Patent Application
20160251731
This invention generally relates to hydrocarbon exploration, e.g., oil and gas exploration, oil pollution monitoring and management, and microbiology. In alternative embodiments, the invention provides products of manufacture and compositions, e.g., nucleic acid probes, for use as identifying agents or indicators to detect the presence of a hydrocarbon in a sample, e.g., in marine sediments, muds, sands and the like, or in a solution, e.g., an aqueous solution, such as fresh water, underground water or seawater. In alternative embodiments, the invention provides compositions, e.g., nucleic acid probes, for use as sensors and/or identifying agents to detect the presence of a hydrocarbon in a sample (e.g., in fresh water, underground water or seawater, or a marine mud, sand or sediment), where the presence of the hydrocarbon indicates e.g., the presence of a subsurface oil, petroleum or gas accumulation or deposit. In alternative embodiments, the invention provides compositions and methods for use as tools for offshore oil exploration activities.
Commercially relevant accumulations of oil and/or gas reside in geologic features that prevent their further migration, so-called trap structures. The seals of these traps are rarely perfect and leakage occurs. In cases where substantial amounts of petroleum escape, both liquid and gaseous components migrate upward through faults and fractures until they reach the surface. These type of seeps are referred to as ‘prolific’ or ‘macroseeps’ and often are laterally displaced significant distances from their source. Microseeps, in contrast, result from low molecular weight gases (e.g. methane, ethane, propane) escaping from petroleum reservoirs that migrate vertically with little or no lateral displacement creating a diffuse plume overlying the source.
The presence of surface hydrocarbon seeps has been used as an exploration tool for oil/gas reservoirs ever since wells have been drilled. Given the often significant lateral displacement of prolific seeps as a result of travelling through faults, these type of seeps are used as a general (basin-wide) indication of hydrocarbons and to gain clues as to the geochemical character (e.g. API gravity) and the source/age of the resource.
A number of challenges confront the scientist tasked with interpreting geochemical data from potential seep sites. Some of these challenges relate to the ephemeral nature of seeps (diurnal, seasonal variations) and to the effects of microbes actively metabolizing seeping hydrocarbons.
In alternative embodiments, the invention provides products of manufacture and compositions, e.g., nucleic acid probes and primers, for use as identifying agents or indicators to detect the presence of a hydrocarbon in a sample, e.g., an environmental sample, e.g., a marine sediment, sand or mud, or a solution, e.g., an aqueous solution, such as fresh water, underground water or seawater. In alternative embodiments, the invention provides compositions, e.g., nucleic acid probes, for use as a sensor, e.g., a bioindicator, to detect the presence (e.g., immediate or nearby) of a hydrocarbon in a sample, e.g., in fresh water, underground water or seawater, where the presence of the hydrocarbon indicates e.g., the presence of a subsurface oil, petroleum or gas accumulation, deposit or leak or spill. The identified or detected hydrocarbon can be a vertically migrating hydrocarbon, e.g., vertically migrating in fresh water, underground water or seawater or sand, shale or mud. In alternative embodiments, the invention provides compositions and methods for use as tools for offshore oil exploration activities.
In alternative embodiments, the invention provides isolated, synthetic or recombinant nucleic acids comprising or consisting of:
(a) a nucleic acid or a nucleic acid sequence as set forth in Table 1, Table 2, Table 3 or Table 4;
(b) a nucleic acid or a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or complete (100%) sequence homology to a nucleic acid or a nucleic acid sequence as set forth in Table 1, Table 2, Table 3 or Table 4;
(c) a nucleic acid or a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or complete (100%) sequence homology to a nucleic acid or a nucleic acid sequence: as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192 SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199 or SEQ ID NO:200 (hereinafter referenced as SEQ ID NO: 1 to SEQ ID NO:200); or
(d) a nucleic acid or a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or complete (100%) sequence homology to a nucleic acid or a nucleic acid sequence: as set forth in any one of SEQ ID NO:201 to SEQ ID NO:583,
and optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection,
and optionally the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d “nr pataa” -F F, and all other options are set to default.
In alternative embodiments, the invention provides isolated, synthetic or recombinant nucleic acids comprising or consisting of a nucleic acid sequence capable of specifically (selectively) hybridizing (hybridizes under stringent conditions to) to a nucleic acid of the invention, or a nucleic acid sequence as set forth in Table 1, Table 2, Table 3 or Table 4, or a nucleic acid or nucleic acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO:200 or SEQ ID NO:201 to SEQ ID NO:583,
wherein optionally the stringent conditions include a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes.
In alternative embodiments, the nucleic acid sequence capable of specifically (selectively) hybridizing to (hybridizes under stringent conditions to) a nucleic acid of the invention, or a nucleic acid sequence as set forth in Table 1, Table 2, Table 3 or Table 4, comprises or consists of:
(a) a member of an amplification primer pair, a polymerase chain reaction (PCR) primer pair, ligase chain reaction (LCR) pair, or a qPCR primer pair capable of amplifying a nucleic acid sequence as set forth in Table 2; or,
(b) a hybridization probe sequence capable of specifically (selectively) hybridizing to a nucleic acid or nucleic acid sequence of the invention, or as set forth in Table 1, Table 2, Table 3 or Table 4, or a nucleic acid or nucleic acid sequence as set forth in any one of SEQ ID NO:1 to SEQ ID NO:200 or SEQ ID NO:201 to SEQ ID NO:583.
In alternative embodiments, a nucleic acid of the invention can further comprise a detectable moiety or an enzyme. In alternative embodiments, the detectable moiety comprises a radioactive probe, a fluorescent molecule (e.g., a fluorescent label or a fluorophore, e.g., a coumarin, resorufin, xanthene, benzoxanthene, cyanine or bodipy analog), a quantum dot or a colloidal quantum dot (QD) (e.g., a QDOT™ nanocrystal, Life Technologies, Carlsbad, Calif.), and/or an epitope or binding molecule (e.g. a ligand).
In alternative embodiments, a nucleic acid of the invention can further comprise, or can be immobilized or conjugated or bound to, a solid or semi-solid surface. The solid or semi-solid surface comprises or consists of an array, a biochip, a chip, a bead, a gel, a liposome, a fiber, a film, a membrane, a metal, a resin, a polymer, a ceramic, a glass, an electrode, a microelectrode, a graphitic particle, or a microparticle or a nanoparticle.
