Abstract:
The present invention provides for methods and systems for Electronic DNA sequencing, single molecule DNA sequencing, and combinations of the above, providing low cost and convenient sequencing.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 13/397,581, filed Feb. 15, 2012, which is a continuation-in-part of PCT International Application No. PCT/US11/54769, filed Oct. 4, 2011, which claims priority to and benefit of U.S. Provisional Patent Application No. 61/389,490, filed Oct. 4, 2010, U.S. Provisional Patent Application No. 61/389,484, filed Oct. 4, 2010, U.S. Provisional Patent Application No. 61/443,167, filed Feb. 15, 2011, and U.S. Provisional Patent Application No. 61/491,081 filed May 27, 2011, each of which is hereby incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention provides for methods and systems for Electronic DNA sequencing, single molecule DNA sequencing, and combinations of the above, providing low cost and convenient sequencing. 
     BACKGROUND OF THE INVENTION 
     Methods for quick and cost effective DNA sequencing (e.g., at high-throughput) remain an important aspect of advancing personalized medicine and diagnostic testing. Some known systems for DNA sequencing require that DNA samples be transferred between various subsystems (e.g., between the nucleic acid isolation subsystem and the amplification subsystem), thus resulting in inefficiencies and potential contamination. Some known methods for DNA sequencing employ optical detection, which can be cumbersome, expensive, and can limit throughput. Other systems utilize some forms of electronic sensing, but the sensor and sequencing flow cell are one-time use disposables, which substantially increase the cost to the user, and limits the complexity of the sensor which may be cost effectively manufactured, as it will be thrown out after a single use. Some systems utilize amplification methods within the same flow cell, in which the sequencing is performed, binding the amplified directly to the flow cell, preventing reuse. Other systems utilize emulsion PCR, wherein beads and samples are mixed into small emulsions utilizing low concentrations. Due to Poisson distribution, most of the beads and sample do not come together in an emulsion with a single bead and a single sample, and are thus lost. The cost of the beads and amplification is a substantial portion of the cost of the sequencing, and most of that cost is thrown away without ever generating any useful data. The current system enables utilization of virtually all of the sample, thus reducing the cost to the user. 
     Current DNA sequencing systems typically need whole genome amplification in order to have sufficient sample, as the sample is very inefficiently utilized. Such whole genome amplification methods typically introduce significant amounts of bias in amplification in different portions of the genome, and require higher levels of coverage to overcome said bias. Methods for localizing samples, and reagents into a volume wherein a desired reaction or binding may occur is another aspect which is envisioned for the system, which may eliminate or reduce the need for whole genome amplification, and thus reduce the coverage needed. 
     Thus, a need exists for improved systems and methods for extracting, amplifying and sequencing polynucleotides. 
     SUMMARY OF THE INVENTION 
     The embodiments described herein relate to systems and methods for sequencing polynucleotides. In some embodiments the polynucleotides to be sequenced may be individual double or single stranded polynucleotides, which may be circularized in some embodiments, as opposed to clonal polynucleotides. 
     In one aspect, the invention provides a method for conducting nucleic acid amplification and/or sequencing. The method comprises applying an electric field for confinement of a biological material to an environment, and conducting nucleic acid amplification and/or nucleic acid sequencing on the biological material. The confinement of the environment from an external environment via the electric field has the effect of isolating the biological material into a plurality of regions. The confinement creates a virtual well facilitating amplification and/or detection, and preventing contamination between virtual wells. In various embodiments, the biological material is associated with a plurality of beads, and the beads are held in place by a localized magnetic field in each of the plurality of regions. In certain embodiments, amplification within the virtual wells generates a clonal population of DNA associated with each of the beads, or on the surface of a sensor. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a schematic representation of a cross section of an under-etched NanoNeedle Sensor. 
         FIG. 2  shows a schematic representation of an isometric view of several under-etched NanoNeedle Sensors. 
         FIG. 3  shows a cross sectional view of a schematic representation of several NanoNeedle Sensors with attached polymerase and target DNA. 
         FIG. 4  is a photomicrograph of several under-etched NanoNeedle Sensors. 
         FIG. 5  is a photomicrograph of several interdigitated under-etched NanoNeedle Sensors. 
