HK1216764B - Apparatus for high throughput sequencing of nucleic acids - Google Patents

Description

Methods for high-throughput nucleic acid sequencing

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/983,886, filed October 30, 2007, entitled “Apparatus For High Throughput Sequencing Of Nucleic Acids,” under 35 U.S.C. § 119e, the contents of which are incorporated herein by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTS MADE AS A RESULT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

not applicable

Reference to a Sequence Listing, Table or Computer Program Listing Annex Submitted on a CD-ROM

not applicable

Background of the Invention

The present invention generally relates to the field of automated optical detection of nucleic acids. The present invention generally relates to a scalable reaction and detection system for automated high-throughput sequencing of nucleic acids.

The advent of the Human Genome Project necessitated the development of improved methods for sequencing nucleic acids such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Sequencing the entire 3,000,000,000 bases of the human genome has provided the basis for identifying the genetic basis of numerous diseases. However, much work remains to identify the genetic variants associated with each disease, and the current cost of sequencing 6,000,000,000 bases for each individual (the size of a diploid human genome) remains both extremely difficult and prohibitively expensive.

With the development of DNA sequencing systems, many companies have taken on the challenge of high-throughput DNA sequencing. While such systems have reduced the cost and increased the efficiency of DNA sequencing, they are typically self-contained units with multiple interdependent components. Such single-unit sequencing systems have many limitations, including limited scalability, time lags in introducing innovation in specific components, and the direct dependence of the overall system's functionality on each component.

Flow cells for sequencing reactions and analysis are known. Examples of such flow cells include those containing any matrix used for sequencing reaction performance, such as those described in more detail herein, and those described in U.S. Patent Nos. 5,958,760, 6,403,376, 6,960,437, 7,025,935, 7,118,910, 7,220,549, 7,244,559, 7,264,929, WO 01/35088, and disclosed U.S. Patent Application No. 2007/0128610.

The present invention solves the limitations of the known prior art.

definition

To gain sufficient background on the current technology, it is helpful to understand the following technical terms.

"Amplicon" refers to the product of a polynucleotide amplification reaction, i.e., a population of polynucleotides replicated from one or more starting sequences. Amplicons can be generated by a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification, rolling circle amplification, and the like (see, e.g., U.S. Patent Nos. 4,683,195, 4,965,188, 4,683,202, 4,800159, 5,210,015, 6,174,670, 5,399,491, 6,287,824, and 5,854,033; and U.S. Publication No. 2006/0024711).

"Array" or "microarray" refers to a solid support having a surface, preferably but not exclusively a planar or substantially planar surface, which carries a collection of sites containing nucleic acids such that each site of the collection is spatially defined and does not overlap with other sites of the array; that is, the sites are spatially separated. An array or microarray may also include a non-planar interrogatable structure such as a bead or a hole having a surface. The oligonucleotides or polynucleotides of the array may be covalently bound to the solid support, or it may be non-covalently bound. Conventional microarray technology is reviewed in, for example, Schena (2000), Microarrays: A Practical Approach (IRL Press, Oxford). As used herein, a "random array" or "random microarray" is a type of microarray in which the identity of the oligonucleotides or polynucleotides cannot be distinguished based on their position, at least initially, but can be determined by specific biochemical detection techniques for the array. See, e.g., U.S. Patent Nos. 6,396,995, 6,544,732, 6,401,267, and 7,070,927; World Intellectual Property Organization Publications WO 2006/073504 and 2005/082098; U.S. Publication Nos. 2007/0207482 and 2007/0087362.

"Hybridization" refers to the process by which two single-stranded polynucleotides non-covalently associate to form a stable double-stranded polynucleotide. The term "hybridization" can also refer to triple-stranded hybridization. The resulting double-stranded polynucleotide is (typically) a "hybrid" or "duplex." "Hybridization conditions" typically include a salt concentration of less than about 1 M, more typically less than about 500 mM, and less than about 200 mM. "Hybridization buffer" refers to a buffered salt solution, such as 5× SSPE. The hybridization temperature can be as low as 5°C, but is typically above 22°C, more typically above about 30°C, and preferably above about 37°C. Hybridization is typically performed under stringent conditions—conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer fragments may require higher hybridization temperatures to achieve specific hybridization. Because other factors (including the base composition and length of the complementary strands, the presence of organic solvents, and the extent of base mismatching) can affect the stringency of hybridization, the combination of parameters is more important than the absolute measurement of any one parameter alone. Stringent conditions are generally selected to be about 5°C lower than the Tm for the specific sequence at a defined ionic strength and pH. Exemplary stringent conditions include a salt concentration of at least 0.01 M and no more than 1 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of at least 25°C. For example, 5× SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-specific probe hybridization. For more information on stringent conditions, see, e.g., Sambrook, Fritsche, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), and Anderson, Nucleic Acid Hybridization, 1st ed., BIOS Scientific Publishers Limited (1999).

"Hybridizing specifically to" or "specifically hybridizing to" or similar expressions refers to the binding, duplexing or hybridization of a molecule substantially or only to one or more specific nucleotide sequences when such sequences are present in a complex mixture (e.g., total cellular DNA or RNA) under stringent conditions.

"Ligation" means the formation of a covalent bond or linkage between the termini of two or more nucleic acids (e.g., oligonucleotides and/or polynucleotides) in a template-driven reaction. The nature of the bond or linkage can vary widely, and the ligation can be enzymatic or chemical. As used herein, ligation is generally performed enzymatically to form a phosphodiester linkage between the 5' carbon of the terminal nucleotide of one oligonucleotide and the 3' carbon of the other oligonucleotide. A variety of template-driven ligation reactions are described in the following references: Whitely et al., U.S. Patent 4,883,750; Letsinger et al., U.S. Patent 5,476,930; Fung et al., U.S. Patent 5,593,826; Kool, U.S. Patent 5,426,180; Landegren et al., U.S. Patent 5,871,921; Xu and Kool, Nucleic Acids Research, 27:875-881 (1999); Higgins et al., Methods in Enzymology, 68:50-71 (1979); Engler et al., The Enzymes, 15:3-29 (1982); and Namsaraev, U.S. Patent Publication 2004/0110213. Enzymatic ligation typically occurs in a ligase buffer, which is a buffered salt solution containing any divalent cations, cofactors, etc. required for the particular ligase employed.

"Mismatch" refers to a base pair between any two bases other than the Watson-Crick base pairs G-C and A-T, namely A, T (or U in RNA), G, and C. The eight possible mismatches are A-A, T-T, G-G, C-C, T-G, C-A, T-C, and A-G.