In alternative embodiments, the invention provides amplification primer pairs or amplification pairs, polymerase chain reaction (PCR) primer pairs, ligase chain reaction (LCR) pairs, or qPCR primer pairs, comprising or consisting of:
(a) a primer pair as set forth in Table 2, or one member of a primer pair as set forth in Table 2,
(b) a primer pair comprising or consisting of: SEQ ID NO: 1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:19 and SEQ ID NO:20; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:43 and SEQ ID NO:44; SEQ ID NO:45 and SEQ ID NO:46; SEQ ID NO:47 and SEQ ID NO:48; SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:51 and SEQ ID NO:52; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:55 and SEQ ID NO:56; SEQ ID NO:57 and SEQ ID NO:58; SEQ ID NO:59 and SEQ ID NO:60; SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64; SEQ ID NO:65 and SEQ ID NO:66; SEQ ID NO:67 and SEQ ID NO:68; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:71 and SEQ ID NO:72; SEQ ID NO:73 and SEQ ID NO:74; SEQ ID NO:75 and SEQ ID NO:76; SEQ ID NO:77 and SEQ ID NO:78; SEQ ID NO:79 and SEQ ID NO:80; SEQ ID NO:81 and SEQ ID NO:82; SEQ ID NO:83 and SEQ ID NO:84; SEQ ID NO:85 and SEQ ID NO:86; SEQ ID NO:87 and SEQ ID NO:88; SEQ ID NO:89 and SEQ ID NO:90; SEQ ID NO:91 and SEQ ID NO:92; SEQ ID NO:93 and SEQ ID NO:94; SEQ ID NO:95 and SEQ ID NO:96; SEQ ID NO:97 and SEQ ID NO:98; SEQ ID NO:99 and SEQ ID NO:100; SEQ ID NO:101 and SEQ ID NO:102; SEQ ID NO:103 and SEQ ID NO:104; SEQ ID NO:105 and SEQ ID NO:106; SEQ ID NO:107 and SEQ ID NO:108; SEQ ID NO:109 and SEQ ID NO:110; SEQ ID NO:111 and SEQ ID NO:112; SEQ ID NO:113 and SEQ ID NO:114; SEQ ID NO:115 and SEQ ID NO:116; SEQ ID NO:117 and SEQ ID NO:118; SEQ ID NO:119 and SEQ ID NO:120; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO: 125 and SEQ ID NO:126; SEQ ID NO:127 and SEQ ID NO:128; SEQ ID NO:129 and SEQ ID NO:130; SEQ ID NO:131 and SEQ ID NO:132; SEQ ID NO:133 and SEQ ID NO:134; SEQ ID NO:135 and SEQ ID NO:136; SEQ ID NO:137 and SEQ ID NO:138; SEQ ID NO: 139 and SEQ ID NO:140; SEQ ID NO:141 and SEQ ID NO:142; SEQ ID NO:143 and SEQ ID NO:144; SEQ ID NO: 145 and SEQ ID NO:146; SEQ ID NO: 147 and SEQ ID NO: 148; SEQ ID NO: 149 and SEQ ID NO:150; SEQ ID NO:151 and SEQ ID NO:152; SEQ ID NO: 153 and SEQ ID NO:154; SEQ ID NO:155 and SEQ ID NO:156; SEQ ID NO:157 and SEQ ID NO:158; SEQ ID NO:159 and SEQ ID NO:160; SEQ ID NO:161 and SEQ ID NO:162; SEQ ID NO:163 and SEQ ID NO: 164; SEQ ID NO: 165 and SEQ ID NO: 166; SEQ ID NO: 167 and SEQ ID NO:168; SEQ ID NO:169 and SEQ ID NO:170; SEQ ID NO:171 and SEQ ID NO:172; SEQ ID NO:173 and SEQ ID NO:174; SEQ ID NO:175 and SEQ ID NO: 176; SEQ ID NO:177 and SEQ ID NO:178; SEQ ID NO:179 and SEQ ID NO:180; SEQ ID NO:181 and SEQ ID NO:182; SEQ ID NO:183 and SEQ ID NO:184; SEQ ID NO:185 and SEQ ID NO:186; SEQ ID NO:187 and SEQ ID NO:188; SEQ ID NO:189 and SEQ ID NO:190; SEQ ID NO:191 and SEQ ID NO: 192; SEQ ID NO: 193 and SEQ ID NO: 194; SEQ ID NO: 195 and SEQ ID NO: 196; SEQ ID NO: 197 and SEQ ID NO: 198; or, SEQ ID NO: 199 and SEQ ID NO:200;
(c) all of the primer pairs as set forth in Table 2; or (d) all of the primer pairs of (b).
In alternative embodiments, at least one member of the primer pair further comprises a detectable moiety. In alternative embodiments, the detectable moiety comprises a radioactive probe, a fluorescent molecule (e.g., a fluorescent label or a fluorophore, e.g., a coumarin, resorufin, xanthene, benzoxanthene, cyanine or bodipy analog), a quantum dot or a colloidal quantum dot (QD) (e.g., a QDOT™ nanocrystal, Life Technologies, Carlsbad, Calif.), and/or an epitope or binding molecule (e.g. a ligand).
In alternative embodiments, at least one member of the primer pair, or both members of the primer pair, further comprise, or are immobilized or conjugated or bound to, a solid or a semi-solid surface. The solid or semi-solid surface can comprise or consist of an array, a biochip, a chip, a bead, a gel, a liposome, a fiber, a film, a membrane, a metal, a resin, a polymer, a ceramic, a glass, an electrode, a microelectrode, a graphitic particle, or a microparticle or a nanoparticle.
In alternative embodiments, the invention provides products of manufacture, arrays, biochips, chips, beads, gels, liposomes, fibers, films, membranes, metals, resins, polymers, ceramics, glasses, electrodes, microelectrodes, graphitic particles, or microparticles or nanoparticles, comprising a nucleic acid of the invention, or a plurality of or all of the nucleic acids of the invention, or an amplification primer pair, polymerase chain reaction (PCR) primer pair, a ligase chain reaction (LCR) pair, or a qPCR primer pair of the invention, or all amplification primer pairs, polymerase chain reaction (PCR) primer pairs, a ligase chain reaction (LCR) pairs or qPCR primer pairs of the invention.
In alternative embodiments, the invention provides kits comprising a nucleic acid of the invention, or a plurality of or all of the nucleic acids of the invention, or an amplification primer pair, a polymerase chain reaction (PCR) primer pair, a ligase chain reaction (LCR) pair, or a qPCR primer pair of the invention, wherein optionally the kit comprises or is a PCR, LCR or qPCR kit, and optionally the nucleic acid, amplification primer pair, polymerase chain reaction (PCR) primer pair, ligase chain reaction (LCR) pair or qPCR primer pair is contained or stored in a solution, a test tube or a container.
In alternative embodiments, the invention provides methods of detecting, identifying, quantifying and/or indicating the presence of a hydrocarbon in a sample, comprising:
(a) obtaining or providing one sample or a set of samples,
wherein optionally the sample is an aqueous sample, a fresh water sample or a sea water sample, or a sediment, sand, shale or mud, or a marine sediment, sand, shale or mud, or a solution,
or optionally the samples comprise fresh water, underground water or seawater, or a production water, or an aqueous sample or a marine sediment, sand, shale or mud are taken from or prepared from a core sample;
(b) detecting, determining, quantifying and/or characterizing the presence of a nucleic acid in the sample or samples, wherein the detecting, determining, characterizing or quantifying (measuring) the presence of the nucleic acid in the sample or samples indicates the presence of, or quantifies or estimates the amount of, the hydrocarbon in the sample or solution,
and the nucleic acid detected, characterized or quantified comprises or consists of a nucleic acid of the invention, and/or
the nucleic acid is detected, characterized or quantified using:
wherein optionally the determining, quantifying and/or characterizing the presence of a nucleic acid in the sample or samples is by a method comprising an amplification, a polymerase chain reaction (PCR), a qPCR and/or a hybridization;
wherein optionally identifying, quantifying and/or characterizing a nucleic acid in the sample or samples also by correlation identifies, quantifies or indicates the presence of a hydrocarbon in the solution.
wherein detecting, quantifying, determining and/or characterizing the nucleic acid in the sample or samples quantifies, identifies or detects the presence of the hydrocarbon in the sample.
In alternative embodiments of the methods, each test sample is assayed for the presence of a plurality of, or many independent, bioindicators that are positively correlated with the presence of one or more hydrocarbons, wherein optionally the bioindicator comprises a nucleic acid of the invention.
In alternative embodiments of the methods, a test sample is assayed for the presence of one or more, or a plurality of, microbial bioindicator sequences or nucleic acids that are positively and negatively associated with the presence of a hydrocarbon, wherein optionally the microbial bioindicator sequence or nucleic acid comprises a nucleic acid of the invention.