         FIG. 6  shows an array of electronic sensors with a set of electrodes used for concentration or confinement of multiple charged moieties above the sensors. 
       FIG  7 . shows magnetic or electric or electromagnetic retention of a clonal bead held in place for sequencing over a sensor. 
         FIG. 8A  depicts an image of a portion of a sequencing system that includes the nanosensors. 
         FIG. 8B  shows a schematic illustration of a nanosensor, and a series of nanosensors in electrical communication with the microfluidic channels of a sequencing system. 
         FIG. 9  shows a schematic illustration of an array of nanosensors within a sequencing system. The on-chip amplification is optional. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     As used herein, “bead” may mean beads, moieties or particles that are spherical or non-spherical, wherein said beads, moieties or particles may be porous or solid or a mixture of solid and porous, and can include magnetic beads that are may be paramagnetic, super-paramagnetic, diamagnetic, or ferromagnetic. 
     As used herein, “bead capture features” may mean features that can temporarily hold a single bead in a fixed position relative to the sensor and can include local magnetic structures on the substrate, depressions which may utilize an external magnet, local magnetic structures, Van der Waals forces, or gravity as forces that fix the position of a bead. Optionally, the bead may be bound in place using covalent or non-covalent binding. 
     As used herein, “clonal” may mean that substantially all of the populations of a bead or particle are of the same nucleic acid sequence. In some embodiments there may be two populations associated with a single sample DNA fragment, as would be desired for “mate pairs,” “paired ends”, or other similar methodologies; the populations may be present in roughly similar numbers on the bead or particle, and may be randomly distributed over the bead or particle. 
     As used herein, “confinement” may mean when a molecule generated (such as DNA) at one bead or particle stays associated with the same bead or particle so as to substantially maintain the clonal nature of the beads or particles. 
     As used herein “isolate” may mean the prevention of migration, diffusion, flow, or other movement, from one virtual well to another virtual well as necessary to maintain the clonal nature of the beads or particles. 
     As used herein, “localized magnetic feature” may mean a magnetic feature created on a substantially planar substrate to hold individual beads on said substantially planar substrate. 
     As used herein, “localized magnetic field” may mean a magnetic field that substantially exists in the volume between the north pole of a first magnetic region and the south pole of a second magnetic region or substantially exists in the volume between the north and south poles of a single magnetic region. 
     As used herein, “nanosensor” may mean a sensor designed to detect beads or particles less than one of 0.1, 1, 5, 10 or 20 micrometers as measured on the diameter or the long axis for non spherical beads or particles. Alternatively, the sensor may be sensitive to moieties associated with said beads or particles, or with reaction products or byproducts wherein the reaction includes a moiety associated with said bead or particle. Said moieties may include DNA fragments, hydrogen ions, or other ions which are counter ions and thus associated with said beads or particles or moieties bound or associated with said beads or particles. Nanosensors can include “NanoBridge, “NanoNeedle or ISFET sensors. 
     As used herein, “particle” can mean a non bead moiety such as a molecule, an aggregation of molecules, molecules bound to a solid particle, or particles, and other forms known in the art. 
     As used herein, “single phase liquid” is a liquid with relatively uniform physical properties throughout, including such properties as density, index of refraction, specific gravity, and can include aqueous, miscible aqueous and organic mixtures but does not include non miscible liquids such as oil and water. Among the physical properties not considered to potentially cause a liquid to not be considered a single phase liquid include local variations in pH, charge density, and ionic concentration or temperature. 
     As used herein, “Substantially planar” shall allow small pedestals, raised sections, holes, depressions, or asperity which does not exceed 40 μm relative to the local plane of the device. Variations due to warpage, twist, cupping or other planar distortions are not considered to constitute a portion of the permitted offset. Protrusions or depressions which are not essential for the uses as described herein but which exceed 40 μm do not preclude a device from being considered substantially planar. Fluidic channels and or structures to generate said fluidic channels which have dimensions of greater than 40 μm also do not preclude a device from being considered substantially planar. 
     As used herein, “virtual wells” refers to local electric field or local magnetic field confinement zones where the species or set of species of interest, typically DNA or beads, generally does not migrate into neighboring “virtual wells” during a period of time necessary for a desired reaction or interaction. 