"Polymerase chain reaction" or "PCR" refers to a reaction for in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary strands of DNA. In other words, PCR refers to a reaction for preparing multiple copies or replicas of a target nucleic acid flanked by primer binding sites, such a reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing a primer to the primer binding site, and (iii) extending the primer by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled in a thermal cycler through different temperatures optimized for each reaction condition. The specific temperature, duration, and rate of change between reactions depend on many factors well known to those skilled in the art, as exemplified by references such as McPherson et al., eds., PCR: A Practical Approach and PCR 2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in conventional PCR using Taq DNA polymerase, the double-stranded target nucleic acid can be denatured at a temperature >90°C, the primers can be annealed at a temperature in the range of 50-75°C, and the primers can be extended at a temperature in the range of 72-78°C. As described above, the term "PCR" encompasses derivative forms of this reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, and the like. Reaction volumes range from several hundred nanoliters (e.g., 200 nL) to several hundred microliters (e.g., 200 μL).

"Nucleic acid" and "oligonucleotide" are used herein to refer to polymers of nucleotide monomers. As used herein, the term may also refer to double-stranded forms. The monomers that make up nucleic acids and oligonucleotides can specifically bind to natural polynucleotides via regular patterns of monomer-monomer interactions, such as Watson-Crick type base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, and the like, to form duplex or triplex forms. Such monomers and their internucleoside linkages may be naturally occurring, or may be analogs thereof, such as naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs may include peptide nucleic acids, locked nucleic acids, phosphorothioate internucleosidic linkages, bases containing linking groups that allow attachment of labels such as fluorophores or haptens, and the like. Whenever the use of an oligonucleotide or nucleic acid requires enzymatic processing, such as extension using a polymerase, ligation using a ligase, and the like, it will be understood by those skilled in the art that the oligonucleotide or nucleic acid in those cases will not contain certain analogs of internucleoside linkages, sugar moieties, or bases at any or certain positions (when such analogs are incompatible with enzymatic reactions). Nucleic acids typically range in size from a few monomeric units (e.g., 5-40, in which case they are generally referred to as "oligonucleotides") to hundreds of thousands or more monomeric units. Whenever a nucleic acid or oligonucleotide is represented by a sequence of (uppercase or lowercase) letters, such as "ATGCCTG," it is understood that the nucleotides are in 5'→3' order from left to right, and that "A" represents deoxyadenylic acid, "C" represents deoxycytosine, "G" represents deoxyguanosine, and "T" represents deoxythymine, "I" represents deoxyinosine, and "U" represents uracil, unless otherwise indicated or apparent from the context. Unless otherwise indicated, terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Nucleic acids typically comprise natural nucleosides (e.g., deoxyadenylic acid, deoxycytosine, deoxyguanosine, deoxythymidine for DNA, or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleoside analogs, such as modified bases, sugars, or internucleoside linkages. For those skilled in the art, if the activity of the enzyme has a specific oligonucleotide or nucleic acid substrate requirement, such as single-stranded DNA, RNA/DNA duplexes, etc., then the selection of the appropriate composition of the oligonucleotide or nucleic acid substrate is well within the knowledge of those skilled in the art, especially guidance from monographs such as Sambrook et al., Molecular Cloning, 2nd Edition (Cold Spring Harbor Laboratory, New York, 1989) and similar references.

"Primer" refers to an oligonucleotide, either natural or synthetic, that can serve as a starting point for nucleic acid synthesis when forming a duplex with a polynucleotide template and that extends from its 3' end along the template to form an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers are typically extended by a DNA polymerase. Primers typically range in length from 9 to 40 nucleotides, or in some embodiments, from 14 to 36 nucleotides.

"Probe" as used herein refers to an oligonucleotide, either natural or synthetic, that is used to query a nucleic acid of unknown sequence for a complementary sequence. Hybridization of a specific probe to a target polynucleotide indicates a specific sequence within the target polynucleotide sequence that is complementary to the probe.

"Readout" refers to one or more parameters of a measurement and/or detection that can be expressed as a number, numerical value, or other indicia for evaluation. In some contexts, a readout can refer to the actual digital representation of the data so collected or recorded. For example, a readout of a fluorescence intensity signal from a microarray refers to the location and fluorescence intensity of the signal generated at each hybridization site on the microarray; thus, such a readout can be recorded or stored in a variety of ways, such as an image of the microarray, a numerical table, and the like.

"Solid support" and "support" are used interchangeably to refer to a substance or group of substances having one or more rigid or semi-rigid surfaces. A microarray typically comprises at least one planar solid support, such as a microscope slide.

As used herein, the term "Tm" means "melting temperature." This melting temperature refers to the temperature at which half of a population of double-stranded nucleic acid molecules dissociates into single strands. Several equations for calculating the Tm of nucleic acids are known in the art. As shown in standard references, a simple estimate of the Tm value can be calculated using the following equation: Tm = 81.5 + 0.41 (% G + C), when the nucleic acid is present in a 1 M NaCl aqueous solution (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawi, H.T. & SantaLucia, J., Jr., Biochemistry 36:10581-94 (1997)) include alternative calculation methods that take into account structural and environmental as well as sequence characteristics when calculating Tm.

By way of explanation, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

When a range of values is provided, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly dictates otherwise, to the nearest tenth of the unit of the lower limit) and any other indicated or intermediate values within the range are encompassed herein. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed herein, subject to any specifically excluded limits in the range. When the range includes one or both of two limits, ranges excluding one or both of those included limits are also encompassed herein.

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art are not described to avoid obscuring the present invention.

In general, unless otherwise indicated, the molecular biology and sequencing analyses involved in the present invention are, in their basic aspects, routine methods within the skill of those skilled in the relevant art. These techniques are fully described in the literature, see, for example, Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982) and Sambrook, Russell & Sambrook, Molecular Cloning: A Laboratory Manual (2001). The nucleic acid chemistry, biochemistry, genetics and molecular biology terminology and notation used herein follows standard monographs and texts in the field, e.g., Kornberg and Baker, DNA Replication, 2nd ed. (W. H. Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984), etc.

SUMMARY OF THE INVENTION

According to the present invention, a system for performing nucleic acid sequencing for genomic analysis includes, for example, separate functional units for sample preparation and for observation, and the system can be configured in a manner that allows different sequencing reaction components to be used interchangeably with separate equipment components for optical image acquisition and/or analysis to minimize obstacles in sample preparation and data extraction running at different speeds.

The invention provides a high-throughput system for the sequence determination of unknown sequence nucleic acid. System according to the present invention comprises a plurality of assemblies based on purpose, separation, and said assembly is physically loosely coupled (coupled) in such a system, and is reversibly integrated, for sequence query and analysis. The loose coupling and reversible integration properties of this system provide higher efficiency and versatility in the use of multiple system components, thereby allowing the optimization of the system based on time requirement and each component capacity. This allows improved scalability, increases the ease of improvement to the system, and than the currently available fully integrated system of this area, has the creation of a plurality of system configurations of enhanced user flexibility.