In alternative embodiments of the methods, an RNA is extracted from the sample or samples, and the RNA converted to DNA prior to PCR amplification and/or hybridization, wherein optionally the RNA is ribosomal RNA, or optionally the RNA converted to DNA using a reverse transcriptase enzyme.
In alternative embodiments the methods further comprise characterizing and/or identifying one, all or substantially most of the microbes in the sample or samples, wherein optionally the microbial composition is determined by a chemical or analytical method, and optionally the chemical or analytical method comprises a fatty acid methyl ester analysis, a membrane lipid analysis and/or a cultivation-dependent method.
In alternative embodiments the invention provides methods of detecting the presence of a subsurface hydrocarbon, petroleum, oil or gas accumulation or deposit, or the presence of a petroleum or hydrocarbon seep, spill, pollutant or leak, comprising:
(a) obtaining or providing one samples or a set of samples,
wherein optionally the sample or samples are from, or comprise, a marine sediment, shale, sand or mud, or an aqueous source, or seawater, fresh water or production fluid, and optionally the sample or samples comprise a fresh water, underground water or seawater source, or a production water, or the marine sediment, sand or mud, or aqueous sample is taken from or prepared from a core sample, and optionally the seep is a thermogenic hydrocarbon seep or a macroseep or a microseep;
(b) determining, detecting and/or characterizing the presence of a nucleic acid in the sample or samples, wherein the presence of a nucleic acid in the sample or samples indicates the presence of a subsurface hydrocarbon, petroleum, oil or gas accumulation or deposit, or a leak, pollutant, seep or spill,
and the nucleic acid detected, characterized or quantified comprises or consists of a nucleic acid of the invention, and/or
the nucleic acid is detected, characterized or quantified using:
In alternative embodiments of the methods, each sample is assayed for the presence of a plurality of, or many independent, bioindicators that are positively correlated with the presence of one or more hydrocarbons. In alternative embodiments of the methods, the sample is assayed for the presence of one or more, or a plurality of, microbial bioindicator sequences that are positively and negatively associated with the presence of hydrocarbons.
In alternative embodiments of the methods, an RNA is extracted from samples and converted to DNA by methods well known in the art (e.g. using reverse transcriptase), prior to PCR amplification and/or hybridization, wherein optionally the RNA is ribosomal RNA.
In alternative embodiments the methods further comprise characterizing and/or identifying one, all or substantially most of the microbes in the sample or samples, wherein optionally the microbial composition is determined by a chemical or analytical method, and optionally the chemical or analytical method comprises a fatty acid methyl ester analysis, a membrane lipid analysis and/or a cultivation-dependent method.
In alternative embodiments, the invention provides kits comprising a kit of the invention and instructions comprising a method of the invention.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
In one embodiment, the invention provides compositions and products of manufacture, e.g., nucleic acid primers and probes, for use as identifying agents or indicators to detect the presence of a hydrocarbon in a sample, e.g., a solution, e.g., an aqueous solution, or an environmental sample such as fresh water, underground water or seawater or sand, shale or mud. In alternative embodiments, the invention provides compositions and products of manufacture, e.g., nucleic acid primers and probes, for use as bioindicators and biodetectors to detect the presence of (e.g., immediate or nearby) vertically migrating (e.g., in fresh water, underground water or seawater) hydrocarbons that e.g., can indicate the presence of subsurface petroleum, oil or gas accumulations or deposits, or leaks or spills. In one embodiment, the invention provides methods for making and using the compositions of the invention.
In alternative embodiments, the invention provides compositions, e.g., nucleic acid probes, for use as indirect bioindicator assays to detect the presence of a hydrocarbon in a sample, e.g., an aqueous sample such as water or seawater (and methods for using them), e.g., to detect seep sites, e.g., seeping hydrocarbons, which can be a “prolific” or “macroseep” or a “microseep”, or to detect leaks or spills. In alternative embodiments, use of compositions and methods of the invention has advantages over direct chemical analysis. Thus, compositions and methods of the invention can be used to interpret geochemical data from potential seep sites. In alternative embodiments, compositions and methods of the invention are used to overcome challenges related to the ephemeral nature of seeps (e.g., which include diurnal, seasonal variations) and the effects of microbes actively metabolizing seeping hydrocarbons.
A study was conducted to characterize microbial communities associated with thermogenic hydrocarbon seeps in the Green Canyon block of the Gulf of Mexico (GOM). One of the goals of the project was to identify microbes that could themselves be used as bioindicators to detect the immediate, or nearby, presence of vertically migrating hydrocarbons that would indicate the presence of subsurface petroleum accumulations. A collection of 16S rRNA gene sequences was found comprising individual bioindicator sequences that each displayed significant statistical associations with certain hydrocarbons. The organisms these sequences identify also may possess value for chemical transformation (upgrading) of heavy oil or enhanced oil recovery.
In alternative embodiments, the invention provides synthetic, recombinant and isolated nucleic acids, including amplification primer pairs and probes, e.g., hybridization probes, for detecting or quantifying a hydrocarbon in a sample such as water, fresh water, seawater, mud, shale or sand, or for detecting the presence of a subsurface petroleum, oil or gas accumulation or deposit, or for detecting the presence of a petroleum seep or leak or spill, and generally practicing methods of the invention.
The nucleic acids of the invention, or used to practice methods this invention, can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. In practicing the methods of the invention, homologous genes can be modified by manipulating a template nucleic acid, as described herein. The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
General Techniques
The synthetic, recombinant and isolated nucleic acids of the invention, or used to practice methods this invention, whether RNA (e.g., rRNA), antisense nucleic acid, cDNA, genomic DNA, vectors, viruses and the like, may be isolated, or initially isolated, from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
Alternatively, nucleic acids of the invention, or used to practice methods this invention, can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066. In alternative embodiments, nucleic acids used to practice this invention, or nucleic acids of this invention, can comprise entirely, or in part, any non-naturally-occurring oligonucleotide analogue, e.g., thioate-type oligonucleotides, or synthetic oligos comprising unsubstituted purin-9-yl, unsubstituted 2-oxo-pyrimidin-1-yl or a substituted purin-9-yl, e.g., as described in U.S. Pat. App. Pub. No. 20090149404. In alternative embodiments, a ribose sugar of one or more of a nucleotide used to practice this invention is replaced with another moiety, e.g., a non-carbohydrate, e.g., a cyclic carrier, e.g., as described in U.S. Pat. App. Pub. No. 20100069471. In alternative embodiments, nucleic acids used to practice this invention, or nucleic acids of this invention, can comprise entirely, or in part, any peptide nucleic acids (PNA), e.g., any polyamide nucleic acid (PNA) derivative, e.g., as described in U.S. Pat. App. Pub. No. 20100022016; PNA binds to complementary DNA and RNA even at low salt concentration.
In alternative embodiments, nucleic acids used to practice methods of this invention, or nucleic acids of this invention, can comprise (partially or entirely) peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units; or can comprise phosphorothioate linkages, e.g., as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). In alternative embodiments, nucleic acids used to practice this invention, or nucleic acids of this invention, can comprise (partially or entirely) synthetic DNA backbone analogues comprising phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids.
Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Amplification of Nucleic Acids
In alternative embodiments, nucleic acids of the invention, or used to practice methods this invention, are used in amplification reactions to detect nucleic acids in a sample, e.g., an aqueous sample, such as an environmental sample (such as fresh, sea or ground water, sand, mud, shale and the like) e.g., to detect and/or quantify the presence of a hydrocarbon in the sample, e.g., in a subsurface petroleum, oil or gas accumulation or deposit, or the presence of a petroleum seep, spill or leak. Alternatively, nucleic acids of the invention, or used to practice methods this invention, themselves can be made or reproduced by amplification. Amplification can also be used to clone or modify the nucleic acids of the invention, or used to practice methods this invention.
