detection apparatus and method for imaging a substrate

专利摘要:
INTEGRATED OPTOELECTRONIC READING HEAD AND FLUID CARTRIDGE USEFUL FOR NUCLEIC ACID SEQUENCING. It is a detection apparatus having a read head that includes a plurality of microfluorometers positioned to simultaneously acquire a plurality of wide-field images in a common plane; and (b) a translation stage configured to move the readhead along a substrate that is in the common plane. The substrate may be a flow cell that is included in a cartridge, the cartridge also including a housing for (i) a sample reservoir; (ii) a fluidic line between the sample reservoir and the flow cell; (iii) several reagent reservoirs in fluid communication with the flow cell, (iv) at least one valve configured to mediate fluid communication between the reservoirs and the flow cell; and (v) at least one pressure source configured to move liquids from the reservoirs to the flow cell. The detection device and cartridge can be used together or independently.
公开号:BR112014024789B1
申请号:R112014024789-7
申请日:2013-02-13
公开日:2021-05-25
发明作者:Dale Buermann；John A. Moon；Bryan Crane；Mark Wang；Stanley S. Hong；Jason Harris；Matthew Hage；Mark J. Nibbe
申请人:Illumina, Inc；
IPC主号:

专利说明:

[001] This application is based on and claims the benefit of U.S. Provisional Application No. 61/619,784, filed April 3, 2012, and incorporated herein by reference. FUNDAMENTALS
[002] The modalities of this disclosure refer, in general, to apparatus and methods intended for fluid manipulation and optical detection of samples, for example, in nucleic acid sequencing procedures.
[003] Our genome provides a master plant to predict many of our predispositions, such as preferences, talents, susceptibility to diseases and reaction to therapeutic drugs. The genome of an individual human being contains a sequence of more than 3 billion nucleotides. Differences in just a fraction of these nucleotides convey many of our unique characteristics. The research community has been making significant advances in unraveling the resources that constitute the master plant and, with that, a more comprehensive understanding of how the information in each master plant is related to human health. However, our understanding is far from a conclusion and prevents the movement of information from research labs to clinics where the hope is that one day each of us will have a copy of our own personal genome so that we can reunite with our physicians to determine the appropriate choices for a healthy lifestyle or an appropriate course of treatment.
[004] The current hurdle is a matter of yield and scale. A key component of unraveling the master plant for any individual is determining the exact sequence of the 3 billion nucleotides in its genome. Techniques are available to accomplish this, but these techniques typically take many days and thousands of dollars to devise. Additionally, the clinical relevance of any individual's genomic sequence is a matter of comparing the unique features of its genomic sequence (ie, its genotype) to reference genomes that are correlated with known traits (ie, phenotypes). The issue of scale and yield becomes evident when considering that reference genomes are created based on the correlations of genotype to phenotype that emerge from research studies that typically use thousands of individuals to be statistically valid. Thus, billions of nucleotides can eventually be sequenced for thousands of individuals to identify any clinically relevant correlations between genotype and phenotype. Multiplying further by the number of diseases, drug responses, and other clinically relevant features, the need for fast and inexpensive sequencing technologies becomes even more apparent.
[005] A reduction in sequencing costs is needed that results in large genetic correlation studies carried out by research scientists and that makes sequencing accessible in the clinical setting for the treatment of individual patients making decisions with lifelong consequences. The embodiments of the invention presented herein satisfy that need and provide other advantages as well. BRIEF SUMMARY
[006] The present disclosure provides a detection apparatus that includes (a) a carriage including a plurality of microfluorometers, wherein each of the microfluorometers has an objective configured for detection of wide-field images, wherein the plurality of microfluorometers is positioned to simultaneously acquire a plurality of wide-field images on a common plane, each of the wide-field images coming from a different area of the common plane; (b) a translation stage configured to move the carriage in at least one direction parallel to the common plane; and (c) a sampling stage configured to maintain a substrate in the common plane.
[007] The present disclosure further provides a method for imaging a substrate, including the steps of (a) providing a substrate including fluorescent features on a surface; (b) acquiring a plurality of wide-field images of a first portion of the surface using a plurality of microfluorometers, wherein each of the microfluorometers acquires a wide-field image from a different location on the surface, wherein the plurality of microfluorometers is attached to a car; and (c) translate the carriage in a direction parallel to the surface and repeat (b) for a second portion of the surface. The method can use any apparatus shown anywhere in this document, but need not be limited in all embodiments.
[008] A fluidic cartridge is also provided which includes (a) a flow cell having an optically transparent surface, an inlet and an outlet; and (b) a carcass made from a material that is optically opaque and impervious to aqueous liquids, wherein the carcass maintains: (i) a sample reservoir; (ii) a fluidic line between the sample reservoir and the flow cell inlet; (iii) a plurality of reagent reservoirs in fluid communication with the flow cell through the flow cell inlet, (iv) at least one valve configured to mediate fluid communication between the reservoirs and the flow cell inlet; and (v) at least one pressure source configured to move liquids from the sample reservoir or reagent reservoirs to the flow cell through the inlet of the flow cell, wherein an optically transparent window interrupts the housing and a port The inlet port interrupts the housing, where the inlet port is in fluid communication with the sample reservoir, and where the optically transparent surface is positioned on the window.