Having system components loosely coupled and reversibly integrated provides many benefits, including facilitating any repairs that need to be made to a single component of the system without disrupting other components of the overall system. Furthermore, the strategy of coupling independent system components facilitates the introduction of any improvements to a single component, thereby promoting the use of new innovations and providing the latest technological innovations to the overall system.

In certain embodiments of the present invention, higher throughput can be achieved by using multiple components for each of the activities required to perform nucleic acid sequencing. For example, using multiple optical detection instruments and/or multiple sequencing reaction components can greatly increase the number of sequences determined and reduce the time required to do so.

In one embodiment, a single reaction device and a single optical detection and analysis instrument for sequencing are provided, wherein the reaction device and the optical instrument are physically loosely coupled and reversibly integrated.

In another embodiment, multiple biochemical components and a single optical detection instrument are provided for different sequencing reaction components, such as components directed to sequencing by synthesis and components directed to sequencing by probe connection. The sequencing reaction components of such a system can be maintained as separate units, each unit physically reversibly interconnected with the optical imaging system. This allows a single system to utilize different sequencing technologies and benefit from the advantages of multiple different sequencing methods in a single device configuration. The optical instrument can be provided in a single system with an analytical component, or they can be configured as two separate components of the entire system.

In a specific embodiment, the system may include three compartmentalized components: (i) a fluidics system for storing and transferring detection and processing reagents, such as probes, wash solutions, etc.; (ii) a reaction platform for performing biochemical sequencing reactions in a series of reaction chambers or in one or more flow cells; and (iii) separate illumination and detection systems for capturing optical images of the sequencing reactions and analysis of these images.

The reaction platform for biochemical sequencing reactions preferably has a plurality of reaction units including individual flow cells and a mechanism for transferring each flow cell from the reaction apparatus to an illumination and detection system after the biochemical sequencing reaction is completed.

In a preferred aspect of various embodiments, the flow cell comprises an array of nucleic acids of unknown sequence attached to a solid surface (e.g., a glass slide or a flexible material such as a film or membrane). In another embodiment, each flow cell comprises an array of nucleic acids of unknown sequence attached to beads, which are optionally attached to a solid or semi-solid surface.

In one aspect of an embodiment of the present invention, the sequencing reaction component of the system provides a plurality of flow cells for processing samples. In a preferred aspect, each flow cell comprises a substantially sealed chamber having a fluid inlet and a fluid outlet for respectively introducing and removing fluids used in the sequencing reaction.

In a specific embodiment, two or more sequencing reaction platforms can be interconnected with a single optical imaging system, and the imaging system can record and analyze the individual sequencing information from each reaction unit. In a specific aspect, each reaction unit and flow cell on multiple reaction platforms is designed to implement the same high-throughput nucleic acid sequencing biochemistry on multiple flow cells. On the other hand, different reaction platforms and flow cells are designed to make different biochemical methods suitable for high-throughput nucleic acid sequencing, and each reaction platform is optimized to implement a specific flow cell sequencing reaction. The ability to reversibly connect the optimized sequencing reaction platform and flow cell biochemical reaction unit (each designed to supply the specific biochemistry of the sequencing method) to a single irradiation and analysis system provides the optimal utilization of space and run time, and is more cost-effective than having a separate complete system for each potential biochemical sequencing application.

In particular aspects of certain embodiments, the inner surface of each flow cell is defined in part by the surface of the support carrying the sample, an arrangement which has the advantage of minimizing the number of components involved in assembly of the flow cell.

In a specific embodiment, the flow cells of a particular sequencing reaction unit each contain an array of target nucleic acids of unknown sequence by sandwiching glass and a gasket between two solid planar surfaces. One surface has an opening of sufficient size to allow imaging and an indexing pocket for a coverslip. The other surface has an indexing pocket for the gasket, a fluidic interface, and an optional temperature control system.

In one specific aspect of the invention, a flow cell designed for use with a sequencing reaction unit comprises a 1" square, 170 mm thick coverslip. In a preferred embodiment, the flow cell has a surface that has been derivatized to bind to a large number of biological structures of unknown sequence for high-throughput, genome-scale sequencing.

In certain specific aspects of the invention, the flow cell can include a fluidic interface connected to a device capable of effecting the entry or exit of fluid from the flow cell, such as a syringe pump.

In another specific aspect of the present invention, the flow cell includes an interface connected to a mixing chamber, which is optionally equipped with a liquid level sensor. Solutions required for the sequencing reaction are dispensed into the chamber, mixed as needed, and then drawn into the flow cell. In a preferred aspect, the chamber is conical in nature and acts as a funnel. In certain aspects of embodiments of the present invention, each flow cell includes a temperature control subsystem having the ability to maintain a temperature within a range of about 5-95°C, or more particularly 10-85°C, and can change the temperature at a rate of about 0.5-2°C per second.

In yet another aspect of certain embodiments of the present invention, the system further provides an automated apparatus for processing samples, particularly biological samples, supported on a support, the apparatus comprising: a support gripping device for gripping one or more supports, the sample on the or each support being present within a respective substantially sealed chamber; a fluid delivery device for delivering a processing fluid to the or each chamber; a waste collection device for removing fluid from the or each chamber; and computer control means for monitoring sequencing reactions. The apparatus is preferably used in conjunction with one or more flow cells as defined above.

The present invention will be better understood by those skilled in the art upon reading the process details as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the basic format of the sequencing reaction platform of the present invention.

FIG2 is a diagram illustrating a first embodiment of a system including a sequencing reaction platform and an optical imaging device.

FIG3 is a diagram illustrating a second embodiment of a system including a sequencing reaction platform and an optical imaging device.

FIG4 is a diagram showing a third embodiment of a system including a sequencing reaction platform including a telescopic arm and an optical imaging device.

FIG5 is a diagram illustrating a system including sequencing reaction platforms configured in parallel.

FIG6 is a diagram showing details of a system according to the present invention.

Detailed Description of the Invention

FIG1 shows a schematic side view of an exemplary sequencing reaction platform having a reaction workspace, with a longitudinal dimension of X, a width dimension of Y, and a height dimension of Z.

Figure 2 shows a schematic top view of a first sequencing reaction platform according to a preferred aspect of an embodiment of a sequencing system of the present invention. A platform of this nature is also disclosed in U.S. Patent No. 7,264,432. The reaction platform 3 comprises a flow cell 2 placed on separate solid supports 2' and located on at least one substantially horizontal table 4 having a longitudinal dimension X and a width dimension Y. The reaction platform 3 includes at least one track 5 extending parallel to the X-direction and at least one displacement unit 6 having a transport device 9 that can move along the track 5 with the displacement unit to transfer the target in the X-direction. The transport device 9 is implemented here as a carrier plate 11 (which can move along the track 5 with the displacement unit 6) and a motorized gripping mechanism 8 for gripping and moving each separate support 2 to a connected optical characterization tool 7. Using the gripping mechanism 8, the support 2' and flow cell 2 are dragged onto the carrier plate 11 and transferred along the X-direction to the observation tool 7, i.e., the imaging device, using the transport device 9.