In alternative embodiments, amplification reactions are used to quantify the amount of nucleic acid in a sample (such as the amount of a specific rRNA sequence in a sample), to label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample. In one aspect of the invention, RNA isolated from a sample is amplified, or reverse transcribed and then amplified.
In alternative embodiments, in addition to the amplification primers described herein, skilled artisan can select and design equivalent oligonucleotide amplification primers to practice the methods of this invention. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J. Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
In practicing the invention, any apparatus for nucleic acid, e.g., DNA, amplification, e.g., for qualitative and/or quantitative measurements, can be used, e.g., as described in U.S. Pat. App. Pub. No. 20100075312. For example, practicing the invention can comprise methods or compositions as described in U.S. Pat. No. 5,994,056, which describes an approach to PCR in which there is simultaneous amplification and detection. Alternatively, practicing the invention can comprise using methods or compositions as described in U.S. Pat. No. 6,586,233, which describes an arrangement for convectively-driven thermal cycling to perform a polymerase chain reaction (PCR). Alternatively, practicing the invention can comprise using quantitative PCR (qPCR) arrays as described in e.g., U.S. Pat. App. Pub. No. 20090142759, describing qPCR assays.
Alternatively, practicing the invention can comprise using real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR); or multiplex qPCR, real-time PCR, and/or reverse transcription quantitative PCR (RT-qPCR).
Hybridization of Nucleic Acids
In alternative embodiments, the invention provides nucleic acids that hybridize under stringent conditions (or selective, or highly selective) to polynucleotides whose presence in a sample detects or indicates the presence of a hydrocarbon, e.g., a subsurface petroleum, oil or gas accumulation or deposit, or the presence of a petroleum seep or leak or spill, or quantifies the presence of a hydrocarbon in the sample. The stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions. In one aspect, it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid binds to a desired target.
In alternative embodiments, nucleic acids of the invention are designed to hybridize under high stringency comprising conditions of about 50% formamide at about 37° C. to 42° C.; or designed to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30° C. to 35° C.; or are designed to hybridize under high stringency comprising conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA); or to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35° C.
In alternative embodiments, following hybridization, the hybridized nucleic acids are washed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide. In alternative embodiments, hybridization is conducted at 30% formamide; or hybridization is conducted at 10% formamide.
In alternative embodiments, hybridization is carried out in buffers, such as SSC, e.g., 6×SSC, e.g. containing formamide, e.g. at a temperature of 42° C. In alternative embodiments, the concentration of formamide in the hybridization buffer is reduced. In alternative embodiments, following hybridization, a filter may be washed with 6×SSC, 0.5% SDS at 50° C.
In alternative embodiments, selection of a hybridization format is not critical—it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid remains bound (hybridized) to a desired target. In alternative embodiments wash conditions include, e.g.: a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50° C. or about 55° C. to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C. for about 15 minutes; or, a salt concentration of about 0.2×SSC at a temperature of at least about 50° C. or about 55° C. to about 60° C. for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2×SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausubel for a description of SSC buffer and equivalent conditions.
Determining the Degree of Sequence Identity
The invention provides isolated, synthetic or recombinant nucleic acids comprising sequences having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or complete (100%) sequence identity (homology) to a nucleic acid or a nucleic acid sequence as set forth in Table 1, Table 2, Table 3 or Table 4, or SEQ ID NO:1 to SEQ ID NO:200, or SEQ ID NO:201 to SEQ ID NO:583.
The extent of sequence identity (homology) may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters. In alternative embodiments, the sequence identify can be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400 consecutive residues, or the full length of the nucleic acid. Algorithms and programs used to practice this invention include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).
A “comparison window” includes reference to a segment of any one of the number of contiguous residues. For example, in alternative embodiments of the invention, contiguous residues ranging anywhere from 20 to the full length of an exemplary sequence of the invention are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. If the reference sequence has the requisite sequence identity to an exemplary sequence of the invention, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence of the invention, that sequence is within the scope of the invention. In alternative embodiments, subsequences ranging from about 20 to 600, about 50 to 200, and about 100 to 150 are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequence for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. In one aspect, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”).
In one embodiment, to determine if a nucleic acid has the requisite sequence identity to be within the scope of the invention, the NCBI BLAST 2.2.2 programs is used. default options to blastp. There are about 38 setting options in the BLAST 2.2.2 program. In this exemplary aspect of the invention, all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a “-F F” setting is used, which disables filtering. Use of default filtering often results in Karlin-Altschul violations due to short length of sequence. The default values used in this exemplary aspect of the invention, include:
Nucleic acids, e.g., the probes, of the invention can be immobilized to or applied to an array, chip, biochip and the like. Arrays, chips etc. can be used to screen for or monitor samples (e.g., environmental samples such as fresh water, sea water, mud, sand and the like) for practicing a method of the invention, e.g., identifying and/or indicating the presence of a hydrocarbon in a marine sediment, sand, mud or solution.
In alternative aspects, “arrays” or “microarrays” or “biochips” or “chips” of the invention comprise a plurality of target elements (e.g., positive controls or negative controls) in addition to a nucleic acid (e.g., probe) of the invention; each target element can comprises a defined amount of one or more nucleic acids immobilized onto a defined area of a substrate surface.
The present invention can be practiced with any known “array,” also referred to as a “microarray” or “nucleic acid array” or “bioarray” or “biochip,” or variation thereof. Arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., genomic nucleic acid or mRNA transcripts.
In practicing the methods of the invention, any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp. 21:25-32. See also published U.S. patent applications Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.
Nucleic acid sequences of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. In alternative embodiments, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid sequences of the invention, e.g., an exemplary sequence of the invention. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
In alternative embodiments, the invention provides a computer readable medium having recorded thereon at least one nucleic acid sequence of the invention. Computer readable media include magnetically readable media, optically readable media, electronically readable media, magnetic/optical media, flash drives and flash memories. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, a flash memory, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM), or any type of media known to those skilled in the art.
The invention provides kits comprising compositions and methods of the invention, including instructions for use thereof. In alternative embodiments, the invention provides kits comprising a composition (e.g., a probe of the invention), a product of manufacture, or mixture (e.g., comprising a probe of the invention) or a culture of cells (e.g., expressing probes of the invention), of the invention; wherein optionally the kit further comprises instructions for practicing a method of the invention.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.
This Example describes characterization of microbial communities associated with thermogenic hydrocarbon seeps in the Green Canyon block of the Gulf of Mexico (GOM). One of the goals of the project was to identify microbes that could themselves be used as bioindicators to detect the immediate, or nearby, presence of vertically migrating hydrocarbons that would indicate the presence of subsurface petroleum accumulations. A collection of 16S rRNA gene sequences was found comprising individual bioindicator sequences that each displayed significant statistical associations with certain hydrocarbons. The organisms these sequences identify also may possess value for chemical transformation (upgrading) of heavy oil or enhanced oil recovery.
In this study, piston core samples of marine sediment were collected over a number of well-defined seep features in the Gulf of Mexico (GOM). Many of the cores contained obvious oil staining and methane hydrates. A number of molecular biological and genomics tools were utilized to characterize the microbial communities present in these samples including serial analysis of ribosomal DNA (SARD), 454 pyrosequencing and Sanger sequencing of 16S rRNA gene libraries.