     In some embodiments, a NanoNeedle, NanoBridge, ChemFET or ISFET may be fabricated such that the sensor is created on the surface of a substrate such as silicon, fused silica, glass or other similar material. In other embodiments, the sensor may be fabricated such that it projects vertically or horizontally above the substrate, such that the sensor is more accessible to the fluid and reagents. The greater accessibility to fluid and reagents may decrease the time needed for a sequencing reaction to occur, allow lower concentrations of reagents to be utilized, and increase the sensitivity of the sensor by increasing the surface area associated with the active area of the sensor. 
     As shown in  FIG. 1 , a NanoNeedle sensor structure  100  may be fabricated with a Silicon substrate  101 , and may have a 800 nm deep channel  102  etched in said substrate. A silicon oxide layer of 200 nm thickness  103  may be fabricated on the substrate, followed by a conductive p+ silicon layer of 80 nm thickness  104 , followed by a silicon oxide layer of 30 nm thickness  105 , followed by a conductive p+ silicon layer of 80 nm thickness  106 , followed by a silicon oxide layer of 20 nm thickness  107 . The channel may be created after the structure is fabricated. The structure may be generated such that an oxide layer or a resist layer covers all sections which are to be retained in the final structure. A chemical wet etch, a plasma etch, or a vapor phase etch may be utilized to remove the silicon or other similar substrate from under the structure. The conductive tip of the structure may then be exposed using an ion milling step. 
     All of the thickness may be varied, as may the materials. The channel in the substrate may alternatively be fabricated using an oxide layer, with a resist layer in the volume of the channel. The layers of Oxide and conductors may then be fabricated on top of the oxide and resist, obviating the need to under-etch the structure.  FIG. 4  Illustrates a single ended NanoNeedle array fabricated in a manner similar to that schematically depicted in  FIG. 1 . 
     As shown in  FIG. 2  such a structure may have sensors  201  on both sides of a channel  202  formed in a substrate  203 . Polymerase and or target DNA  204  may be attached to the active area of the sensor. The sensor itself may be used to electrophoretically and or dielectrophoretically localize the polymerase and or target DNA to the active area of the sensor. The target DNA may be a single double stranded, single stranded DNA target, or a circularized DNA target, or a local amplification may be done in place on the active area of the sensor, as described in PCT/US11/54769, which is hereby incorporated by reference.  FIG. 5  Illustrates an interdigitated NanoNeedle array fabricated in a manner similar to that schematically depicted in  FIG. 2 . 
     Nucleotides or probes  205  may be then be provided, and a sequencing by synthesis process, or a sequencing by ligation process may commence. 
     To improve the sensitivity of either the Nanoneedle or the Nanobridge, a local amplifier may be provided. The amplifier may be either a BJT or an FET. The sensor can be fabricated as a narrow structure, and can be etched under the structure so that both sides are accessible to changes in pH, or to changes in conductivity. The surface of the device may rough, permitting greater surface area for binding of sample molecules. The surface associated with the electrodes of a NanoNeedle may be gold or platinum, or may be platinum black, iridium oxide, or Ppy/PSS to increase the surface area and the associated double layer capacitance. 
     Electric concentration of ions may be effected, concentrating the DNA, polymerase, primers nucleotides and other reagents as needed to the active area of the NanoNeedle or Nanobridge sensor. Said concentration allow much more of the sample to be attached or associated with each sensor, mitigating the need for whole genome amplification. 
     In a further embodiment, an electronic sensing subsystem may detect a change in conductivity, either in a bulk solution, across the surface of the sensor (either from moieties bound to the sensor or from moieties within the Debye length of the surface of the sensor), across the surface of a bead or particle (either from moieties bound to the bead or particle or from moieties within the Debye length of the surface of the bead or particle), or a combination thereof In a yet further embodiment, an electronic sensing subsystem may detect a change in charge near or on the surface of the sensor, near or on the surface of a bead or particle. For example, the electronic sensing subsystem may detect charge changes within the Debye length of the surface of the sensor, or bead or particle, or of moieties bound to the surface or bead or particle. 