The received separated support 2' with the flow cell 2 can be assigned to its initial position on the work area 4 according to the position X of the carrier plate 11. This detection of the position X of the carrier plate 11 and the movement path of the gripping mechanism 8 for gripping the object (initial position Y of the object) is performed by suitable sensors for detecting linear movement (not shown), which are known to those skilled in the art from the relevant technical field. The processing of the information from these sensors, the drive control for the movement of the carrier plate 11 in the X direction and the gripping mechanism 8 in the Y direction, and the assignment of the information of the initial position X/Y of the object are preferably performed by a suitable programmable controller implemented by a digital computer (not shown), which is also an integrated part of the present system.

Since all samples contained in a flow cell will be variable to some extent in the sequencing of unknown nucleic acids, identification of all flow cell supports 2 across the entire platform 3 is desirable and beneficial. This may also be important for tracking individual sequences across a series of flow cells using software applications. The defined position and orientation of the flow cells on the reaction platform allows identification of each set of sequenced samples and, consequently, sample tracking for later cross-verification and assembly purposes.

In specific aspects of these embodiments, the flow cell 2 and the support 2' are formed as a single integrated structure. In the specific embodiment illustrated in FIG3 , the system 3 further provides a characterization tool 12, such as a barcode reader. The characterization tool can read the identification element of one or more supports 2' and determine the identity of the sample in the corresponding flow cell. This identification is preferably performed while the support 2' is dragged onto the carrier plate 11 of the carrier device 9.

Figure 4 illustrates a support 2' comprising a flow cell 2 that is transferred in the Z direction to an imaging system that is not in the same plane as the reaction platform 3. The clamping mechanism comprising an element 8 at one position and an element 8' at another position is implemented here as a telescopic arm; as an alternative, it can also be implemented as an articulated arm. The carrier device 9 can be rotated at an angle, preferably +180° and/or -180°, with reference to the Z axis perpendicular to the horizontal working field 4. A further optional embodiment of the clamping mechanism (not shown) includes a track running in the Y direction with a crawler, for example, which can be raised and/or lowered to grab and/or display the carrier. When using this carrier device 9, the support 2' comprising the flow cell 2 can be transferred in the X direction and then displayed by the clamping mechanism 8 at a position within the field of view of the observation tool 7, which is different from the initial position of the target object in the working area 4, which is obviously the area where chemistry is performed before observation. At the same time, when the clamping mechanism 8 is removed, preferably the identity of the sample and/or object is detected again and the new X/Y position of the flow cell 2 and support 2' is stored in an associated computer component of the system.

From the foregoing description, it can be seen that the support 2' can not only be grasped, transferred in a plane, and repositioned using the gripping mechanism 8', but the support 2' can also be transferred from one plane to a plane above or below it in the Z direction and positioned there for further analysis using the illumination, detection, and analysis components of the system of the present invention. When these transfer tasks are performed, it is beneficial, but not absolutely necessary, for each target object to be identified or characterized using the characterization tool 12 (Figure 3).

More than two working platforms can be combined into a high-level system, as shown in Figure 5. The working areas 4, 4' can be placed parallel to each other, end to end in series or overlapped, and can be rotated at any angle in the horizontal plane (not shown).

One aspect of the present invention supports automated sequencing of reaction components in a timely and efficient manner. This process can involve multiple sequencing reaction system components optimized for biochemical interrogation of nucleic acids of unknown sequence. Various biochemical sequencing reactions can be adapted for use with the system of the present invention, including but not limited to hybridization-based methods such as those disclosed in U.S. Patent Nos. 6,864,052, 6,309,824, and 6,401,267 and U.S. Patent Publication No. 2005/0191656; hybridization-based methods such as those disclosed in U.S. Patent Nos. 6,210,891, 6,828,100, 6,833,246, 6,911,345; the article by Ronaghi et al. (1998), Science, 281:363-365; and the sequencing by synthesis method disclosed in Li et al. Proc. Natl. Acad. Sci., 100:414-419 (2003); and the ligation-based method disclosed in, for example, international patent applications WO1999019341, WO2005082098, WO2006073504 and the article by Shendure et al. (2005), Science, 309:1728-1739.

In certain embodiments, the sequencing reaction component of the system includes one or more flow cells 2 (e.g., reaction chambers) ( FIG. 6 ), in which the actual biochemical sequencing reaction occurs. In a preferred embodiment of the invention, the flow cells 2 of the sequencing reaction component of the system include chambers within a support structure 2′, such as optical microscope slides 20, 22, separated by spacer members 23, 24, 26, 28 inserted into the flow cell chamber 2. Each chamber has an inlet 30, an outlet 32, and a surface having exemplary regions 34, 36 that have been fabricated or treated to allow nucleic acids to adhere thereto when injected through the inlet 30 by liquid transport. The flow cell optionally includes nucleic acids or primers attached to the surface regions 34, 36 of the flow cell, either as a random array or in a predetermined array of micro-sites, so that the identity of each nucleic acid can be monitored as the reaction progresses. Nucleic acids or primers may be attached to the surface such that at least a portion of the nucleic acids or primers are individually optically soluble when viewed through the walls of the support structure 2'.

In a preferred embodiment, flow cell 2 comprises the solid support or at least support (backing), airtight chamber that has the unknown sequence nucleic acid fixed thereon.Flow cell 2 is preferably connected with support locking piece (desk or box), to place solid support or support in this system sequencing reaction assembly.Flow cell 2 can be arranged for example side by side or one in front and one behind on sequencing reaction system assembly. When solid support 2 ' comprises/is microscope slide 22, support locking piece can have such size usually, and promptly it is applicable to conventional size slide (for example, being generally about 25.4mm and taking advantage of 76.2mm slide). When support is film, the size of locking piece will be similar to such size, and promptly it is applicable to conventional size (usually 80mm and taking advantage of 120mm) film, although film is much more variable in size than slide.

The structural aspects of the flow cell are typically held together by an adhesive (associated with the spacer elements 23, 24, 26, 28) or by clamping devices 40, 42. In certain aspects of embodiments of the present invention, the clamping devices 40, 42 can clamp portions of multiple flow cells together. Typically, from one to about twelve or sixteen flow cells can be clamped simultaneously by a single clamping device. The flow cells can be arranged with the clamping devices in a substantially horizontal or substantially vertical manner, although any intermediate position between these two positions is possible.