[009] The present disclosure further provides a sequencing method that includes the steps of (a) providing a fluid cartridge having (i) a flow cell having an optically transparent surface, (ii) a nucleic acid sample, (iii) a plurality of reagents for a sequencing reaction, and (iv) a fluidic system for delivering the reagents to the flow cell; (b) providing a detection apparatus having (i) a plurality of microfluorometers, each of the microfluorometers comprising an objective configured for detecting wide-field images in an image plane in dimensions x and y, and (ii) a sampling stage ; (c) delivering the fluidic cartridge to the sampling stage, where the optically transparent surface is placed on the imaging plane; and (d) perform fluidic operations of a nucleic acid sequencing procedure in the fluid cartridge and detection operations of the nucleic acid sequencing procedure in the detection apparatus, in which (i) the reagents are distributed to the flow cell by the fluidic system , and (ii) the nucleic acid resources are detected by the plurality of microfluorometers. BRIEF DESCRIPTION OF THE DRAWINGS
[010] Figure 1 shows an optoelectronic detection device (left) and a fluid cartridge (right) useful for nucleic acid sequencing.
[011] Figure 2 shows an optical sketch for an individual microfluorometer having orthogonal excitation and emission beam paths.
[012] Figure 3 shows an optical sketch for a microfluorometer.
[013] Figure 4 shows an arrangement of four microfluorometers in relation to a flow cell having two channels.
[014] Figure 5 shows an autofocusing device that can be used in a microfluorometer.
[015] Figure 6 shows in Panel A: top views of an array of four channels in a flow cell (left) and a linear array of objectives in a single row (right), and in Panel B: a flow cell having eight channels (left) and an array of eight objectives in two linear rows of four.
[016] Figure 7 shows top views of a six-channel arrangement in a flow cell (left) and a hexagonal objective packaging arrangement in two rows (right).
[017] Figure 8 shows a perspective view of an array of eight microfluorometers for a detection device.
[018] Figure 9 shows a bottom plan view of an array of eight microfluorometers for a detection device.
[019] Figure 10 shows an optical sketch for an individual microfluorometer having parallel excitation and emission beam paths.
[020] Figure 11 shows a perspective view of a Y-stage type for a detection apparatus.
[021] Figure 12 shows a bottom perspective view of a Y stage for a detection apparatus.
[022] Figure 13 shows a top perspective view of a Y stage that maintains an array of eight microfluorometers.
[023] Figure 14 shows an electrical block diagram for a detection device.
[024] Figure 15 shows an exploded view of a fluidic cartridge with a flow cell.
[025] Figure 16 shows a fluidic map for a fluidic cartridge.
[026] Figure 17 shows a four-sample injection rotary valve.
[027] Figure 18 shows a fluidic map for a reagent reuse system that uses a unidirectional flow and two valves.
[028] Figure 19 shows a fluidic map for a reagent reuse system that uses an alternating flow and a single valve. DETAILED DESCRIPTION
[029] The present disclosure provides methods and apparatus for high resolution detection of flat areas such as those present on substrate surfaces. A particularly useful application is optically based on image form of a biological sample that is present on a surface. For example, the methods and apparatus presented in this document can be used to image nucleic acid resources that are present in nucleic acid arrays, such as those used in nucleic acid sequencing applications. A variety of nucleic acid sequencing techniques that utilize optically detectable samples and/or reagents can be used. These techniques are particularly well suited to the methods and apparatus of the present disclosure and therefore highlight many advantages for particular embodiments of the invention. Some of the advantages are presented for illustrative purposes, and although nucleic acid sequencing applications are exemplified, the advantages can also be extended to other applications.
[030] In relation to some of the examples presented here, the notable features of many nucleic acid sequencing techniques are: (1) the use of multicolor detection (eg generally four different fluorophores are used, one for each of the different nucleotide types A, C, G and T (or U) present in nucleic acids), (2) distribution of large numbers of different fragments from a nucleic acid sample (eg fragments from a genome sample, sample of RNA, or derivatives thereof) on the surface of an array and (3) repeated cycles of fluid processing and imaging of the arrays. The embodiments of the methods and apparatus disclosed herein are particularly useful for nucleic acid sequencing in that they can provide the capability of high resolution imaging of multi-color, multi-repeat array surfaces. For example, the modalities presented here allow an image of a surface to be taken at a resolution that is in the micron range of hundreds, tens or even a single digit. In this way, one can decide on nucleic acid resources having an average nearest neighbor center-to-center spacing that is less than 100 microns, 50 microns, 10 microns, 5 microns or less. In particular modalities, wide-field images of surfaces can be acquired, including, for example, images that cover an area of 1 mm2 or greater of an array. Images can be acquired in multiple colors simultaneously or sequentially, for example, to identify fluorescent markers uniquely associated with different types of nucleotides. Furthermore, images can be acquired sequentially for multiple cycles of a sequencing technique. Images from a given area of the array can be reliably compared from each cycle to determine the sequence of color changes detected for each nucleic acid resource in the array. The sequence of color changes can successively be used to infer the sequences of the nucleic acid fragments in each resource.
[031] In particular embodiments, an apparatus of the present disclosure includes one or more microfluorometers. Each of the microfluorometers can include an excitation radiation source, a detector and an objective to form an integrated sub-unit of a readhead. Other optical components may be present in each microfluorometer. For example, a beam splitter may be present to provide a compact epifluorescent detection configuration whereby the beam splitter is positioned to direct excitation radiation from the excitation radiation source to the objective and direct the radiation from emission from the objective to the detector.
