rDNA amplicon mix in defined quantities just prior to fragmentation and 2) probes complimentary to a pre-labeled oligonucleotide added into the hybridization mix. The first control collectively tested the fragmentation, biotinylation, hybridization, staining and scanning efficiency. It also allowed the overall fluorescent intensity to be normalized across all the arrays in an experiment. The second control directly assayed the hybridization, staining and scanning.
A study was done on diverse and dynamic bacterial population in urban aerosols utilizing an array system of certain embodiments. Air samples were collected using an air filtration collection system under vacuum located within six EPA air quality network sites in both San Antonio and Austin, Tex. Approximately 10 liters of air per minute were collected in a polyethylene terephthalate (Celanex), 1.0 μm filter (Hoechst Calanese). Samples were collected daily over a 24 h period. Sample filters were washed in 10 mL buffer (0.1M Sodium Phosphate, 10 mM EDTA, pH 7.4, 0.01% Tween-20), and the suspension was stored frozen until extracted. Samples were collected from 4 May to 29 Aug. 2003.
Sample dates were divided according to a 52-week calendar year starting Jan. 1, 2003, with each Monday to Sunday cycle constituting a full week. Samples from four randomly chosen days within each sample week were extracted. Each date chosen for extraction consisted of 0.6 mL filter wash from each of the six sampling sites for that city (San Antonio or Austin) combined into a “day pool” before extraction. In total, for each week, 24 filters were sampled.
The “day pools” were centrifuged at 16,000×g for 25 min and the pellets were resuspended in 400 μL sodium phosphate buffer (100 mM, pH 8). The resuspended pellets were transferred into 2 mL silica bead lysis tubes containing 0.9 g of silica/zirconia lysis bead mix (0.3 g of 0.5 mm zirconia/silica beads and 0.6 g of 0.1 mm zirconia/silica beads). For each lysis tube, 300 μL buffered sodium dodecyl sulfate (SDS) (100 mM sodium chloride, 500 mM Tris pH 8, 10% [w/v] SDS), and 300 μL phenol:chloroform:isoamyl alcohol (25:24:1) were added. Lysis tubes were inverted and flicked three times to mix buffers before bead mill homogenization with a Bio101 Fast Prep 120 machine (Qbiogene, Carlsbad, Calif.) at 6.5 m s−1 for 45 s. Following centrifugation at 16,000×g for 5 min, the aqueous supernatant was removed to a new 2 mL tube and kept at −20° C. for 1 hour to overnight. An equal volume of chloroform was added to the thawed supernatant prior to vortexing for 5 s and centrifugation at 16,000×g for 3 min. The supernatant was then combined with two volumes of a binding buffer “Solution 3” (UltraClean Soil DNA kit, MoBio Laboratories, Solana Beach, Calif.). Genomic DNA from the mixture was isolated on a MoBio spin column, washed with “Solution 4” and eluted in 60 μL of 1× Tris-EDTA according to the manufacturer's instructions. The DNA was further purified by passage through a Sephacryl S-200 HR spin column (Amersham, Piscataway, N.J., USA) and stored at 4° C. prior to PCR amplification. DNA was quantified using a PicoGreen fluorescence assay according to the manufacturer's recommended protocol (Invitrogen, Carlsbad, Calif.).
The 16S rRNA gene was amplified from the DNA extract using universal primers 27F.1, (5′ AGRGTTTGATCMTGGCTCAG) (SEQ ID NO: 1) and 1492R, (5′ GGTTACCTTGTTACGACTT) (SEQ ID NO: 2). Each PCR reaction mix contained 1× Ex Taq buffer (Takara Bio Inc, Japan), 0.8 mM dNTP mixture, 0.02 U/μL Ex Taq polymerase, 0.4 mg/mL bovine serum albumin (BSA), and 1.0 μM each primer. PCR conditions were 1 cycle of 3 min at 95° C., followed by 35 cycles of 30 sec at 95° C., 30 sec at 53° C., and 1 min at 72° C., and finishing with 7 min incubation at 72° C. When the total mass of PCR product for a sample week reached 2 μg (by gel quantification), all PCR reactions for that week were pooled and concentrated to a volume less than 40 μL with a Micron YM100 spin filter (Millipore, Billerica, Mass.) for microarray analysis.