Analysis of the GOM SARD profile data identified about 20,000 unique types of microbes inhabiting offshore hydrocarbon seeps. About 600 of these were found to be associated with hydrocarbon seep components and represented a significant opportunity to develop new petroleum bioindicators. The detection of a given 16S rRNA gene sequence serves as a proxy for the presence of microbes that harbor that specific gene sequence. The DNA sequences from several of these microbes were utilized to develop quantitative polymerase chain reaction (qPCR) assays to detect their presence in marine sediments. A subset of these molecular bioindicator sequences were utilized in qPCR assays to detect the presence gasoline-range hydrocarbons in a geochemically blinded set of 77 marine sediments. The assays correctly predicted the presence of these hydrocarbons in 76/77 samples, thus demonstrating the accuracy and value of reagents of the invention as a new tool for offshore oil exploration activities.
A total of 33 piston cores (6 m) were collected across the seep field. Each core was sub-sampled at 3 intervals per core (i.e. top, middle, bottom). A total of 93 subsamples were collected from the piston cores. Some intervals were not obtained for samples with significant methane hydrates present. Expansion of methane hydrates as the piston cores were raised from high pressure of the seafloor resulted in sample loss in some cases. Samples from each interval were divided up to be sent to different labs for specific geochemical analysis. Subsamples for microbiological analysis were treated aseptically, transferred to sterile containers and immediately frozen at −20° C. These samples were kept frozen until they were processed for DNA extraction at Taxon's facility (Taxon Biosciences, Inc., Tiburon, Calif.).
A subset of 16 samples was chosen for a detailed microbial community profiling to comprise a gradient of the level of hydrocarbons present. Our laboratory was only provided the geochemical data for these 16 ‘unblinded’ samples. Geochemical data for the remaining 77 samples was withheld from our lab in order to create a geochemically ‘blinded’ set of samples. One objective the project was to test whether the bioindicators sequences identified by correlation to hydrocarbons in the 16 unblinded samples could be used to accurately predict the presence of hydrocarbons in the 77 unblinded samples.
All of the samples were from the lowest interval except for those from two cores where the top, middle and lower intervals were sampled from two complete piston cores. These two cores comprised a negative control core taken from outside the seep area and a highly positive core.
Genomic DNA was extracted from the samples by a bead beating procedure e.g., as described by Ashby, M. N.; J. Rine, et al. (2007). “Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities.” Appl Environ Microbiol 73(14): 4532-42, and was utilized to construct three types of 16S rRNA gene profiles including Sanger sequencing of clone libraries, 454 pyrosequencing utilizing Roche's Titanium chemistry and SARD. All of these approaches began with PCR amplification of a portion of the 16S rRNA gene using the primers TX9 and 1391r that corresponds approximately to positions 800 to 1400 (E. coli numbering). This portion of the 16S rRNA gene includes four variable regions (V5-V8). Each of these approaches provides a different level of detail of microbial communities.
Clone libraries were constructed by ligating PCR products into the pUC19™ (Stratagene, San Diego, Calif.) vector. E. coli transformants were picked for plasmid preparation by blue/white screening on X-Gal-containing plates. 960 individual clones (10 plates of 96) were utilized for Sanger sequencing and further analysis. Low quality and short sequences were filtered out as were sequences that failed a chimera check program, e.g., using GREENGENES™, Center for Environmental Biotechnology Lawrence Berkeley National Laboratory, Berkeley, Calif. (Bellerophon, http://greengenes.lbl.gov). Phylogenetic trees were constructed either using the PHYLIP™ software package (Felsenstein, J. 2004. [phylogeny inference package], version 3.63. Department of Genome Sciences, University of Washington, Seattle, Wash.) utilizing neighbor-joining and the KIMURA 2™-parameter distance method or using the NAST aligner available from GREENGENES™, combined with the ARB™ software package (joint initiative of the Lehrstuhl für Mikrobiologie and the Lehrstuhl für Rechnertechnik und Rechnerorganisation/Parallelrechnerarchitektur of the Technische Universität, München, Germany).
Analysis of more than 11,000 Sanger reads of 16S rRNA gene clone libraries revealed the 16 GOM sediment samples harbored significant biodiversity (
Comparison of the bacterial division representation among the 15 GOM sediment samples did not reveal any strong division-level bias toward samples that were located directly on seep features (strongly positive with visible oil staining of the sediment), adjacent to seep features (weakly positive) or outside the seepage area (negative) (
In contrast, Archaeal division representation revealed considerable bias toward and against the sample location relative to seep features (
Representatives from the candidate Archaeal divisions GOM1, 2, 3, and 10 were seen exclusively on seep features with one exception. A single GOM1 sequence was identified from sample 16-25 that was adjacent to a seep feature and was weakly positive. Nevertheless, hundreds of sequences were observed from this candidate division among the ‘on feature’ strongly positive locations. ANME1 division sequences were only found in samples associated with the seep features (weakly or strongly positive). Representatives from the candidate division GOM13 and the division SAGMEG-1 were found with a strong bias against samples with oil and gas hydrates present.
SARD libraries were also constructed from the 15 GOM samples as described previously (Ashby, Rine et al. 2007). A total of about 3.5 million V5 sequence tags were identified that comprised about 20,000 distinct or unique sequences. A 2-Dimensional dendrogram showing the distribution of SARD tags revealed non-random distribution among the sediment samples (
In
16S rRNA gene sequences whose distribution correlated with specific hydrocarbons were identified by comparing their abundance in the set of GOM samples to the levels of hydrocarbons. Often clusters of related sequences (clades) were identified.
Quantitative PCR (qPCR) primers were designed by aligning the collection of 16S rRNA gene sequences that were correlated with a specific hydrocarbon type in the sediment samples. qPCR primers were chosen such that they were: 1) located within variable regions, 2) were of a sufficient length to confer an annealing temperature of approximately 63° C.; and 3), did not show any perfect matches to sequences present in GenBank using BLASTn (see e.g., Zheng Zhang et al. (2000), “A greedy algorithm for aligning DNA sequences”, J. Comput. Biol. 7(1-2):203-14). Primers were designed to 8 distinct composite 16S rRNA gene sequences that correlated with gasoline-range hydrocarbons.
Alignment of 16S rRNA gene sequences whose distributions among the samples were correlated with gasoline-range hydrocarbons. A consensus sequence is of each group is included in the alignment. The primers (oligonucleotides) designed to selectively amplify each group of sequences is indicated on the top line of the alignment, as indicated below in Table 1 (for ease of viewing, the reverse primer is shown as its reverse-complement).
In alternative embodiments, the invention provides nucleic acids comprising or consisting of the nucleic acids of Table 1, including the amplification probes (amplification primer pairs) described in Table 1, including substantially complementary probes which can amplify the same sequences as set forth in Table 1 as the described amplification primer pairs. In alternative embodiments, the invention provides nucleic acids comprising or consisting of the nucleic acids substantially complementary to the sequences of Table 1 such that they can be used as hybridization probes to identify, quantify, and/or isolate the sequences of Table 1 by sequence complementary hybridization.
For example, in one embodiment, an amplification primer pair of the invention comprises or consists of AG GGGATATCAA CTCCTCCGTG TCG (SEQ ID NO:1) and ATCACTCCGTGGCCACCCGTTG CAAC (SEQ ID NO:2), whose “reverse complement is: GGGTGGCCAC GGAGTGAT (SEQ ID NO:201), see the “PTM03” amplification primer pair; and Table 2).