[032] An advantage of using an integrated microfluorometer design is that the microfluorometer can be conveniently moved, for example, in a scanning operation, to allow imaging of a substrate that is larger than the microfluorometer's field of view. In particular embodiments, several microfluorometers can be combined to form a readhead. Various settings for the readhead combination will be presented below and can be selected to suit a particular shape for a substrate that is to be imaged, while maintaining the relatively compact size for the readhead as a whole. The relatively small size and low mass of the readhead in several embodiments of the present disclosure results in a relatively low inertia so that the readhead goes into sleep mode quickly after being moved, thus favoring a quick scan of a array of nucleic acid or other substrate. In some cases, microfluorometers can be attached to a cart so that they are not independently movable in at least some dimensions during the course of an analytical application, such as a nucleic acid sequencing cycle. For example, multiple microfluorometers can be permanently fixed so that they are not moveable independently of each other in the x and y dimensions (where at least one between x and y is the sweep direction). Microfluorometers can, however, be independently actuated in the z dimension to provide independent focus control. Reducing the degrees of freedom between several different microfluorometers of an apparatus of the present disclosure provides protection against loss of alignment during transportation, handling, and use of the apparatus.
[033] In some embodiments, multiple microfluorometers that are present in a readhead or carriage may have a dedicated autofocus module. Correspondingly, each microfluorometer can be focused independently. In some modalities, a particular autofocusing module in a readhead, although dedicated to the actuation of a particular microfluorometer, can nevertheless receive information from at least one other autofocusing module in the readhead and information from that readhead module particular autofocus and at least one other autofocus module can be used to determine an appropriate actuation to obtain the desired focus for the particular microfluorometer. Thus, the focus for any given microfluorometer can be determined by consensus between two or more microfluorometers present in the same readhead or carriage.
[034] In particular embodiments, a sample to be detected in a method or apparatus presented herein may be provided in a cartridge format. For example, the cartridge can include a substrate to be detected along with other fluidic components used to process the substrate for detection. Taking the more specific example of a nucleic acid sequencing application, the cartridge may include a flow cell capable of presenting an array of nucleic acid resources to a sensing device, and optionally one or more of the reservoirs for maintain sequencing reagents, reservoirs to hold sample preparation reagents, reservoirs to hold waste products generated during sequencing, and/or pumps, valves, and other components capable of moving fluids through the flow cell. Such a fluidic cartridge can provide the advantages of a convenient and compact format for storing and processing a sample and nucleic acid sequencing reagents.
[035] In particular embodiments, a fluidic cartridge can be configured to allow the reuse of one or more reagents. For example, the fluidic cartridge can be configured to deliver a reagent to a flow cell, then remove the reagent from the flow cell, and then reintroduce it to the flow cell. One advantage of reusing reagents is that it reduces scrap and reduces the costs of processes that use expensive reagents and/or reagents that are delivered in high concentrations (or in high quantities).
[036] The fluidic cartridges of the present disclosure can provide a modularity advantage whereby different samples can be fluidly processed in a first module (i.e., the fluidic cartridge) that is in optical communication with a second module (e.g., a microfluorometer, a readhead or a detection device). A fluidic cartridge may contain sufficient sample(s), reagents, and fluidic hardware for a complete fluidic processing procedure (eg, a nucleic acid sequencing procedure) and the fluidic cartridge may be delivered to a detection device. Once the fluidic and detection procedures are complete, the fluidic cartridge can be removed so that the detection apparatus is ready for another procedure. Because the fluidics and detection modules are separable, the present system allows multiple samples to be evaluated while avoiding the risk of cross-contamination between samples. This provides advantages for modalities where sensing components are relatively expensive and technically difficult to assemble, avoiding unnecessary maintenance, decontamination or disposal of optical components that may be necessary when the fluidic components and optical components are not modular.
[037] Figure 1 shows an exemplary optical scanning device 1 that explores the advantages of integrated optoelectronics and cartridge-based fluidics that are provided by the various modalities presented here. Exemplary device 1 includes a housing 2 that contains various fixed components including, for example, optical components, computational components, power sources, fans, and the like. A screen 3 present, for example, on the front face of housing 2 works as a graphical user interface that can provide various types of information, such as operational status, status of an analytical procedure (for example, a sequencing cycle) being performed , data transfer status to or from device 1, instructions for use, alerts, or the like. A cartridge receptacle 4 is also present on the front face of housing 2. As shown, cartridge receptacle 4 can be configured as a slot having a protective door 5. A status indicator 6, in the form of an indicator light on the frame of the cartridge receptacle in this example, is present and can be configured to indicate the presence or absence of a cartridge in device 1. For example the indicator light 6 can toggle between on and off or change color to indicate the presence or absence of a cartridge. A power control button 7 is present on the front face of housing 2 in this example as it is identifying inscriptions 8 such as the name of the manufacturer or instrument.
[038] In Figure 1, there is also shown an exemplary fluidic cartridge 10 that can be used to provide a sample and reagents to device 1. The fluidic cartridge 10 includes a housing 11 that protects various fluidic components, such as reservoirs, fluidic connections, pumps, valves, and the like. A flow cell 12 is integrated with the fluidic cartridge at a position where it is in fluid communication with the reagents within the housing. The housing 11 has an opening 13 through which a face of the flow cell 12 is exposed so that it can optically interact with the optical scanning device 1 when the fluidic cartridge 10 is placed in the cartridge receptacle 4. The cartridge housing 11 also includes a sample port 14 for introducing a target nucleic acid sample. A barcode 15 or other machine readable inscriptions may optionally be present on the cartridge housing 11, for example, to provide tracking and sample management. Other inscriptions 16 may also be present on the housing for convenient identification by a human user, for example, to identify the manufacturer, analytical analysis supported by the fluidic cartridge, batch number, expiration date, safety notices, and the like.