Alternatively, or in addition to clamping, the flow cell may have a biasing structure that connects the flow cell assembly. The biasing structure may include one or more spring biasing members 46, 48, 50, 52. In a particular embodiment, the holder is attached to the clamping member by a spring-loaded mounting pin, so that formation of the flow cell compresses the spring of the spring-loaded mounting pin, which thereby connects the flow cell assembly.

In other specific aspects of embodiments of the present invention, the force applied to the flow cell structure by the clamping device and/or the biasing device helps to ensure a liquid-tight seal between the support and the support locking member.

In certain aspects, it is generally preferred that the flow cell further include a sealing device to assist in the formation of a substantially closed chamber. The sealing device can be an integral part of the support locking member, or can be provided as a separate component of the flow cell. The sealing device typically includes a gasket, which can be made of silicone rubber or other suitable material. In one embodiment, the sealing device includes an O-ring gasket, which is typically in the shape of a frame edge located in a groove in a portion of the support locking member. In an optional embodiment, the sealing device includes a flat frame edge gasket (about 100 to 150 μm thick). In other specific aspects, the gasket or other spacer material can be adhered with an adhesive.

Gasket types can either be discarded after a single use (if, for example, contaminated with a radioactive probe) or reused when needed. Flat gasket embodiments are particularly suitable for use as disposable gaskets that are discarded after a single use. Obviously, the thickness of the gasket (which can be easily changed by exchanging gaskets) can partially determine the volume of the substantially enclosed chamber.

In another aspect of the invention, when small quantities are used in sequencing reactions, flow cell components are directly connected through the use of adhesives. The adhesive is preferably incorporated into a surface that provides optimal adhesion between the various flow cell components (e.g., a slide comprising an array and a coverslip).

The fluid inlet 30 allows the introduction of the desired fluid into the substantially enclosed chamber to process the sample on the support. Typically, such fluids are buffers, solvents (e.g., ethanol/methanol, xylene), reagents (e.g., solutions containing primers or probes), etc. The fluid outlet allows the processing fluid to be removed from the sample (e.g., for washing, or to allow subsequent addition of reagents). Preferably, when the support is being processed, their orientation is such that the fluid inlet is located at the bottom of the substantially enclosed chamber and the fluid outlet is located at the top of the substantially enclosed chamber.

Typically, in the case where the nucleic acid sample is supported on a glass slide 22, the substantially enclosed chamber has a volume of 50 μl to 300 μl, preferably 100-150 μl. Such a small volume allows for economical use of reagents and (where temperature regulation is involved) a fast thermal response time. In the case where the sample is supported on a membrane, the chamber will typically be larger (up to 2-3 mls).

In certain aspects, the flow cell 2 can be configured to amplify a sample attached to a support (e.g., rolling amplification or polymerase chain reaction amplification). In such an embodiment, the flow cell must have an opening to allow for the addition of further reagents. The opening must be designed so that it is transient, and the flow of any new liquid must be very tightly controlled to prevent any leakage from the flow cell and to prevent contamination of the flow cell when any new reagents are added.

In certain aspects of certain embodiments, such as those related to PCR or other reactions requiring tightly controlled temperature regulation, the flow cell is equipped with a temperature control device to allow for rapid heating and cooling (e.g., thermal cycling) of the sample and PCR mixture. Typically, the flow cell is equipped with an electrical heating element or a Peltier device. The flow cell can also be configured (e.g., by providing a cooling device) to provide improved air cooling. Temperature control within the range of 3°C to 105°C is sufficient for most applications.

Can relate to many arrangements for suitable fluid transfer devices. In a preferred embodiment, many reservoirs of treatment fluids (such as buffer, staining agent, etc.) are provided, and each reservoir is connected to an aspiration mechanism. Preferred aspiration mechanisms include but are not limited to syringe pumps 60, such as those manufactured by HooK and Tucker (Croydon, Surrey, Britain), or Kloen with a stroke volume of 1mL to 10mL. Such a pump 60 can be provided for each treatment fluid reservoir, or a single pump can be provided to utilize a multi-port valve layout to aspirate fluid from each of a plurality of reservoirs to a plurality of injection needles 62, 64, 66, 68 that can be arranged with inlet 30.

Each syringe pump 60 can on the contrary be connected to central manifold (central manifold) 70 (such as universal connector) such as by universal connector.Preferably, central manifold 70 leads to selectivity multi-outlet valve 72, so that when needed, when a plurality of samples are just being processed simultaneously, each sample can be processed with different treatment fluids or the combination of treatment fluids.Suitable selectivity multi-outlet valve is a rotary valve, such as the 10 outlet rotary valves supplied by Omnifit (Cambridge, Britain).Thus, each outlet from multi-outlet valve 72 can be connected to an independent flow cell.One or more filters can be incorporated into when needed.Usually, the filter is located between each reservoir and its associated syringe pump.

Each syringe pump 60 may be driven individually by computer control, or two or more pumps may be driven simultaneously to provide two or more treatment fluid mixtures. Controlling the operating rate of each pump 60 will thereby control the composition of the resulting treatment fluid mixture.

In alternative embodiments, the fluid delivery device comprises two or more piston/HPLC type pumps, and each pump is supplied by multiple treatment fluid reservoirs through multi-inlet valves. Suitable pumps can be obtained from, for example, Anachem (Luton, Bed, Britain). The multi-inlet valve will be a rotary valve. Each pump will lead to a rotary mixer for those skilled in the art, thereby allowing the variable composition mixture to be produced for treatment fluids when needed.

In certain aspects, the treatment fluid or treatment fluid mixture then passes through an inline filter and then through a selective multi-valve outlet (such as a rotary valve) before being injected into the flow cell.

As an alternative to the usual "parallel" supply of treatment fluids defined above, the treatment fluids may be supplied "serially" so that, for example, liquid passes from one substantially closed chamber to another. This embodiment has the advantage of minimizing the amount of reagents required.

In aspects of the present invention comprising one or more valves, typically, the valve will be a three-way valve having two inlets and an outlet leading to a substantially enclosed flow cell. One of the valve inlets is indirectly supplied by a treatment fluid reservoir. The second inlet is supplied by a local reservoir, typically a syringe, pipette or micropipette (generally 100-5000 μl volume). The local reservoir can be controlled by a computer control device or can be manually controlled. Local reservoirs are typically used in situations where reagents are rare or expensive. The provision of such a local reservoir minimizes the amount of reagents required, simplifies cleaning, and provides additional flexibility so that each flow cell can be handled individually when needed.

In specific aspects of certain embodiments of the present invention, "flow" for flow cell reactions is achieved by gravity, such as by placing the flow cell at an angle, or by the use of an adsorbent material applied to the flow cell outlet 32. In other aspects of the embodiments, flow is generated using either mechanical or electrical means, such as by introducing a vacuum device into the outlet edge of the flow cell. The flow cell in such embodiments can be substantially closed or can have both an inlet and an outlet available to transfer fluid through the flow cell.