In another embodiment, an amplification primer pair of the invention comprises or consists of GGGCGTAA ACGCTGTGGG CTTA (SEQ ID NO:3) and TGGATGGGTTTCGGGATTGCCTTCAC (SEQ ID NO:4), whose “reverse complement is: GTGAAGGCAA TCCCGAAACC CATCCA (SEQ ID NO:202) (see the “PTM04” amplification primer pair; and Table 2).
In an embodiment, an amplification primer pair of the invention comprises or consists of CGTAA ACGCTGCCCG CTTG (SEQ ID NO:5) and TCGAAGATAGCAACTAAGAGCGAG (SEQ ID NO:6), whose “reverse complement is: CTCG CTCTTAGTTG CTATCTTCGA (SEQ ID NO:203) (see the “PTM05” amplification primer pair; and Table 2).
In another embodiment, an amplification primer pair of the invention comprises or consists of G CTATGTGTCG GGAGATCCAC GT (SEQ ID NO:7) and TCGGGATCGGTACTCTTTGTTCCG (SEQ ID NO:8), whose “reverse complement is: CGGAA CAAAGAGTAC CGATCCCGA (SEQ ID NO:204) (see the “PTMO6” amplification primer pair; and Table 2).
In one embodiment, an amplification primer pair of the invention comprises or consists of TGCTAG CTTGGTGTTG GATAACCTA (SEQ ID NO:9) and CGGACTTGAAAATAGCAACTGAAGATGG (SEQ ID NO: 10); whose “reverse complement is: CCA TCTTCAGTTG CTATTTTCAA GTCCG (SEQ ID NO:205) (see the “PTM07” amplification primer pair; and Table 2).
In one embodiment, an amplification primer pair of the invention comprises or consists of CTCTGTG TCGAAGCTAA CGCCTTAA (SEQ ID NO: 11) and CAGGATTTCTGGGCAGTTTCGTCAG (SEQ ID NO: 12); whose “reverse complement is: CTGA CGAAACTGCC CAGAAATCCT G (SEQ ID NO:206) (see the “PTM08” amplification primer pair; and Table 2).
In one embodiment, an amplification primer pair of the invention comprises or consists of TCGA CCCCTTCTGT GCCGCA (SEQ ID NO:13) and ACCTTCCTCCGCATTATCTGCGA (SEQ ID NO: 14); whose “reverse complement is: TCGCAGA TAATGCGGAG GAAGGT (SEQ ID NO:207) (see the “PTM10” amplification primer pair; and Table 2).
In one embodiment, an amplification primer pair of the invention comprises or consists of GATGTTCA CTTGGTGTCG GTCGCAC (SEQ ID NO:15) and TTGCAACTCTCTGTACCTTCCATTGTAG (SEQ ID NO: 16); whose “reverse complement is: CT ACAATGGAAG GTACAGAGAG TTGCAA (SEQ ID NO:2xx) (see the “PTM11” amplification primer pair; and Table 2).
The composite (or consensus) gasoline-range bioindicator sequences were compared with sequences in the public database GenBank to identify known related sequences (
qPCR assays were performed with SYBR™ Green (Invitrogen, Carlsbad, Calif.) in a ABI 7900HT™ instrument. Melt curves of the products were used to identify reactions with low Tm products. Cloned 16S rRNA genes from the bioindicator strains were used as copy control standards. The qPCR data, expressed as copies per gram of sediment, underwent further data transformation. This included adding a small value (e.g. 1/100th lowest value in table) to each cell in the table, log transforming the data and convert to Z-scores. Z-scores were determined by subtracting the mean and dividing by the standard deviation. Z-score units are expressed as number of standard deviations above (positive) or below (negative) the mean. These units are intuitive and enable combining of Z-scores from different bioindicators (through averaging) to report a single consensus value.
The qPCR assays were designed to detect specific 16S rRNA gene sequences whose sample distribution among the subset of 16 sediment samples was correlated with specific hydrocarbons (e.g. gasoline-range hydrocarbons) (
These assays were performed on both the set of 16 unblinded samples and the 77 blinded GOM samples to determine whether they could predict the presence of gasoline-range hydrocarbons (
These assays revealed the relationship between the abundance of the bioindicators and the abundance of gasoline range hydrocarbons in the 16 unblinded GOM samples was binary in nature rather than linear. Thus, the bioindicators identified the presence of these hydrocarbons, but did not provide information as to the amounts. Examination of the bioindicator levels in the 77 blinded samples revealed, as was the case with the unblinded samples, two groups of samples with either high (Z-Score>1.25) or low (Z-Score<0.8) bioindicator values. The presence of gasoline range hydrocarbons in the unknown blinded samples were predicted based upon having bioindicator Z-score values above or below 1.0. This metric correctly predicted the predicted the presence of these hydrocarbons in 75/77 samples. One of the sample not predicted correctly had a bioindicator Z-score value that was borderline (Z-Score approximately 0.8). The other sample not correctly predicted may have been the result of incorrect geochemical determination of the presence of gasoline-range hydrocarbons. This possibility is supported by the observation that other gasoline-range hydrocarbon species (besides the 14-carbon molecules used in the test) were more consistent with the bioindicator value for this sample.
In one embodiment of the invention, each test sample is assayed for the presence of many independent bioindicators that are positively correlated with the presence of hydrocarbons. Microbes may exhibit different types of positive correlations to a geochemical parameter (e.g. linear, curvilinear, threshold, etc.) by virtue of the specific relationship. These are well known in the art and are described e.g., by Ashby, M. (2003). Methods for the survey and genetic analysis of populations, U.S. Pat. No. 6,613,520.
The sequence count data is expressed as absolute sequence counts per gram of sediment or per microgram of DNA recovered, as Z-scores (no. of standard deviations above/below the mean) with or without first log transforming the sequence count data.
Representative sequences from microbial divisions that were negatively correlated with the presence of hydrocarbons in sediment (e.g. GOM13 and SAGMEG-1 divisions) also have value as bioindicators for the presence of hydrocarbons. Demonstrating that a test (unknown) sediment sample BOTH harbors microbes that are positively correlated with the presence of hydrocarbons AND does not harbor microbes that are negatively correlated with hydrocarbons is a more robust association than the case of a sample only harboring microbes that are positively correlated with hydrocarbons.
In one embodiment of the invention, a test sample is assayed for the presence of microbial bioindicator sequences that are positively and negatively associated with the presence of hydrocarbons. The data could be expressed as absolute sequence counts per gram of sediment or per microgram of DNA recovered, as Z-scores (no. of standard deviations above/below the mean) or as ratios of these numbers derived from the positively correlated bioindicators divided by the negatively correlated bioindicators.
Alternative embodiments comprise methods of obtaining the bioindicator sequence data include qPCR, DNA sequencing technologies including, but not limited to, pyrosequencing (Roche), SOLEXA™ sequencing (Illumina), SOLiD™ (Applied Biosystems), Single Molecule Real Time (SMRT™) sequencing (Pacific Biosciences), Ion PGM™ (Ion Torrent), or hybridization-based methods of DNA detection such as gene chips. Any method that has the ability to capture and record greater than 100 variations in sequence and number of occurrences of 16S rRNA genes present in a sample is adequate to practice this invention.
In another embodiment, RNA is extracted from samples and converted to DNA by methods well known in the art (e.g. using reverse transcriptase), prior to PCR amplification of the 16S rRNA genes present in the sample. RNA is much less stable than DNA and will provide temporal information as to whether the microbes were active, or recently active, when the sample was collected. For example, microbes may persist in the environment in a dormant or dead state in some circumstances. Collection of 16S rRNA gene bioindicator data from both isolated DNA and from isolated RNA will provide both quantitative information (DNA) as well as whether the microbes were active (RNA). The combination of both RNA and DNA measurements will therefore allow one to distinguish active seep from dormant seep and dormant seep from recent organic matter (ROM) background.