[039] The device shown in Figure 1 is an example. Other exemplary embodiments of the methods and apparatus of the present disclosure that may be used alternatively or in addition to the example of Figure 1 are presented in greater detail below.
[040] A detection apparatus is provided herein, having (a) a carriage including a plurality of microfluorometers, wherein each of the microfluorometers includes an objective configured for detection of wide-field images, wherein the plurality of microfluorometers is positioned to simultaneously acquire a plurality of widefield images on a common plane, each of the widefield images coming from a different area of the common plane; (b) a translation stage configured to move the car in at least one direction parallel to the common plane; and (c) a sampling stage configured to maintain a substrate in the common plane.
[041] A detection apparatus (or an individual microfluorometer) of the present disclosure can be used to obtain one or more images at a resolution sufficient to distinguish features on a micron scale. For example, a microfluorometer that is used in a detection device may have a resolution that is sufficient to distinguish features that are separated by at most 500 μm, 100 μm, 50 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 µm or 1 µm. A lower resolution is also possible, for example a resolution that distinguishes features that are separated by more than 500 µm.
[042] A detection apparatus (or an individual microfluorometer) of the present disclosure is well suited for high resolution detection of surfaces. Correspondingly, arrays having features with an average spacing in the micron range are especially useful substrates. In particular embodiments, a detection device or microfluorometer can be used to obtain one or more images of an array having center-to-center spacing capabilities for nearest neighbors that, on average, are equal to or less than 500 μm, 100 μm, 50 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm or 1 μm. In many embodiments, the resources of an array are non-contiguous being separated, for example, by less than 100 µm, 50 µm, 10 µm, 5 µm, 1 µm, or 0.5 µm. However, the resources do not need to be separated. Preferably, some or all of the features of an array can be contiguous with each other.
[043] One can use any of a variety of arrays (also referred to as "microarrays") known in the art. A typical array contains resources, each having an individual probe or a population of probes. In the latter case, the probe population at each site is typically homogeneous having a single probe species. For example, in the case of a nucleic acid array, each resource can have multiple nucleic acid species each having a common sequence. However, in some modalities, populations in each resource of an array can be homogeneous. Similarly, protein arrays can be resourced with a single protein or population of proteins typically, but not always, having the same amino acid sequence. Probes can be attached to the surface of an array, for example, through covalent attachment of the probes to the surface or through non-covalent interaction(s) of the probes with the surface. In some embodiments, probes such as nucleic acid molecules can be affixed to a surface through a gel layer as described, for example, in US 2011/0059865 A1, which is incorporated herein by reference.
[044] Exemplary arrays include, without limitation, a BeadChip Array available from Illumina®, Inc. (San Diego, California, USA) or others, such as those where probes are attached to microspheres that are present on a surface (by example, microspheres in wells on a surface) such as those described in US Pat. 6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO 00/63437, each of which is incorporated herein by reference. Other examples of commercially available microarrays that can be used include, for example, an Affymetrix® GeneChip® microarray or other microarray synthesized according to techniques sometimes referred to as VLSIPSTM (Very Large Scale Immobilized Polymer Synthesis) technologies. An identified microarray can also be used in an apparatus or system in accordance with some embodiments of the invention. An exemplary identified microarray consists of a CodeLinkTM Array available from Amersham Biosciences. Another useful microarray is one manufactured using inkjet printing methods, such as the SurePrintTM Technology available from Agilent Technologies.
[045] Other useful arrangements include those used in nucleic acid sequencing applications. For example, arrays having genomic fragment amplicons (generally referred to as clusters) are particularly useful, such as those described in Bentley et al, Nature 456:5359 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 , or US 2008/0108082 , each of which is incorporated herein by reference. Another type of array useful for nucleic acid sequencing is an array of particles produced from an emulsion PCR technique. Examples are described in Dressman et al, Proc. Natl. Academic Sci. USA 100:8817-8822 (2003), WO 05/010145, US 2005/0130173 or US 2005/0064460, each of which is incorporated herein by reference in its entirety. Although the above arrays have been described in the context of sequencing applications, it will be understood that the arrays can be used in other modalities including, for example, those that do not include a sequencing technique.
[046] Be configured for detection of an array or other sample, one or more microfluorometers that are present in a detection device can be configured for wide field detection. The field diameter for an individual microfluorometer can, for example, be at least 0.5mm, 1mm, 2mm, 3mm, 4mm, 5mm or larger. By choosing suitable optical components, the field diameter can be limited to a maximum area and as such the field diameter cannot be, for example, greater than 5 mm, 4 mm, 3 mm, 2 mm or 1 mm. Correspondingly, in some modalities, an image taken by an individual microfluorometer may have an area that is in a range of 0.25 mm2 to 25 mm2.
[047] In addition to being configured for wide field detection, a microfluorometer can be configured to have a numerical aperture (NA) that is greater than 0.2. For example, the NA of an objective used in a microfluorometer of the present disclosure may be at least equal to 0.2, 0.3, 0.4, or 0.5. Alternatively or additionally, it may be desirable to restrict the objective NA to not greater than 0.8, 0.7, 0.6, or 0.5. The methods and apparatus presented here are particularly useful when detection occurs through an objective having an NA between 0.2 and 0.5.