The pooled PCR product was spiked with known concentrations of synthetic 16S rRNA gene fragments and non-16S rRNA gene fragments according to Table S1. This mix was fragmented using DNAse I (0.02 U/μg DNA, Invitrogen, CA) and One-Phor-All buffer (Amersham, N.J.) per Affymetrix's protocol, with incubation at 25° C. for 10 min., followed by enzyme denaturation at 98° C. for 10 min. Biotin labeling was performed using an Enzo® BioArray™ Terminal Labeling Kit (Enzo Life Sciences Inc., Farmingdale, N.Y.) per the manufacturer's directions. The labeled DNA was then denatured (99° C. for 5 min) and hybridized to the DNA microarray at 48° C. overnight (>16 hr). The microarrays were washed and stained per the Affymetrix protocol.
The array was scanned using a GeneArray Scanner (Affymetrix, Santa Clara, Calif., USA). The scan was recorded as a pixel image and analyzed using standard Affymetrix software (Microarray Analysis Suite, version 5.1) that reduced the data to an individual signal value for each probe. Background probes were identified as those producing intensities in the lowest 2% of all intensities. The average intensity of the background probes was subtracted from the fluorescence intensity of all probes. The noise value (N) was the variation in pixel intensity signals observed by the scanner as it read the array surface. The standard deviation of the pixel intensities within each of the identified background cells was divided by the square root of the number of pixels comprising that cell. The average of the resulting quotients was used for N in the calculations described below.
Probe pairs scored as positive were those that met two criteria: (i) the intensity of fluorescence from the perfectly matched probe (PM) was greater than 1.3 times the intensity from the mismatched control (MM), and (ii) the difference in intensity, PM minus MM, was at least 130 times greater than the squared noise value (>130 N2). These two criteria were chosen empirically to provide stringency while maintaining sensitivity to the amplicons known to be present from sequencing results of cloning the San Antonio week 29 sample. The positive fraction (PosFrac) was calculated for each probe set as the number of positive probe pairs divided by the total number of probe pairs in a probe set. A taxon was considered present in the sample when over 92% of its assigned probe pairs for its corresponding probe set were positive (PosFrac >0.92). This was determined based on empirical data from clone library analyses. Hybridization intensity (hereafter referred to as intensity) was calculated in arbitrary units (a.u.) for each probe set as the trimmed average (maximum and minimum values removed before averaging) of the PM minus MM intensity differences across the probe pairs in a given probe set. All intensities <1 were shifted to 1 to avoid errors in subsequent logarithmic transformations. When summarizing chip results to the sub-family, the probe set producing the highest intensity was used.
To compare the diversity of bacteria detected with microarrays to a known standard, one sample week was chosen for cloning and sequencing and for replicate microarray analysis. One large pool of SSU amplicons (96 reactions, 50 μL/reaction) from San Antonio week 29 was made. One milliliter of the pooled PCR product was gel purified and 768 clones were sequenced at the DOE Joint Genome Institute (Walnut Creek, Calif.) by standard methods. An aliquot of this pooled PCR product was also hybridized to a microarray (three replicate arrays performed). Sub-families containing a taxon scored as present in all three array replicates were recorded. Individual cloned rRNA genes were sequenced from each terminus, assembled using Phred and Phrap (S9, S10, S11), and were required to pass quality tests of Phred 20 (base call error probability <10−2.0) to be included in the comparison.
Sequences that appeared chimeric were removed using Bellerophon (S2) with two requirements; (1) the preference score must be less than 1.3 and (2) the divergence ratio must be less than 1.1. The divergence ratio is a new metric implemented to weight the likelihood of a sequence being chimeric according to the similarity of the parent sequences. The more distantly related the parent sequences are to each other relative to their divergence from the chimeric sequence, the greater the likelihood that the inferred chimera is real. This metric uses the average sequence identity between the two fragments of the candidate and their corresponding parent sequences as the numerator, and the sequence identity between the parent sequences as the denominator. All calculations are made using a 300 base pair window on either side of the most likely break point. A divergence ratio of 1.1 was empirically determined to be the threshold for classifying sequences as putatively chimeric.