In another specific aspect of an embodiment of the present invention, fluid enters the flow cell at the bottom, moves upward and flows out of the flow cell through a fluid outlet at the top. However, in a preferred aspect, fluid enters the flow cell from the top and is carried through the reaction by gravity, thereby flowing out of the flow cell through a fluid outlet at the bottom. The fluid outlet can drain into a common collection pipe, and the pipe drains into a collection container. Desirably, the container is removable from the equipment to allow regular emptying and/or cleaning.

According to the present invention, to accommodate a variety of incompatible reaction rates and volumes of material to be processed, the sequencing reaction assembly is substantially modular, such that, if a large number of flow cells and/or supported samples need to be processed, additional components can be easily added to the existing equipment. In such an embodiment, the observation assembly and sequencing reaction assembly of the system can preferably accept modular flow cell arrays, whether the samples are supported on slides or membranes.

The reversible integration of the sequencing reaction assembly into the system can include a connection to a computer control device that can coordinate the various activities of the functional components of the system. The computer control device can optionally control two or more of the following parameters: the selection of which pump(s) to actuate; the absolute volume and flow rate of the processing fluid through the activated pump(s); the selection of which flow cell to supply the processing fluid; the temperature of the sample supported within the device; the movement of the flow cell from the sequencing reaction device of the system to the imaging assembly; and the timing of various events.

The present invention also relates to the manufacture of the flow cell and/or device of the present invention and the use of the flow cell and/or device of the present invention in processing samples on a support, so that the present invention provides: a method for processing samples on a support using the flow cell and/or automated sequencing reaction device defined above; a method for preparing a flow cell; and a method for preparing a loosely coupled, reversibly integrated system comprising sequencing reaction components according to the present invention.

The present invention provides a detection component for the identification of sequencing reaction component results of the system of the present invention. The detection system for the signal can depend on the label portion used, and the label portion can be defined by existing chemistry. Any detection method suitable for the type of label used and that can be used in the detection component of the system of the present invention can be used. Therefore, exemplary detection methods include radioactivity detection, light absorption detection (such as ultraviolet-visible light absorption detection), light emission detection (such as fluorescence or chemical irradiation). Optical settings include near-field scanning microscopy, far-field confocal microscopy, wide-field surface illumination, light scattering, dark field microscopy, light conversion, single-photon and/or multi-photon excitation, spectral wavelength recognition, fluorophore identification, evanescent wave illumination and total internal reflection fluorescence (TIRF) microscopy.

The nucleic acid molecules of labeling can be detected on the substrate by scanning all or part of each substrate synchronously or sequentially (depending on the scanning method used). For fluorescent labeling, the selected area on the substrate can be sequentially scanned one by one or line by line with fluorescence microscopy equipment, such as Fodor (U.S. Patent number 5,445,934) and Mathies et al. (U.S. Patent number 5,091,652). Guidance can be found in the literature on the application of such techniques for analyzing and detecting nanoscale structures on surfaces, as shown by the following references: Reimer et al., eds., Scanning Electron Microscopy: Physics of Image Formation and Microanalysis, 2nd ed. (Springer, 1998); Nie et al., Anal. Chem., 78: 1528-1534 (2006); Hecht et al., Journal Chemical Physics, 112: 7761-7774 (2000); Zhu et al., eds., Near-Field Optics: Principles and Applications (World Scientific Publishing, Singapore, 1999); Drmanac, International Patent Publication WO 2004/076683; Lehr et al., Anal. Chem., 75: 2414-2420 (2003); Neuschafer et al., Biosensors & Bioelectronics, 18: 489-497 (2003); Neuschafer et al., US Patent 6,289,144, etc.

A specific imaging technique used in the present invention is total internal reflection fluorescence (TIRF) microscopy, which can be used to visualize single fluorophores (dNTPs labeled with Cy-3 or Cy-5). TIRF microscopy uses total internal reflection excitation light, and detection is generally performed using evanescent wave illumination and TIRF microscopy. An evanescent light field can be established on the surface to image, for example, fluorescently labeled nucleic acid molecules. When a laser beam is totally reflected at the interface between a liquid and a solid matrix (such as glass), the excitation beam only penetrates a short distance of the liquid. In other words, the light field does not end abruptly at the reflective interface, but its intensity decreases exponentially with distance. This surface electromagnetic field is called an "evanescent wave" and can selectively excite fluorescent molecules near the interface in the liquid. The weak evanescent light field at the interface provides low background and facilitates visible wavelength detection of single molecules with a high signal-to-noise ratio. Examples of this technique are disclosed by Neuschafer et al., US Patent 6,289,144; Lehr et al. (cited above); and Drmanac, International Patent Publication No. WO 2004/076683.

EPI-fluorescence illumination can also be employed in the detection assembly of the present invention.EPI-fluorescence microscopy is a technique that involves staining with special types of tissue stains called fluorochromes that are incorporated upon hybridization to fluorescently labeled complementary DNA sequences.

Both TIRF and EPI illumination allow the use of virtually any light source. The light source can be raster, expanded beam, coherent, incoherent, and derived from a single or multispectral source. In a specific aspect of the embodiment, imaging can be performed using a Zeiss confocal microscope 200 (Zeiss Axiovert 200) with a 100× objective lens and a 1.3 megapixel Hamamatsu-er-ag or similar system components using TIRF or EPI illumination.

Fluorescence resonance energy transfer (FRET) can also be used as a detection scheme. In the context of sequencing, FRET is generally described in Braslavasky et al., Proc. Nat'l Acad. Sci., 100:3960-3964 (2003), incorporated herein by reference. Essentially, in one embodiment, a donor fluorophore is attached to a primer, polymerase, or template. The nucleotide added for incorporation into the primer includes an acceptor fluorophore that is activated by the donor when the two are in proximity.

A suitable illumination and detection system for fluorescence-based signals is a Zeiss confocal microscope 200 equipped with a TIRF slide coupled to an 80 mW 532 nm solid-state laser. The slide is illuminated by an objective lens positioned at the correct TIRF illumination angle. TIRF can also be accomplished without the use of an objective lens by illuminating the substrate via a prism optically coupled to the substrate. Planar waveguides can also be used to implement TIRF on substrates.

One embodiment of the present imaging system contains a 20× lens with a 1.25 mm field of view, with detection then being accomplished using a 10 megapixel camera. Such a system images approximately 1.5 million nucleic acid molecules attached to a pattern array at a 1 micron pitch. In this configuration, there are approximately 6.4 pixels per nucleic acid molecule. The number of pixels per nucleic acid molecule can be adjusted by increasing or decreasing the field of view of the objective lens. For example, a 1 mm field of view will generate a pixel value of 10 per nucleic acid molecule, and a 2 mm field of view will generate a pixel value of 2.5 per nucleic acid molecule. The field of view can be adjusted relative to the magnification and NA of the objective lens to generate the lowest pixel count nucleic acid molecules that can be resolved by the optics and image analysis software. Imaging speed can be improved by reducing the magnification power of the objective lens, using a grid pattern array, and increasing the number of pixels of the collected data in each image.