[048] In array detection modalities, a detection device (or individual microfluorometer) can be configured to obtain a digital image of the array. Typically, each pixel in the digital detection apparatus (or individual microfluorometer) will collect signal from no more than a single resource in any given image acquisition. This setting minimizes unwanted 'crosstalk' between features in the image. The number of pixels that detect signal from each resource can be adjusted based on the size and shape of the resources that were imaged and based on the configuration of the digital detection device (or individual microfluorometer). For example, each feature can be detected in a given image by no more than about 16 pixels, 9 pixels, 4 pixels, or 1 pixel. In particular modalities, each image can use, on average, 6.5 pixels per feature, 4.7 pixels per feature or 1 pixel per feature. The number of pixels used per feature can be reduced, for example, by reducing variability in the position of the features in the array pattern and narrowing the tolerance for aligning the detection apparatus to the array. Taking as an example a digital detector that is configured to use less than 4 pixels per resource, image quality can be improved by using an array of ordered nucleic acid resources instead of an array of randomly distributed nucleic acid clusters .
[049] It will be appreciated that a detection apparatus having multiple microfluorometers can detect an area of a common plane that is approximately equivalent to the number of microfluorometers multiplied by the wide field area detected by each microfluorometer. Areas do not need to be contiguous. For example, 2 or more microfluorometers can be positioned to detect discrete regions of a common plane that are separated by an undetected area. However, if desired, multiple microfluorometers can be positioned to detect areas that are contiguous but not overlapping. In alternative embodiments, a detection apparatus having multiple microfluorometers can detect an area of a common plane that is substantially smaller than the number of microfluorometers multiplied by the wide field area detected by each microfluorometer. This can result, for example, when multiple microfluorometers are positioned to detect areas that have at least a partial overlap. As presented in greater detail elsewhere in this document, multiple images need not be acquired in a format that is used to reconstruct, or still supports the same, a complete image of an array or other common plane that has been detected.
[050] An exemplary optical sketch for a microfluorometer 100 is shown in Figure 2. The microfluorometer 100 is directed to a flow cell 170 having an upper layer 171 and a lower layer 173 which are separated by a fluid loaded channel 175. In the configuration shown, the topsheet 171 is optically transparent and the microfluorometer 100 is focused to an area 176 on the inner surface 172 of the topsheet 171. In an alternative configuration, the microfluorometer 100 can be focused onto the inner surface 174 of the bottomsheet 173. or both surfaces may include array features to be detected by the microfluorometer 100.
[051] The microfluorometer 100 includes an objective 101 that is configured to direct excitation radiation from a radiation source 102 to flow cell 170 and direct emission from flow cell 170 to a detector 108. In the exemplary sketch, excitation radiation from radiation source 102 passes through a lens 105, then through a beam splitter 106 and then through the objective on its way to flow cell 170. In the embodiment shown, the source radiation includes two light-emitting diodes (LEDs) 103 and 104, which produce radiation at different wavelengths from each other. Emission radiation from flow cell 170 is captured by objective 101 and reflected by the beam splitter through conditioning optics 107 and detector 108 (e.g., a CMOS sensor). The beam splitter 106 functions to direct the emission radiation in a direction that is orthogonal to the path of the excitation radiation. The objective position can be moved in the z dimension to change the focus of the microfluorometer. The microfluorometer 100 can be moved back and forth in the y direction to capture images of various areas of the inner surface 172 of the upper layer 171 of the flow cell 170.
[052] Figure 3 shows an exploded view of an exemplary microfluorometer for purposes of demonstrating the functional arrangement of various optical components. Two excitation sources are shown, including a green LED (LEDG) and a red LED (LEDR). The excitation light passes through a green LED collector lens (L6) and a red LED collector lens (L7), respectively. A retractable LED mirror (Ml) reflects the green excitation radiation to a dichroic combiner (F5) which reflects the green excitation radiation through an excitation filter (F2), then through a diode beam splitter. laser (F3) then through an excitation field stop (FS) then through a group of L2 excitation projection lenses to an excitation/emission dichroic (F4) which reflects the green excitation radiation through of a group of stationary objective lenses (L3) and of a group of objective lenses of translation (L4) to the surface of a flow cell (FC). The red excitation radiation passes from the red LED collector lens (L7) to the dichroic combiner (F5), after which the red excitation radiation follows the same path as the green excitation radiation to the flow cell surface ( FC). As shown in the figure, focusing is performed by moving the group of translational objective lenses (L4) up and down (ie, along the z dimension). Emission from the flow cell surface (FC) passes again through the translational objective lens group (L4), and then through the stationary objective lens group (L3) to the excitation/emission dichroic (F4) which passes the emission radiation to the emission projection (LI) lens group through the emission filter and then to the CMOS image sensor (SI). A laser diode (LD) is also directed through a group of laser diode coupling lenses (L5) to the laser diode beam splitter (F3) which reflects the laser diode radiation across the field stop. excitation (FS), the excitation projection lens group (L2), the excitation/emission dichroic (F4), the stationary objective lens group (L3) and the translation objective lens group (L4) to the cell flow (FC).