Similarity of clones to array taxa was calculated with DNADIST (S12) using the DNAML-F84 option assuming a transition:transversion ratio of 2.0 and an A, C, G, T 16S rRNA gene base frequency of 0.2537, 0.2317, 0.3167, 0.1979, respectively. We calculated these parameters empirically from all records of the ‘Greengenes’ 16S rRNA multiple sequence alignment over 1,250 nucleotides in length. The Lane mask (S13) was used to restrict similarity observations to 1,287 conserved columns (lanes) of aligned characters. Cloned sequences from this study were rejected from further analysis when <1,000 characters could be compared to a lane-masked reference sequence. Sequences were assigned to a taxonomic node using a sliding scale of similarity threshold (S14). Phylum, class, order, family, sub-family, or taxon placement was accepted when a clone surpassed similarity thresholds of 80%, 85%, 90%, 92%, 94%, or 97%, respectively. When similarity to nearest database sequence was <94%, the clone was considered to represent a novel sub-family. A full comparison between clone and array analysis is presented in Table S2.
Primers targeting sequences within particular taxa/sub-families were generated by ARB's probe design feature (S15). Melting temperatures were constrained from 45° C. to 65° C. with G+C content between 40 and 70%. The probes were chosen to contain 3′ bases non-complementary to sequences outside of the taxon/sub-family. Primers were matched using Primer3 (S16) to create primer pairs (Table S3). Sequences were generated using the Takara enzyme system as described above with the necessary adjustments in annealing temperatures. Amplicons were purified (PureLink PCR Purification Kit, Invitrogen) and sequenced directly or, if there were multiple unresolved sequences, cloned using a TOPO pCR2.1 cloning kit (Invitrogen, CA) according to the manufacturer's instructions. The M13 primer pair was used for clones to generate insert amplicons for sequencing at UC Berkeley's sequencing facility.
To determine whether changes in 16S rRNA gene concentration could be detected using the array, various quantities of distinct rRNA gene types were hybridized to the array in rotating combinations. We chose environmental organisms, organisms involved in bioremediation, and a pathogen of biodefense relevance. 16S rRNA genes were amplified from each of the organisms in Table S4. Then each of these nine distinct 16S rRNA gene standards was tested once in each concentration category spanning 5 orders of magnitude (0 molecules, 6×107, 1.44×108, 3.46×108, 8.30×108, 1.99×109, 4.78×109, 2.75×1010, 6.61×1010, 1.59×1011) with concentrations of individual 16S rRNA gene types rotating between arrays such that each array contained the same total of 16S rRNA gene molecules. This is similar to a Latin Square design, although with a 9×11 format matrix.
A taxon (#9389) consisting only of two sequences of Pseudomonas oleovorans that correlated well with environmental variables was chosen for quantitative PCR confirmation of array observed quantitative shifts. Primers for this taxon were designed using the ARB (S15) probe match function to determine unique priming sites based upon regions detected by array probes. These regions were then imputed into Primer3 (S16) in order to choose optimal oligonucleotide primers for PCR. Primer quality was further assessed using Beacon Designer v3.0 (Premier BioSoft, CA). Primers 9389F2 (CGACTACCTGGACTGACACT) (SEQ ID NO: 3) and 9389R2 (CACCGGCAGTCTCCTTAGAG) (SEQ ID NO: 4) were chosen to amplify a 436 bp fragment.