       FIG. 6  includes a schematic illustration of a nanosensor  604  and a series of nanosensors  610  associated with the micro fluidic channels in electrical communication with the sequencing system. The nanosensors may have clonal DNA  602  bound or associated directly thereto, and may have electrodes or magnetic elements  608  associated with each nanosensor. In other embodiments the sensor may detect changes in the charge of the clonal DNA on the bead, changes in the counter ions associated with said clonal DNA, or byproducts which result from an incorporation. The nanosensor  604  may further include a signal amplifier  606  for on-chip signal amplification. The nanosensors  604  may further include any of the known insulator materials, such as SiO 2  Al 2 O 3 , SiN, and TaO 2 . In certain embodiments, the nanosensors may comprise coaxial and/or parallel conductive layers, separated by an insulator layer. The conductive layers may be formed from any suitable material, such as gold, platinum, aluminum, carbon, or polysilicon. 
     The (magnetic) beads and DNA fragments may be conveyed into the sequencing system. As shown in  FIG. 7 , the sequencing system may include a series of nanosensors  708  in communication with the microfluidic channels defined within the sequencing system. The beads or particles  702  may be positioned over said sensors  708  by magnetic or electrode elements  710 , which may form localized magnetic fields in some embodiments and may form localized electric fields in other embodiments, wherein both the sensors  708  and magnetic elements may be configured in association with a substrate  712 . The beads or particles  702  may have clonal DNA  704  bound or associated thereto. Reagents, which may include nucleotides, primers, magnesium and polymerase  706  may then be provided to initiate a sequencing reaction. In other embodiments, when magnetic or electrode elements  710  are magnetic elements, they may be either permanent magnetic elements or electromagnetic elements. 
       FIG. 8A  shows a photomicrograph of an array of nanosensors, and a zoomed in view of a single nanosensor  802 . Impedance measurements may be used by such a nanosensor for detecting incorporated nucleotides. The impedance measurement detects the release of H+ion pyrophospate or local change in charge resulting from the polymerization reaction. Generally, the frequency of operation may be selected for maximum change in the impedance over the course of the reaction relative to the impedance at the beginning of the reaction. For example, for some geometries, the frequency may be around 0.1 to 9 KHz. In alternative geometries, the frequency may be 10 KHz or greater. In some embodiments, the nanosensor may be implemented with a single pair of electrodes with or without a pH-sensitive material (e.g., redox sensitive material) to detect the H+ion release or pH change of the reaction. The impedance measurement may be taken, as an example, by determining the current while sweeping from −A to +A volt or the reverse, with periodic sub-signals. A pulse wave with smaller amplitude than A, and a frequency of about 25 Hz or above, can be applied. A measurement of the current during a voltage sweep may indicate a change of pH in the solution proximate the nanosensor.  FIG. 8B  shows a schematic illustration of an array of said nanosensors  802 , wherein an on chip amplifier  804  may be associated with each nanosensor. 
       FIG. 9  is a schematic illustration of an array of nanosensors  902  within the sequencing system. The nanosensor may comprise two electrodes  904 , separated by a dielectric  906 . Although shown in  FIG. 9  as including an array of nanosensors, in other embodiments, the measurement can be done with a single electrode pair to detect the change of ionic construction or pH through impedance, charge, or current measurement. 
       FIG. 3  describes and illustrates a device and method whereby a single DNA molecule  307  can be sequenced by a Nanoneedle biosensor array  300 . A polymerase enzyme  306  may be attached to a sensor  301 . A DNA sample with associated primers may then be caused to enter the volume with said polymerase attached sensors, utilizing for example, pressure induced flow, electrophoretic induced flow and or migration, or similar means. A single molecule from the DNA sample  307  may then be bound by a polymerase attached to a sensor  301  in a sensor array  300 . Additional single DNA molecules  307  may also be bound by other polymerases  306  bound to sensors  301  in the sensor array  300 . 
     In one embodiment, one of the four native dNTPs  302  is then flowed into the channel volume  304  with the sensors. If the dNTP is complementary to the next base in the sample DNA  307 , it may be bound and incorporated. The Nanoneedle sensor  301  may then detect the resulting change in the local charge of the extended primer DNA, permitting detection of the incorporation event, at each appropriate position of the sensor array  300 . If the sample has more than one base in a row which is complementary to the type of dNTP  302  which has been introduced into the channel volume  304  with said sensors  301 , a second or subsequent binding and incorporation of a dNTP  302  may be detected by said Nanoneedle sensors  301 . The dNTPs  302  may then be washed out of the channel volume  304  containing the sensors  301 . 