[053] As demonstrated by the exemplary modalities of Figure 2 and Figure 3, each of the microfluorometers can include a beam splitter and a detector, in which the beam splitter is positioned to direct excitation radiation from a source of excitation radiation to the objective and direct emission radiation from the objective to the detector. As shown in the figures, each microfluorometer can optionally include an excitation radiation source, such as an LED. In this case, each microfluorometer can include a dedicated radiation source, so the readhead includes multiple radiation sources, each separated into individual microfluorometers. In some embodiments, two or more microfluorometers can receive excitation radiation from a common radiation source. In this way, two or more microfluorometers can share a radiation source. In an exemplary configuration, a single radiation source can direct radiation to a beam splitter that is positioned to separate the excitation radiation into two or more beams and direct the beams to two or more respective microfluorometers. Additionally or alternatively, excitation radiation may be directed from a radiation source to one, two or more microfluorometers through one or more optical fibers.
[054] It will be understood that the particular components shown in the figures are exemplary and can be replaced by components of similar function. For example, one can use any of a variety of radiation sources in place of an LED. Particularly useful radiation sources are arc lamps, lasers, semiconductor light sources (SLSs), or laser diodes. LEDs can be purchased, for example, from Luminus (Billerica, Massachusetts, USA). Similarly, a variety of detectors are useful including, but not limited to, a load-coupled device (CCD) sensor; photomultiplier tubes (PMT's); or complementary semiconductor metal oxide sensor (CMOS). A particularly useful detector is a 5-megapixel CMOS sensor (MT9P031) available from Aptina Imaging (San Jose, California, USA).
[055] Figure 2 and Figure 3 provide exemplary modalities of a microfluorometer that includes two excitation sources. This setting is useful for detecting at least two fluorophores that are excited at different wavelengths, respectively. If desired, a microfluorometer can be configured to include more than two excitation sources. For example, a microfluorometer can include at least 2, 3, 4 or more different excitation sources (ie, sources that produce different wavelengths from each other). Alternatively or additionally, beam splitters and optical filters can be used to expand the range of excitation wavelengths available from an individual radiation source. Similarly, multiple radiation sources and/or optical filtering of split excitation beams can be used for modalities where several microfluorometers share the excitation from one or more radiation sources. As discussed in greater detail elsewhere in this document, the availability of multiple excitation wavelengths is particularly useful for sequencing applications that utilize several different fluorophore labels.
[056] Figure 4 shows an exemplary arrangement of four microfluorometers in a single readhead 150. The four microfluorometers are arranged in a decal sketch with respect to channels 161 and 162 of a flow cell 160. In the arrangement shown, two of the microfluorometers (corresponding to detectors 108a and 108c) are configured to image separate regions of a first channel 161 and the other two microfluorometers (corresponding to detectors 108b and 108d) are configured to image separate regions of a second channel 162. As per shown, the microfluorometers (corresponding to detectors 108a and 108c) are offset relative to the microfluorometers (corresponding to detectors 108b and 108d) in the x dimension so that the two pairs of microfluorometers can detect adjacent channels 161 and 162 respectively. Microfluorometers have an orthogonal emission and excitation path (as shown in Figure 2) with radiation sources 102 positioned on the same side of the read head, opposite the flow cell 160. Two of the detectors 108a and 108c are positioned on a first side of the read head and the other detectors 108b and 108d are positioned on the opposite side, both sides being orthogonal to the side where the excitation sources are positioned. In the exemplary embodiment shown in Figure 4, the four radiation sources are in thermal contact with a single large heatsink 120. A single large heatsink provides a greater degree of heat dissipation than many configurations that use a single large heatsink. individual heat for each radiation source. However, if desired, individual radiation sources can be thermally coupled to individual heat sinks (see, for example, Figure 8 and the related description below). An advantage of the microfluorometer arrangement shown in Figure 4 is the provision of a compact readhead. Similar advantages can be derived for modalities where the relative positions of the excitation source and detector in each microfluorometer are exchanged (see, for example, Figure 8 and the related description below).
[057] The readhead 150 shown in Figure 4 is positioned for scanning in the y dimension. Dimension y is parallel to the length of flow cell 160 so that movement of read head 150 in a scanning operation results in imaging of areas along the length of flow cell 160. Detectors 108a, 108b, 108c and 108d are positioned on opposite sides of readhead 150, and on opposite sides of flow cell 160, the sides of the flow cell extending along the scan direction. The orientation of scan head 150 relative to flow cell 160 and scan direction is exemplary. Other orientations are also useful, including, for example, the orientation shown in Figure 13 where the detectors are positioned on opposite sides of the readhead, but in a forward and backward position relative to the scan direction.
[058] A microfluorometer, or read head having multiple microfluorometers, can be positioned above a flow cell (in relation to the arrow of gravity action) as exemplified for several modalities presented here. However, it is also possible to place a microfluorometer, or read head, below a flow cell. Correspondingly, a flow cell may be transparent on the upper side, underside or on both sides with respect to the used excitation and radiation emission wavelengths. In fact, in some embodiments, it may be desirable to position microfluorometers on both sides of a flow cell or to position readheads on both sides of a flow cell. Other gravity orientations are also possible, including, for example, a side-by-side orientation between a flow cell and a microfluorometer (or readhead).
[059] A microfluorometer or readhead can be configured to detect the two opposing inner surfaces of a flow cell from a single side of the flow cell. For example, the microfluorometer or read head can employ an optical compensator that is inserted and removed to detect alternate surfaces of the flow cell. Exemplary methods and apparatus for detecting opposing internal surfaces of a flow cell, such as the use of optical compensators, are described in US 8,039,817, which is incorporated herein in its entirety by way of reference. A compensator is optional, for example, depending on the NA and/or optical resolution of the device.