To test the specificity of this primer pair, we used a nested PCR approach. 16S rRNA genes were amplified using universal primers (27F, 1492R) from pooled aerosol genomic DNA extracts from both Austin and San Antonio, Tex. These products were purified and used as template in PCR reactions using primer set 9389F2-9389R2. Amplicons were then ligated to pCR2.1 and transformed into E. coli TOP10 cells as recommended by the manufacturer (Invitrogen, CA). Five clones were chosen at random for each of the two cities (10 clones total) and inserts were amplified using vector specific primers M13 forward and reverse. Standard Sanger sequencing was performed and sequences were tested for homology against existing database entries (NCBI GenBank, RDPII and Greengenes).
To assay P. oleovorans 16S rRNA gene copies in genomic DNA extracts, we performed real-time quantitative PCR (qPCR) using an iCycler iQ real-time detection system (BioRad, CA) with the iQ Sybr® Green Supermix (BioRad, CA) kit. Reaction mixtures (final volume, 25 μl) contained 1× iQ Sybr® Green Supermix, 7.5 pmol of each primer, 25 ug BSA, 0.5 μl DNA extract and DNase/RNase free water. Following enzyme activation (95° C., 3 min), up to 50 cycles of 95° C., 30 s; 61° C., 30 s; 85° C., 10 s and 72° C., 45 s were performed. The specific data acquisition step (85° C. for 10 s) was set above the Tm of potential primer dimers and below the Tm of the product to minimize any non-amplicon Sybr Green fluorescence. Copy number of P. oleovorans 16S rRNA gene molecules was quantified by comparing cycle thresholds to a standard curve (in the range of 7.6×100 to 7.6×105 copies μl−1), run in parallel, using cloned P. oleovorans 16S rRNA amplicons generated by PCR using primers M13 forward and reverse. Regression coefficients for the standard curves were typically greater than 0.99, and post amplification melt curve analyses displayed a single peak at 87.5° C., indicative of specific Pseudomonas oleovorans 16S rRNA gene amplification (data not shown).
To account for scanning intensity variation from array to array, internal standards were added to each experiment. The internal standards were a set of thirteen amplicons generated from yeast and bacterial metabolic genes and five synthetic 16S rRNA-like genes spiked into each aerosol amplicon pool prior to fragmentation. The known concentrations of the amplicons ranged from 4 pM to 605 pM in the final hybridization mix. The intensities resulting from the fifteen corresponding probe sets were natural log transformed. Adjustment factors for each array were calculated by fitting the linear model using the least-squares method. An array's adjustment factor was subtracted from each probe set's ln(intensity).
For each day of aerosol sampling, 15 factors including humidity, wind, temperature, precipitation, pressure, particulate matter, and week of year were recorded from the U.S. National Climatic Data Center (http://www.ncdc.noaa.gov) or the Texas Natural Resource Conservation Commission (http://www.tceq.state.tx.us). The weekly mean, minimum, maximum, and range of values were calculated for each factor from the collected data. The changes in ln(intensity) for each taxon considered present in the study was tested for correlation against the environmental conditions. The resulting p-values were adjusted using the step-up False Discovery Rate (FDR) controlling procedure (S18).
Multivariate regression tree analysis (S19, S20) was carried out using the package ‘mvpart’ within the ‘R’ statistical programming environment. A Bray-Curtis-based distance matrix was created using the function ‘gdist’. The Brady-Curtis measure of dissimilarity is generally regarded as a good measure of ecological distance when dealing with ‘species’ abundance as it allows for non-linear responses to environmental gradients (S19, S21).
Prior to rarefaction analysis a distance matrix (DNAML homology) of clone sequences was created using an online tool at http://greengenes.lbl.gov/cgi-bin/nph-distance_matrix.cgi following alignment of the sequences using the NAST aligner (http://greengenes.lbl.gov/NAST) (S22). DOTUR (S23) was used to generate rarefaction curves, Chao1 and ACE richness predictions and rank-abundance curves. Nearest neighbor joining was used with 1000 iterations for bootstrapping.
DNA yields in the pooled weekly filter washes ranged from 0.522 ng to 154 ng. As only an aliquot of the filter washes was extracted we extrapolate the range of DNA extractable from each daily filter to be between 150 ng and 4300 ng assuming 10% extraction efficiency. Using previous estimates of bacterial to fungal ratios in aerosols (49% bacterial, 44% fungal clones; S24) this range is equivalent to 1.2×107 to 3.5×108 bacterial cells per filter assuming a mean DNA content of a bacterial cell of 6 fg (S25).