To obtain optical signals, a combination of optical fibers or a charged couple device (CCD) can be used in the detection of the sequencing reaction. Thus, in a specific embodiment, the hybridization pattern on the array formed by the sequencing reaction is scanned with a CCD camera (e.g., model TE/CCD512SF, Princeton Instruments, Trenton, NJ.) having appropriate optics such as those described in Yershov et al., Proc. Natl. Aca. Sci. 93:4913 (1996) (Ploem, in Fluorescent and Luminescent Probes for Biological Activity Mason, T.G. Ed., Academic Press, Landon, pp. 1-11 (1993)), which allows for the simultaneous scanning of a very large number of labeled target nucleic acids.

In a specific embodiment, the efficiency of the sequencing system can be improved by the use of multiple imaging system components. For example, in the system imaging component, preferably in the range of 10-16 million pixels, up to 4 or more cameras can be used. Multiple band pass filters and dichroic mirrors can also be used to collect pixel data across up to 4 or more emission spectra. To compensate for the lower light collection ability of the objective lens with reduced magnification, the energy of the excitation light source can be increased. Throughput can be increased by using one or more flow cells with respective cameras so that the imaging system is not idle when hybridizing/reacting the sample. Because the probe technology of the array can be discontinuous, more than one imaging system can be used to collect data from a group of arrays, further reducing the array time.

One illumination mode is to share a common set of monochromatic light sources (about four lasers to obtain 6-8 colors) among the imagers. Each imager collects data at a different wavelength at any given time, and the light source is converted to the imager by an optical conversion system. In such an embodiment, the illumination source preferably produces at least six, but more preferably eight different wavelengths. Such light sources include gas lasers, multiple diode pumped solid state lasers combined through fiber couplers, filtered xenon arc lamps, tunable lasers, or the newer Spectralum Light Engines soon to be offered by Tidal Photonics. Spectralum Light Engines use prisms to separate light from spectral lines. The spectrum is projected onto a Texas Instruments digital light processor, which can selectively reflect any portion of the spectrum into a fiber or optical connector. The system can monitor and calibrate the energy output across individual wavelengths to make them consistent, so as to automatically compensate for intensity differences as the bulb ages or between bulb changes.

During the imaging process, the substrate must remain in focus. Some key factors in maintaining focus are the planarity of the substrate, the orthogonality of the substrate to the focal plane, and the mechanical forces on the substrate that can deform it. Substrate planarity can be well controlled because glass plates with more than 1/4 wave flatness are readily available. Uneven mechanical forces on the substrate can be minimized through proper hybridization chamber design. Orthogonality to the focal plane can be achieved through a well-adjusted, high-precision stage. After each image is acquired, it is analyzed using a fast algorithm to determine whether the image is in focus. If the image is out of focus, the autofocus system will store the position information of the out-of-focus image so that that portion of the array can be reimaged in the next imaging cycle. By mapping the positions of different locations on the substrate, the time required to acquire the substrate image can be reduced.

The measured signals can be analyzed manually or preferably by appropriate computer methods to tabulate the results. The matrix and reaction conditions may include appropriate controls for verifying hybridization integrity and extension conditions and, if necessary, for providing a standard curve for quantification. For example, a control nucleic acid can be added to the sample.

In a large-scale sequencing run, each imager preferably acquires about 200,000 images/day based on a 300 millisecond exposure time of a 16-megapixel CCD. Thus, an instrument design for the illumination and detection components of the system of the present invention may include four imagers, each servicing four sets of quaternary flow cells (a total of 16 flow cells). Each imager may include a CCD detector with 10 megapixels and be used for a roughly 300 millisecond exposure time. When other fluorophores are being imaged, unintentional photobleaching by the light source can be reduced by maintaining low illumination energy and minimal exposure time.

By using an intensified CDD (ICCD), data of nearly identical quality can be acquired with illumination intensities and exposure times orders of magnitude lower than those of a standard CCD. ICCDs are generally available in the 1-1.4 megapixel range. Because they require much shorter exposure times, a 1-megapixel ICCD can acquire ten or more images in the time it takes a standard CCD to acquire one. When used in conjunction with a fast filter wheel and a high-speed flow cell stage, a 1-megapixel ICCD can acquire the same amount of data as a standard 10-megapixel CCD.

In a specific embodiment, an electron multiplying CCD (EMCCD) is used for nucleic acid imaging. EMCCD is a quantitative digital camera technology that can detect single photon events while maintaining high quantum efficiency, which is achieved through the unique electron multiplying structure built into the sensor. Unlike conventional CCDs, EMCCDs are not limited by the readout noise of the output amplifier, even when operating at high readout speeds. This is achieved by adding a solid-state electron multiplying (EM) register to the end of a normal series register; this register multiplies weak signals before any readout noise is added by the output amplifier, thereby making the readout noise negligible. The EM register has hundreds of stages that use a higher-than-normal clock voltage. As charge is transferred through each stage, impact ionization can be exploited to generate secondary electrons, thereby increasing the EM. When this is accomplished across hundreds of stages, the resulting increase can be controlled (by software) from one to hundreds or even thousands of times.

EMCCD systems can be combined with TIFRM technology to image multiple fluorophore labels by integrating a multi-line laser system, preferably a solid-state laser solution with an acousto-optical tunable filter (AOTF). This technology can be easily adapted for FRET analysis, preferably by integrating a suitable beam splitter on the emission side.

A factor to consider in high-resolution and high-speed imaging and sequencing chemistry-related readouts is the fluctuation in results caused by moving parts. Vibration, if not controlled or isolated, can interfere with image capture and result in poor image resolution. To minimize the effects of vibration from moving parts (particularly the carrier 9 with its motorized gripping mechanism 8, 8'), the characterization tool 7, including the optical components, and the reaction platform 3 are intentionally physically loosely coupled. Specifically, they are physically separated from each other by vibration isolators, even when they are operating in parallel. This requires control and sensing mechanisms as part of the carrier 9, as well as position registration mechanisms as part of the characterization tool 7. Various such mechanisms are known in the art. For example, robotics, where electronic eyes, perceptible alignment markers, and the like are used to ensure accurate transfers without introducing excessive vibration into the sensitive field of view of the characterization tool, allows for continuous or near-continuous operation. The goal is to accurately and efficiently acquire and process large amounts of data while linking two or more technologies (including batch processing involving mechanics, electronics, optics, and biochemistry), which have not yet been unified into efficient, continuously operating analytical methods.