[060] A microfluorometer used in an apparatus or method presented in this document may include an autofocus module. Correspondingly, multiple microfluorometers that are present in a readhead can have a dedicated autofocus module. An exemplary 1600 autofocus module is shown in Figure 5. The module includes a 1602 receptacle for a microfluorometer objective (eg, the translation objective lens shown in Figure 3). Receptacle 1602 is secured to a slider bracket 1603 having a lever arm 1604. Lever arm 1604 functionally interacts with a motor 1610 that is configured to move the lever arm up and down (along the z direction). In this way, the 1610 motor actuates the objective movement in the z direction to change the focus. The 1610 motor is a linear actuator that uses a lead screw. Rotation of an internal lead thread under the rotational force of the motor causes the lead nut 1613, through which the lead thread is threaded, to move up and down. Lead nut 1613 is positioned between two bearings 1611a and 1611b. The movement of the advance nut is oriented against spring 1608. The advance nut 1613 is in physical contact with the lever arm 1604 so that the up and down movement of the advance nut actuates the up and down movement. under the 1603 slide holder and, consequently, the objective. A 1609 sensor is located on the underside of the autofocus module separated from the actuator by a 1612 spacer.
[061] The autofocusing module 1600 shown in Figure 5 further includes a structural support having a side body 1607 connected to a back plane 1614 and connected to an upper bend 1606 and a lower bend 1605. of side body box frame 1607. In addition, rigidity is provided by two triangular supports 1615a and 1615b between side body 1607 and back plane 1614. Flexions 1606 and 1605 can be co-molded with the sliding support to provide a high tolerance between sliding bracket 1603 and side body 1607.
[062] As shown by the exemplary modality of Figure 5, an autofocusing module that is used in a microfluorometer can include a detector and an actuator, in which the actuator is configured to change the focus of the microfluorometer in relation to the common plane, and in that the detector is configured to direct actuator movement. Thus, an autofocusing module can include a dedicated detector that directs actuator movement. The dedicated detector can operate in a closed loop with the actuator without the need to communicate data outside the microfluorometer or outside the detection head in order to achieve automatic focusing. Alternatively or additionally, a detector outside the autofocus module, such as the imaging detector that is used for wide-field imaging, can direct the actuator's movement. Therefore, the same detector that is used for wide-field imaging and outputting image data to a processing unit out of the microfluorometer or readhead can also be used to achieve automatic focusing.
[063] In particular embodiments, autofocus modules for two or more microfluorometers in a readhead can be configured to communicate with each other. For example, an autofocusing module for a first microfluorometer of a readhead can be configured to integrate data from an autofocusing module to a second microfluorometer of the device. Thus, the autofocusing module for the first microfluorometer can change the focus of the first microfluorometer based on the perceived focal position of the first microfluorometer and the perceived focal position of the second microfluorometer. Therefore, a detector for an autofocus module can be configured so that it is dedicated to genetically focusing along a readhead while not being configured for analytical image acquisition. Information from two different autofocus modules can be useful in determining the readhead tip tilt. Unwanted tip tilt can be corrected by compensating actuation of one or more microfluorometers based on the tip tilt information.
[064] Although autofocusing has been exemplified in relation to an advancing screw motor, it will be understood that autofocusing modules that use other modes of actuation can be used including, for example, those that use a piezomotor or voice coil motor in place of the lead screw motor exemplified above.
[065] A readhead can include two or more microfluorometers, for example, attached to a carriage. For modalities that utilize a multi-channel flow cell, the readhead can include a series of microfluorometers that correspond to the number of channels in the flow cell. As shown earlier by the example in Figure 4, more than one microfluorometer per flow cell channel may be present. In particular embodiments, a readhead can provide a single microfluorometer per flow channel. In the exemplary arrangement shown in Figure 6, the flow cell has four channels and the readhead has four microfluorometers. The figure shows a top plan view of the flow cell and microfluorometer objectives. For ease of demonstration, microfluorometer components other than objectives are not shown; however, those components can be positioned to achieve a compact design, for example, along the lines exemplified elsewhere in this document. As shown in panel A of Figure 6, the four objectives can be arranged in a linear relationship so that the objectives are tightly conditioned and an imaginary straight line passes through the center point of each objective. The imaginary line can be offset at an angle to the y dimension, with the y dimension being the longest dimension of the flow cell (or sweep direction). The angle can be between 0° and 90° in the x-y quadrant and can be selected to accommodate the spacing of the channels in the flowcell (and the spacing of the objectives in the readhead). Figure 6A shows a relatively small offset angle for a line passing through the tightly conditioned objectives that accommodate the relatively strictly conditioned channels. A larger offset angle can be used to accommodate channels that are separated by greater distances from each other or objectives that are packed more tightly.
[066] Panel B of Figure 6 shows an array of multiple objectives in two lines. In this document, the flow cell includes eight channels and the readhead has eight microfluorometers. The overall packaging of the objectives on the two lines is approximately straight. The arrangement accommodates strictly conditioned objectives and two sets of strictly conditioned channels (ie, a first set of four strictly conditioned channels and a second set of four strictly conditioned channels). In this example, the two sets of tightly wrapped channels are separated by a spacing greater than the spacing separating the individual channels in each set of four. It will be appreciated that the overall packaging of the objectives on the two lines can be shifted from the straight packaging to accommodate different channel arrangements. Additionally, as shown in relation to a single objective line, the offset angle of the imaginary line extending through the centers of both objective lines can be changed and/or the distance between the objectives can be changed to accommodate different arrangements of channel.