E. coli biotin synthetase
E. coli biotin synthetase
E. coli bioC protein
E. coli bioC protein
E. coli dethiobiotin synthetase
B. subtilis dapB, dihydrodipicolinate reductase
B. subtilis dapB, dihydrodipicolinate reductase
Saccharomyces, Gene for actin (Act1p) protein
Saccharomyces, RNA polymerase II mediator complex subunit (SRB4p)
Saccharomyces, TATA-binding protein, general transcription factor (SPT15)
Saccharomyces, Beta subunit of the oligosaccharyl transferase (OST)
Saccharomyces, Ubiquinol-cytochrome-c reductase (RIP1)
Chroococcidiopsis; Unclassified; sf_1
Leptolyngbya; Unclassified; sf_1
Phormidium; Unclassified; sf_1
Plectonema; Unclassified; sf_1
Spirulina; Unclassified; sf_1
Devosia; Unclassified; sf_1
Fulvimarina; Unclassified; sf_1
Shewanella; Unclassified; sf_1
1A sub-family must have at least one taxon present above the positive probe threshold of 0.92 (92%) in all three replicates to be considered present.
2For a clone to be assigned to a sub-family its DNAML similarity must be above the 0.94 (94%) threshold defined for sub-families.
3This is the maximum DNAML similarity measured.
4Both maximum preference score and maximum divergence ratio must pass the criteria below for a clone to be considered non-chimeric.
5Bellerophon preference score, a ratio of 1.3 or greater has been empirically shown to demonstrate a chimeric molecule.
6Bellerophon divergence ratio. This is a new metric devised to aid chimera detection, a score greater than 1.1 indicates a potential chimera.
Actinobacteria,
Actinokineospora
Actinosynnemataceae,
diospyrosa, AF114797
Actinobacteria,
Bifidobacterium
Bifidobacteriaceae,
adolescentis,
Actinobacteria,
Kineosporiaceae, sf_1
Chloroflexi,
Anaerolineae, sf_9
Cyanobacteria,
Geitlerinema sp. PCC
Geitlerinema, sf_1
Cyanobacteria,
Thermosynechococcus
Thermosynechococcus,
elongatus BP-1,
Gammaproteobacteria,
Pseudoalteromonas sp.
Pseudoaltermonadaceae,
Nitrospira,
Nitrospira
Nitrospiraceae, sf_1
moscoviensis,
Planctomycetes,
Planctomyces
Plantomycetaceae,
brasiliensis,
Proteobacteria,
Campylobacteraceae,
Spirochaetes,
Leptospira
Leptospiracea,
borgpetersenii,
Spirochaetes,
Spirochaeta asiatica,
Spirochaetaceae, sf_1
Spirochaetes,
Borrelia hermsii
Spirochaetaceae, sf_3
Arthrobacter oxydans
Bacillus anthracis AMES
Caulobacter crescentus CB15
Dechloromonas agitata CKB
Dehalococcoides ethenogenes 195
Desulfovibrio vulgaris
Francisella tularensis
Geobacter metallireducens GS-15
Geothrix fermentans H-5
Sulfolobus solfataricus
aStain obtained from Hoi-Ying Holman, LBNL.
b Strain obtained from Arthur Friedlander USAMRID.
c Strain obtained from John Coates, UC Berkeley.
d Strain obtained from Lisa Alvarez-Cohen, UC Berkeley.
eStrain obtained from Terry Hazen, LBNL.
0.703
0.665
0.685
0.691
0.571
−0.777
−0.540
0.607
0.486
−0.498
−0.529
0.617
−0.817
−0.507
0.613
−0.829
0.989
0.553
0.570
0.622
0.630
−0.749
−0.523
0.722
−0.495
0.501
−0.508
−0.830
−0.515
−0.843
0.991
Actinokineospora
diospyrosa str.