     In some embodiments, the nucleotides may be native dNTPs. In other embodiments, the dNTPs may be modified, with charge modifying structures. The charge modifying structures may be associated, bound or conjugated to the polyphosphate, and subsequently cleaved as part of the incorporation process, obviating the need for a separate process to cleave, separate, or remove the charge modifying structure. 
     In an alternative embodiment, the charge modifying structure may be utilized as a terminator and thus be associated, bound or conjugated to the 3′ position of the sugar of the dNTP, and may thus act as a terminator. Detection may occur as a result of the process of incorporation, or may result from cleavage of the charge modifying structure. 
     In other embodiments, the charge modifying structure may be associated, bound or conjugated to the 2′ or 4′ positions of the dNTP sugar. In yet further embodiments, the charge modifying structure may be associated, bound or conjugated to the base of the nucleotide. The charge modifying structures may act as terminators, preventing the incorporation of additional dNTPs. 
     The linkage, association or conjugation may be broken as a result of a physical process, such as temperature change, or may be broken as a result of a chemical process, or may be as a result of a photochemical reaction. 
     Said linkage, association of conjugation may be broken after each nucleotide incorporation, or several nucleotides may be incorporated, and the the number of nucleotides which were incorporated may be determined as a result of measuring the amount of charge which was added as a result of said incorporation(s). 
     A different dNTP may then be flowed into the sensor array volume, permitting detection of incorporation events. Subsequent cycles of washing, introduction of each of the four dNTPs one at a time, and detection of incorporation events permit determination of the different sample DNA sequences. 
     In an alternative embodiment, non-natural dNTPs may be utilized. 
     In yet another embodiment, all four nucleotides may be present, and determination as to which nucleotide is incorporated may determined by observation of the kinetics associated with the incorporation reaction. 
     In a further embodiment, two or three nucleotides at a time may be utilized, allowing the addition of multiple bases at a time, and a correspondingly large signal. After completing the extension of the primer, with associated data collection, the extended primer may be melted off, new primer added, and the process of extension may be performed again using a different order of combinations of dNTPs. This process determines which dNTPs do not follow the completion of a previous set of dNTPs, along with information as to the length of the incorporation, wherein said length determination need not be exact. 
     Reversibly terminated dNTPs may be utilized as part of the dNTP pools. If reversibly terminated dNTPs are used, all four dNTPs may be provided, wherein one or more of the dNTPs is a reversibly terminated dNTP. 
     Reverse sequencing may be performed, wherein a polymerase with 3′ to 5′ exonuclease activity is utilized with a dNTP pool that is missing at least one dNTP. The polymerase with 3′ to 5′ exonuclease activity will remove bases back to the next dNTP in the provided dNTP pool, at which point equilibrium will be reached, and no further nucleotides will be removed. The amount of charge present on the DNA, or change in conductivity due to changes in the counter ions, or an increased quantity of hydroxide with concomitant change in pH may be measured during the reaction, or may be measured after the completion of the reaction. 
     In a further embodiment, blunt end ligation may be performed with ligands that have different binding reagents on the 3′ and 5′ ends of said ligands. The electrodes of the NanoNeedle may be conjugated with the complementary reagents for binding e.g. the 3′ end of the ligands may have a thiol group, and one electrode may be fabricated of gold, while the 5′ end of the ligands may have a PNA sequence, and second electrode may have the complement to said PNA sequence. The strand of DNA may then be electrophoretically and or dielectrophoretically concentrated to the area of the NanoNeedle, wherein said DNA strand may then bind with one end associated with one electrode, and the other end associated with the second electrode of the NanoNeedle. Polymerase and primer may provided with the DNA strand, or may be introduced later. Measurement of incorporation events may then result from direct measurement of the impedance of the DNA combined with the much larger conductivity of the counter ions associated with the DNA. 
     In an alternative embodiment, other molecules and assays may be utilized, most particularly those which allow detection of kinetics of single molecule reactions, such as other enzymatic reactions. 