This Example describes an alternative protocol for characterizing microbial communities associated with thermogenic hydrocarbon seeps.
Genomic DNA extracted as described in “Example 1: Characterization of microbial communities associated with thermogenic hydrocarbon seeps” were further prepared as follows. A portion of the 16S rRNA gene was amplified using the TX9/1391 primers as previously described (Ashby et al., 2007 AEM 73(14):4532-4542). Amplicons were agarose gel purified and quantitated using SYBR green (Invitrogen, Carlsbad, Calif.). A second round of PCR was performed using fusion primers that incorporated the ‘A’ and ‘B’ 454 pyrosequencing adapters onto the 5′ ends of the TX9/1391 primers, respectively. The forward fusion primer also included variable length barcodes that enabled multiplexing multiple samples into a single 454 sequencing run. These amplicons were PAGE purified and quantitated prior to combining into one composite library. The resulting library was sequenced using the standard 454 Life Sciences Lib-L emulsion PCR protocol and Titanium chemistry sequencing (Margulies, M., M. Egholm, et al. 2005 “Genome sequencing in microfabricated high-density picolitre reactors.” Nature 437(7057): 376-380). Sequences that passed the instrument QC filters were also subjected to additional filters that required all bases be Q20 or higher and the average of all bases in any read to be Q25 or greater. Furthermore, the TX9 primer was trimmed off of the 5′ end and the sequences were trimmed on the 3′ end at a conserved site distal to the V6 region (ca. position 1067, E. coli numbering). The final sequences were approximately 250 bp in length and included the V5 and V6 regions (V5V6 sequences). The term “V5V6” indicates sequences that include the fifth variable (V5) and sixth variable (V6) regions of the 16S rRNA gene.
The 93 samples profiled from the Green Canyon block of the Gulf of Mexico, resulted in 5,625,371 V5V6 sequences of which 552,568 were unique. The sequences were filtered to only include unique sequences with abundance greater than 0.5% in one of the 93 samples, and those 473 V5V6 sequences were correlated with geochemical data. A total of 198 V5V6 sequences were selected for bioindicator design based on strong correlation to gasoline-range hydrocarbons.
The 198 sequences were aligned with the NAST aligner available from GREENGENES™ and analyzed with the ARB™ software package (joint initiative of the Lehrstuhl für Mikrobiologie and the Lehrstuhl für Rechnertechnik und Rechnerorganisation/Parallelrechnerarchitektur of the Technische Universität, München, Germany). The analysis found 35 groups (clades) of sequences with similarity within a group greater than 97% and 57 sequences that did not cluster and were treated separately. Bioindicator primers were designed as previously described in Example 1 to the consensus sequence of the 35 groups (Table 3), and to each of the 57 unique un-grouped sequences (Table 4) resulting in 92 bioindicator probes (PTM12 through 103, Table 5).
Genomic DNA extracted as described in “Example 1: Characterization of microbial communities associated with thermogenic hydrocarbon seeps” were further prepared as follows. A portion of the 16S rRNA gene was amplified using the TX9/1391 primers as previously described (Ashby et al., 2007 AEM 73(14):4532-4542). Amplicons were agarose gel purified and quantitated using SYBR green (Invitrogen, Carlsbad, Calif.). A second round of PCR was performed using fusion primers that incorporated the ‘A’ and ‘B’ 454 pyrosequencing adapters onto the 5′ ends of the TX9/1391 primers, respectively. The forward fusion primer also included variable length barcodes that enabled multiplexing multiple samples into a single 454 sequencing run. These amplicons were PAGE purified and quantitated prior to combining into one composite library. The resulting library was sequenced using the standard 454 Life Sciences Lib-L emulsion PCR protocol and Titanium chemistry sequencing (Margulies, M., M. Egholm, et al. 2005 “Genome sequencing in microfabricated high-density picolitre reactors.” Nature 437(7057): 376-380). Sequences that passed the instrument QC filters were also subjected to additional filters that required all bases be Q20 or higher and the average of all bases in any read to be Q25 or greater. Furthermore, the TX9 primer was trimmed off of the 5′ end and the sequences were trimmed on the 3′ end at a conserved site distal to the V6 region (ca. position 1067, E. coli numbering). The final sequences were approximately 250 bp in length and included the V5 and V6 regions (V5V6 sequences).
Regarding discovery of the consensus sequences of PTM12 through PTM103, 93 samples were profiled from the Green Canyon block of the Gulf of Mexico, and this resulted in 5,625,371 V5V6 sequences, of which 552,568 were unique. The sequences were filtered to only include unique sequences with abundance greater than 0.5% in one of the 93 samples, and those 473 V5V6 sequences were correlated with geochemical data. A total of 198 V5V6 sequences were selected for bioindicator design based on strong correlation to gasoline-range hydrocarbons.
The 198 sequences were aligned with the NAST aligner available from GREENGENES™ and analyzed with the ARB™ software package (joint initiative of the Lehrstuhl für Mikrobiologie and the Lehrstuhl für Rechnertechnik und Rechnerorganisation/Parallelrechnerarchitektur of the Technische Universität, München, Germany). The analysis found 35 groups (clades) of sequences with similarity within a group greater than 97% and 57 sequences that did not cluster and were treated separately. Bioindicator primers were designed as previously described in Example 1 to the consensus sequence of the 35 groups (Table 3), and to each of the 57 unique un-grouped sequences (Table 4) resulting in 92 bioindicator probes (PTM12 through PTM103, Table 2).
The exemplary sequences of the invention can be used individually or in groups as probes or detection molecules, or in pairs, e.g., as amplification pairs, e.g., as PCR primer pairs, to practice methods of the invention, e.g., methods of detecting the presence of a subsurface petroleum or gas accumulation or deposit, or the presence of a petroleum seep; or, methods of detecting the presence of a hydrocarbon, a petroleum or a gas accumulation, or the presence of a hydrocarbon, a petroleum or a gas pollutant.
In alternative embodiments, when sequences of the invention are used individually (or in groups), e.g., to practice methods of the invention, they can be used in hybridization reactions, e.g., in situ hybridizations, or as probes immobilized on a bead or a semisolid or solid surface, e.g., as probes immobilized on an array, a biochip, a chip, a bead, a gel, a liposome, a fiber, a film, a membrane, a metal, a resin, a polymer, a ceramic, a glass, an electrode, a microelectrode, a graphitic particle, or a microparticle or a nanoparticle. In alternative embodiments, sets of probes are used together in one detection reaction, e.g., one hybridization reaction, or immobilized individually on the same array, biochip, fiber, electrode and the like. For example, four probes, such as SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4 can be used in one detection reaction, or can be immobilized on the same array, biochip, fiber, electrode and the like. In alternative embodiments, all of the sequences (e.g., probes) of the invention are immobilized on the same product of manufacture of the invention, e.g., all can be immobilized on the same array, biochip, chip, bead, gel, liposome, fiber, film, membrane, metal, resin, polymer, ceramic, glass, electrode, microelectrode, graphitic particle, or microparticle or nanoparticle.
In alternative embodiments, sequences of the invention are used as amplification pairs, e.g., as PCR primer pairs, e.g., to practice methods of the invention. In alternative embodiments, sets of amplification (e.g., PCR) primer pairs are used together in one amplification (e.g., PCR) reaction. For example, two amplification pairs, such as SEQ ID NO: 1/2 and SEQ ID NO:3/4 can be used in one detection reaction.