Although the present invention is satisfied by many different forms of embodiments (as detailed with respect to the preferred embodiments of the present invention), it should be understood that the present disclosure is considered to be an example of the principles of the present invention and is not intended to limit the invention to the specific embodiments shown and described herein. Numerous variations can be made by those skilled in the art without departing from the spirit of the invention. The scope of the present invention will be measured only by the claims of any corresponding patent application and its equivalents. The abstract and title should not be interpreted as limiting the scope of the invention, as their purpose is to enable relevant institutions and the public to quickly determine the general nature of the invention. In the claims of any corresponding patent application, unless the term "device" is used, the features or elements cited in the text should not be interpreted as device-plus-function limitations based on 35 U.S.C §112.

Claims (34)

1. An automated method for high-throughput nucleic acid sequencing, comprising the following steps: One or more flow cells are placed on a reaction platform, each flow cell including multiple identifiable reaction sites and multiple nucleic acid samples attached to the multiple reaction sites; The processing reagents are injected into the flow cell to perform a sequencing reaction and produce fluorescent sequencing products; Each flow cell is moved to a position within the field of view of a detection subsystem using a carrier device, the detection subsystem including an imager configured to capture an optical image of the flow cell; Each flow cell is illuminated within the field of view to cause the fluorescent sequencing product to emit distinguishable spectral fluorescence at the distinguishable reaction sites; and Images of fluorescence from the flow cell that can identify reaction sites are captured to identify the nucleic acid sequence of the nucleic acid sample; The vibration isolator prevents vibration from interfering with image capture; and The sample preparation in the reaction step and the data extraction in the capture step occur at different rates. 2. The method of claim 1, wherein the duration of the capture step is shorter than that of the reaction step. 3. The method of claim 1, further comprising placing a plurality of flow cells on a plurality of reaction platforms and transferring each flow cell from the plurality of reaction platforms to a detection subsystem. 4. The method of claim 1, wherein the transfer is performed by a vehicle comprising a computer-controlled clamping mechanism. 5. The method of claim 4, wherein the vehicle includes control and sensing mechanisms, and the imaging subsystem includes a location registration mechanism. 6. The method of claim 1, wherein the transfer of each flow cell comprises reversibly disconnecting the fluid reservoir in the reaction subsystem from the fluid inlet of the flow cell. 7. The method of claim 1, further comprising returning each flow cell to its initial reaction platform after the image has been captured. 8. An automated method for high-throughput nucleic acid sequencing, comprising repeatedly performing the following steps: Multiple flow cells are processed simultaneously on the reaction platform, allowing the nucleic acids immobilized in each flow cell to undergo biochemical sequencing reactions, producing fluorescent sequencing products; and Using a carrier device configured to transfer each flow cell from the fluid processor to the imager, each flow cell on the reaction platform is sequentially transferred to the detection subsystem to capture images of the fluorescent sequencing products. The vibration isolator prevents vibration from interfering with the image capture subsystem. 9. The method of any one of claims 1-8, wherein the detection subsystem is spaced apart from other modules of the system such that vibrations from other modules do not disrupt image capture by the imager. 10. The method of any one of claims 1-8, wherein the detection subsystem is physically isolated from other modules of the system such that vibrations from other modules do not disrupt image capture by the imager. 11. The method of any one of claims 1-8, wherein the vibration isolator is located between the detection subsystem and the reaction subsystem to isolate the vibration of the detection subsystem from that of the reaction subsystem. 12. The method of any one of claims 1-8, wherein each flow cell has a volume of about 50-300 μL. 13. The method of any one of claims 1-8, comprising capturing images from multiple different fluorescent markers in each flow cell within 300 milliseconds. 14. The method of any one of claims 1-8, comprising capturing approximately 200,000 flow cell images per day. 15. A system for high-throughput nucleic acid sequencing, comprising: A reaction subsystem, including at least one reaction platform, is configured to perform a biochemical sequencing reaction on a nucleic acid sample in a reaction unit; A detection subsystem is used to capture optical images of the sequencing reaction for optical image analysis; The connection subsystem is used to transfer multiple nucleic acid samples or reaction units into the detection field of view of the detection subsystem; Vibration isolators prevent vibration from interfering with the image capture by the detection subsystem; The operating rate of the detection subsystem differs from that of the reaction subsystem. 16. The system of claim 15, wherein the reaction platform comprises a plurality of flow cells. 17. The system of claim 16, wherein the flow cell has a plurality of reaction units. 18. The system of claim 15, wherein the connection subsystem includes a carrier device. 19. The system of claim 18, wherein the carrier device further comprises a track. 20. The system of claim 18, wherein the carrier device is operable to transfer multiple nucleic acid samples or reaction units one by one into the detection field of view of the detection subsystem. 21. The system of claim 18, wherein the carrier device is operable to select and transport the reaction unit from a selected platform of at least one separate reaction platform to the detection field of view of the detection subsystem. 22. The system of claim 15, wherein the connecting subsystem is further configured to transfer multiple nucleic acid samples or reaction units one by one into the detection field of view of the detection subsystem. 23. The system of any one of claims 15-22, wherein the vibration isolator is configured to isolate the detection subsystem from vibration. 24. The system of claim 22, wherein the vibration is generated by the moving parts in the reaction subsystem and the carrier device. 25. The system according to any one of claims 15-22, wherein the reaction platform and the detection subsystem are physically loosely connected in the reversibly integrated system. 26. A system for high-throughput nucleic acid sequencing, comprising: A reaction subsystem, including at least one reaction platform, is configured to perform a biochemical sequencing reaction on a nucleic acid sample in a reaction unit; A detection subsystem is used to capture optical images of the sequencing reaction for optical image analysis; The reaction platform includes a track and a displacement unit, wherein the displacement unit has at least one carrier, and the carrier can move along the track with the displacement unit to transfer multiple nucleic acid samples or reaction units into the detection field of view of the detection subsystem. Vibration isolators are used to prevent vibration from interfering with the image capture of the detection subsystem; The operating rate of the detection subsystem differs from that of the reaction subsystem. 27. The system of claim 26, wherein the reaction platform comprises a plurality of flow cells. 28. The system of claim 27, wherein the flow cell comprises a plurality of reaction units. 29. The system of claim 28, wherein the reaction platform further includes a solid support and the flow cell is located on the solid support. 30. The system of claim 26, wherein the carrier includes a carrier plate. 31. The system of any one of claims 26-30, wherein the vibration isolator is configured to isolate the detection subsystem from vibration. 32. The system of claim 31, wherein the vibration is generated by the reaction subsystem and the moving parts in the carrier. 33. The system according to any one of claims 26-30, wherein the reaction platform and the detection subsystem are physically loosely connected in the reversibly integrated system. 34. The system according to any one of claims 26-30, wherein the carrier can move along a track with the displacement unit to sequentially transfer multiple nucleic acid samples or reaction units into the detection field of view of the detection subsystem.