     In a yet further embodiment, ligation may be utilized rather than polymerization. Four pools of probe oligos may be utilized, wherein the first base of each probe in a single probe pool is the same. The probes may be utilize a reversibly terminated tail, or may have a native tail, such that multiple ligations may occur, with concomitant increases in signal levels. In a manner similar to the use of multiple dNTPs and polymerase, more than one pool of oligos (with all probes starting with a single base) may be combined, again with concomitant increase in the number of ligations and signal levels. The second strand may be removed and a new primer introduced wherein the length of said primer may be shorter or longer than the length of the previous primers. 
     In order to permit repeated measurements of the same DNA sample, the DNA sample may be circularized, whilst the polymerase may be a strand displacing polymerase, or may be a polymerase with 5′ to 3′ exonuclease activity. Thus the DNA sample may be repeatedly sequenced by allowing the primer extension reaction to continue for many cycles completely around the circular DNA sample. In a distinct advantage over a system which utilizes detection of fluorophores, the system of the current invention can utilize the full capability of the read length of the polymerase, unhindered by having the read length reduced by phototoxicity. In some embodiments, a strand displacing enzyme may be utilized, thus generating an increase in charge and associated counter ions. In other embodiments a polymerase with 5′ to 3′ exonuclease activity may be utilized, allowing net charge to remain the same, while generating protons and or hydroxide ions, which may be measured as an increase in conductivity, or may be measured as a result of the ions interaction with the surface of an ISFET, ChemFET, or NanoBridge sensor. 
     The polymerase bound to the sensor may be a highly processive polymerase, permitting more bases to be incorporated then might occur with a less processive polymerase. The polymerase may be phi29, T4, F-530, B104, or other highly processive polymerases. The polymerase may be modified, so that it has reduced or no 3′ to 5′ exonuclease activity, or the polymerase may have no or little 3′ to 5′ exonuclease activity in its native form. Similarly, any 5′ to 3′ exonuclease activity may be modified so that it is reduced or virtually eliminated. The polymerase and or DNA may be directly bound to or near the sensor, or may be bound through a linker. 
     In some embodiments the sensor combines pH sensing with electrochemistry detection as a result of the incorporation of a reversibly reducible layer which may be fabricated above the previous sensor design. Such sensors are available from Senova Systems. During a sequencing cycle, a reducing reaction will occur if a base has been incorporated in the bead associated with a sensor. The level of reduction can be measured, and after the completion of the sequencing cycle, a voltage can be impressed on the sensor, causing an oxidation of the surface, returning it to its original state, whereupon it can be utilized for the next sequencing cycle. 
     In yet another embodiment, the attached DNA molecule may have a hairpin primer, wherein a portion of the hairpin primer has a restriction site. Subsequently, after completion of the primer extension and associated determination of the sample DNA sequence, the restriction site may be cleaved by an appropriate endonuclease enzyme or nicking enzyme, and the extended primer may be melted off by changing one of the temperature or pH of the solution in which the sample DNA is solvated. The sample may then be re-sequenced restoring the temperature or pH of the solution in which the sample DNA is solvated to the conditions appropriate for primer extension, including appropriate concentrations of nucleotides and cations. In an alternative embodiment, a strand displacing enzyme, or an enzyme with 5′ to 3′ exonuclease activity may be utilized, obviating the need to remove the second strand. 
     In a further embodiment, a linkage may be provided which may be chemically cleaved, obviating the need for enzymatic cleavage. 
     In other embodiments, a primer may be provided which has a nick site. In still further embodiments, multiple adjacent primers may be provided, obviating the need for a nicking endonuclease. The primers may be complementary to a ligated primer, or may be complementary to a targeted section of DNA. The sequencing primers may comprise all or part of primers used for clonal generation via an amplification reaction, or may comprise regions which are not used as part of the primers for amplification, or may comprise both regions used for primers in an amplification reaction, and a region which is not used for amplification reaction. 
     While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Where methods and/or schematics described indicate certain events, and/or flow patterns, and/or chemical reactions occur in a certain order, the ordering of certain events and/or flow patterns and/or chemical reactions may be modified. While the embodiments have been particularly shown and described, it will be understood that various changes in form and or detail may be made. 
     Although various embodiments have been described as having particular features and/or combinations of components, other embodiments are possible having a combination of any features and/or components as discussed above.