In Table 2, the “PT” number references the consensus sequence from which the primer pair was derived; thus, for example, the exemplary embodiments SEQ ID NO: 1 and SEQ ID NO:2, are a sense and antisense (respectively) nucleic acid primer pair (amplification pair; primer pair sequence) that can be used to amplify, detect and/or quantify a genus of sequences based on the same consensus sequence, in this example, PTOM-03. The number after the “PTOM” designation (for example, for SEQ ID NO: 1 and SEQ ID NO:2, is 834F and 1270R) indicates the residue number of the consensus sequence the forward, or “F” amplification primer, begins (the 5′-most residue) on the sense strand (e.g., 834 for SEQ ID NO: 1), and the residue number of the consensus sequence the reverse amplification primer, or “R”, sequence begins (the 5′-most residue) on the antisense strand (e.g., 1270 for SEQ ID NO:2). To further illustrate, in Table 2: for SEQ ID NO: 1, the 834F residue is in bold (it's a “A” nucleotide) and for SEQ ID NO:2 the 1270R residue is in bold (it's a “G” nucleotide).
In practicing the methods of the invention (e.g., methods of detecting the presence of a subsurface petroleum or gas accumulation or deposit, or the presence of a petroleum seep; or, methods of detecting the presence of a hydrocarbon, a petroleum or a gas accumulation, or the presence of a hydrocarbon, a petroleum or a gas pollutant), or when using the compositions, e.g., the amplification primer pairs of the invention, in polymerase chain reaction (PCR), exemplary (alternative) conditions for PCR include: 20 sec at 94° C.; 25 sec at 63° C. and 30 sec at 72° C. In Table 2, the “TM” is the melting temperature (Tm). In alternative embodiments, Tm melting temperatures are important for determining the appropriate temperatures to use in a protocol such as an amplification reaction (e.g., PCR), or Tm melting temperatures can also be used as a proxy for equalizing the hybridization strengths of a set of molecules, e.g. the oligonucleotide probes of arrays or microarrays of the invention.
Consensus sequences 16S rRNA genes whose distribution among the 16 GOM sediment samples were found to be significantly negatively associated with the presence of hydrocarbons. The two consensus sequences were derived from the Archaeal candidate division GOM13 and the division SAGMEG-1.
In summary, the following are consensus sequences of eight (8) bioindicator sequences, e.g, gasoline-range hydrocarbon bioindicator sequences, of the invention:
Rules for Consensus Sequence:
dash (-)=>60% of sequences have gap there
Other letters (used when a few letters are each seen in >30% of sequences):
M=A or C
R=A or G
W=A or T
S=C or G
Y=C or T
K=G or T
V=A, C, or G
H=A, C, or T
D=A, G, or T
B=C, G, or T
N=G, A, T, or C
UPPER CASE=>95% of sequences are same letter
lower case=>70% of sequences are same letter
dot (.)=<50% of sequences are same letter (note: this applies to “other letters” also)
In Table 3, below, alignment of partial 16S rRNA gene sequences (V5V6 sequences), whose distributions among the samples were correlated with gasoline-range hydrocarbons. The consensus sequence of each group is included in the alignment and primers (oligonucleotides) designed to selectively amplify each group of sequences is indicated on the top line of the alignment. For ease of viewing, the reverse primer is shown as its reverse-complement.
In Table 4, below, lists unique V5V6 sequences (PTM 47 through 103) whose distributions among the samples were correlated with gasoline-range hydrocarbons. Primers (oligonucleotides) designed to amplify each sequence is indicated by bold text and shading. For ease of viewing, the reverse primer is shown not as its actual sequence (which is listed in Table 2), but as its reverse-complement. The term “V5V6” indicates sequences that include the fifth variable (V5) and sixth variable (V6) regions of the 16S rRNA gene.
In summary, PTM 47 through 103, the sequences of Table 4, are 57 sequences that did not group into “clades” having multiple species, or members (although, in one sense, that each define a “clade” but only having one member). PTM 03 to 46 have multiple members in their respective “clades”, and thus each have a true “consensus” sequence.
The methods used to design the PTM 03 to 46 clade primer/probes was different than for the PTM 46 to PTM 103 clade primer/probes. The analysis found 35 groups (clades) of sequences (clades PTM 12 to 46) with similarity within a group greater than 97% and 57 sequences (PTM 47 through 103) that did not cluster and were treated separately. Bioindicator primers were designed as described in Example 1 to the consensus sequence of the 35 groups (Table 3), and to each of the 57 unique un-grouped sequences (Table 4) resulting in 92 bioindicator probes (PTM12 through PTM103, Table 5).
GGCTTTGAGCTGTA
GCTGAGC
GTGGCTACGTGCCACT
TACGGGGGCAT
ATGGCCATGAGCCATG
AGAG
GAGCCGTA
TGGCTACGAGCCACT
AGAG
ACATTCTTTATGGGTGTC
GAATCCACGAGATTCTC
AAGTGGT
GCTTCGAGCCCAAT
GGTGGT
GGGCTCTACGAGAGCGCC
GGTGGT
AGGTCCTACGAGGGCGTC
GTGGT
GGGTCCTACGAGGGCGCC
TGGT
CCACTGCGAGTGGTAC
GGTGGT
TGCGAGTGGTGC
AGAGGTGGC
CATTACTGCGAGTGATGT
GGC
GGTGACCACGAGTTACCG
TGAGAGGAGGT
TCACACTGCGAGTATGAC
AGGT
GCATGTCCGTGAT
GGT
CAGTTTTACTGCGAGTAAAAT
AGGAGGT
ACGGTGCGACCGTGGT
GGT
ATACGGTGCGTCCGTATT
AGGT
CTCCTG
GC
GTATCGACCCCTCCA
CAGGTGGTGC
ATACCTTCTG
TGC
GGTTTAGATACCTTCTG
C
GACCCCTTCTG
GTGC
TCATAATTTGGTGCGTG
CAGGTGCTGC
CACACATATAGATCGGT
GTGATGC
TTGGGGAGGTTGCTTCTCAG
ACAGGTGCTGC
CTTCAAG
CGACCCTTCAAG
ACCCCCTCTG
GATGCTGC
ATCGACCCTCTCCG
ACCCCTCTGT
AGGTGCTGC
GACCCTCTCCG
TGCTGC
CGACCCTTTCTG
GTGCTGC
TATCGACCCTCCTCG
ACCCTCTCTG
GCTGC
GGGGAGTATCGACCCTCTTCG
TATCGACCCTCTCCG
GGTGCTGC
TTGACCCCATCC
GTGCTGC
GGTATAGATTCCCTCC
CAGGTGCTGC
CCATCA
C
TGACACCAGCA
TGCTGC
GGCATATGTCTGCGTCGGA
CTGC
AGTTATTGCTAGATCG
TTTATGCAGGAGT
GCTGC
TAAGCAGGAGTG
TGCTGC
ATATACAGTCAGCGA
CGCCCCTCA
TGGTGC
CCTCTCG
GCTGC
CTCG
TGCTGC
TATTGACCCCTGCT
GACCCCTGCT
GGAGCTTGCTCTTCGGT
GTAGGTG
GAGAC
GGTTTCCGCTTAGT
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application is a continuation of United States 371(c) patent application Ser. No. 13/696,954 filed Apr. 1, 2013 which is a National Stage Entry of International (PCT) Patent Application PCT/US2011/046015 filed Jul. 29, 2011 which claims benefit of priority to U.S. Provisional Patent Application Ser. No. 61/369,616 filed Jul. 30, 2010, which is expressly incorporated by reference herein in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61369616 | Jul 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13696954 | Apr 2013 | US |
| Child | 15158262 | US |