Actinokineospora
diospyrosa str.
Streptomyces sp.
Sporichthya
polymorpha
Sporichthya
polymorpha
Sporichthya
polymorpha
Streptomyces
Streptomyces
Actinomadura
kijaniata
Ornithinimicrobium
humiphilum str.
Pseudonocardia
thermophila str.
Pseudonocardia
thermophila str.
Pseudonocardia
thermophila str.
Pseudonocardia
thermophila str.
Pseudonocardia
thermophila str.
Corynebacterium
otitidis
Corynebacterium
otitidis
Corynebacterium
otitidis
Corynebacterium
otitidis
Brachybacterium
rhamnosum LMG
Brachybacterium
rhamnosum LMG
Brachybacterium
rhamnosum LMG
Streptomyces scabiei str.
Catellatospora
Catellatospora
Catellatospora subsp.
citrea str. IMSNU
Catellatospora
Catellatospora
Catellatospora
Saccharothrix
tangerinus
Saccharothrix
tangerinus
Saccharothrix
tangerinus
Rhodococcus
fascians
propionicum str.
Nonomuraea
terrinata
Thermoactinomyces
intermedius str.
Symploca atlantica str.
Taxeobacter
ocellatus
Bifissio spartinae
Bifissio spartinae
Bifissio spartinae
Bifissio spartinae
Sphingomonas
Sphingomonas
Pleomorphomonas
oryzae str. B-32
Pleomorphomonas
oryzae str. B-32
Sphingomonas adhaesiva
Sphingomonas adhaesiva
Methylobacterium
organophilum
Methylobacterium
organophilum
Methylobacterium
organophilum
Devosia neptuniae
Burkholderia
andropogonis
Pseudomonas
oleovorans
Pseudomonas
oleovorans
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
Bacillus anthracis
Bacillus cohnii, B. psychrosaccharolyticus,
B. benzoevorans
Bacillus megaterium
Bacillus horikoshii
Bacillus litoralis, B. macroides, B.
psychrosaccharolyticus
Staphylococcus saprophyticus, S. xylosus, S.
cohnii
Bacillus anthracis, cereus, thuringiensis,
mycoides + others
Rickettsia prowazekii - rickettsii
Rickettsia australis, R. eschlimannii, R. typhi,
R. tarasevichiae + others
Rickettsia prowazekii
Rickettsia rickettsii, R. japonica, R. honei +
Burkholderia mallei - pseudomallei
Burkholderia pseudomallei, B. thailandensis
Burkholderia mallei
Burkholderia pseudomallei, Burkholderia
cepacia, B. tropica, B. gladioli, B. stabilis, B.
plantarii + others
Clostridum botulinum - perfringens
Clostridium butyricum, C. baratii, C.
sardiniense + others
Clostridium botulinum type C
Clostridium perfringens
Clostridium botulinum type G
Clostridium botulinum types B and E
Francisella tularensis
Tilapia parasite
Francisella tularensis
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present embodiments. The foregoing description and Examples detail certain preferred embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the present embodiments may be practiced in many ways and the present embodiments should be construed in accordance with the appended claims and any equivalents thereof.
The term “comprising” is intended herein to be open-ended, including not only the recited elements, but further encompassing any additional elements.
This application is a continuation of U.S. patent application Ser. No. 12/474,204, filed May 28, 2009, which is a continuation of, and claims the benefit of priority to, PCT Application No. PCT/US2007/024720, filed Nov. 29, 2007, which was written in English, published in English as WO/2008/130394 and designated the United States of America, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/861,834 filed Nov. 30, 2006, both of which are hereby incorporated by reference in their entirety.
This invention was made with Government support under Grant No. HSSCHQ04X00037 from the Department of Homeland Security and Contract No. DE-AC02-05CH11231 from the Department of Energy.
| Number | Date | Country | |
|---|---|---|---|
| 60861834 | Nov 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12474204 | May 2009 | US |
| Child | 14298081 | US | |
| Parent | PCT/US2007/024720 | Nov 2007 | US |
| Child | 12474204 | US |