[067] Figure 7 demonstrates a multiple objective arrangement in which an imaginary line extending through the objective centers is at a 90° angle to the longest dimension of the flowcell (or scan direction). The imaginary line extends along the geometric x axis. In this example, the objectives are in two rows and are hexagonally stowed. Hexagonal packing provides the advantage of maximum compression in the x-y plane. The readhead is shown with six objectives and the flow cell has six channels. It will be appreciated that similar arrangements can be used for a readhead having only four objectives or for readheads having more than six objectives (eg eight objectives as shown in Figure 8, Figure 9, and Figure 13 ). As is evident by visual comparison, the flow cell channels are spaced even further apart in the arrangement in Figure 7 than in the arrangement in Figure 6. However, the channel spacing in both cases is within a useful and convenient range. , for example, for nucleic acid sequencing applications.
[068] As demonstrated by the examples in Figures 6 and 7, each objective in a readhead can be positioned to image at least a portion of an individual flow channel. Each objective can be positioned to image one and only one channel of a flow cell having multiple channels. A single objective can be positioned to image a portion of one and only one channel, for example, when located at a particular y-stage position. Y-dimension scanning can allow all or part of the channel to be imaged through the objective. In some cases, for example, when the field diameter of the objective (or other limiting optical components of a microfluorometer) is smaller than the channel width, the objective can also be swept in the x dimension to image all or part of the channel. Multiple objectives and their respective microfluorometers can be positioned so that many of the objectives are positioned to obtain images for at least a portion of one and only one channel. Naturally, movement of a readhead containing the multiple objectives and their respective microfluorometers can occur in the y and/or x direction to image all or part of each respective channel. These particular configurations are useful for multi-channel flow cells as exemplified above. However, it will be understood that the configurations and fundamentals presented above can be applied to an appropriate arrangement of several individual flow cells, each of which has only a single channel. Additionally, as is the general case for the methods and apparatus presented in this document, the provisions can be applied to substrates other than flow cells.
[069] A perspective view of a 1000 readhead having an array of eight microfluorometers is shown in Figure 8. Each microfluorometer has a compact design similar to that shown in Figure 3. For ease of demonstration, the components of only one of the microfluorometers are marked in Figure 8 and will be described in this document. However, as seen in Figure 8, each of the microfluorometers has similar components and configurations. Two excitation sources are present in each microfluorometer, including a 1040 green LED and a 1030 red LED. Excitation light from the LEDs passes through a 1075 green LED collector lens and a 1076 red LED collector lens, respectively. A 1074 LED retractable mirror reflects green excitation radiation to a 1073 dichroic combiner which reflects green excitation radiation through a 1072 laser diode beam splitter, then through a 1071 excitation projection lens to a dichroic excitation/emission sensor 1060 which reflects the green excitation radiation through a 1010 objective. The red excitation radiation passes from the red LED collector lens 1076 to the dichroic combiner 1073 after which the red excitation radiation follows the same trajectory as the green excitation radiation. The 1010 objective is positioned to collect emission radiation and direct it through the 1060 excitation/emission dichroic which passes the emission radiation to the 1080 CMOS image sensor. A laser diode 1301 is positioned to direct radiation through a group of diode laser coupling lens 1401 to diode laser beam splitter 1072 which reflects laser diode radiation through excitation projection lens 1071, excitation/emission dichroic 1060, and objective 1010. 1600 autofocus is coupled to at least part of the 1010 objective and configured to translate the 1010 objective up and down (ie, along the z dimension). The autofocusing module may, but need not, include components of the autofocusing apparatus exemplified in Figure 5. It will be appreciated that additional optical components may be present in the readhead 1000 including but not limited to those exemplified for the Figure B. Additionally, certain optical components may be absent from the 1000 readhead or modified in the 1000 readhead to suit particular applications. Printed circuit boards 1701 and 1702 can be configured to communicate with detectors, autofocus modules and/or excitation sources.
[070] Figure 9 shows a bottom plan view of the 1000 read head. Again, for ease of demonstration, the components of only one of the microfluorometers are marked in Figure 9 and described in this document. The 1030 red LED is shown in thermal communication with the 1201 heatsink and in optical alignment with the 1076 red LED collector lens. The green LED is dimmed by the 1030 red LED and most of the excitation path is dimmed by the autofocus module 1600 in this view. The 1010 objective is visible conforming to a portion of the 1080 CMOS image sensor; however, most of the emission path is darkened in this view. As is evident from the figures, the objectives are arranged in two rows and packed hexagonally.
[071] The configurations described above exemplify a readhead in which each of the microfluorometers includes at least one radiation source, a beam splitter and a detector, in which the beam splitter is positioned to direct the excitation radiation from from the excitation radiation source to the objective and directing the emission radiation from the objective to the detector, wherein the excitation radiation and emission radiation are directed in mutually orthogonal directions. In the modalities exemplified in Figures 8 and 9, the detectors for several microfluorometers are arranged on a first side of the readhead that is opposite the common plane to which the objectives are focused, a subset of the radiation sources is arranged on a second side of the read head (the second side being orthogonal to the first side and orthogonal to the common plane) and a second subset of the radiation sources is disposed on a third side of the read head (the third side being opposite the second side, orthogonal to the first side and orthogonal to the common plane). Alternatively, and as exemplified in Figure 4, the radiation sources for several microfluorometers are disposed on a first side of the readhead that is opposite the common plane to which the objectives are focused, a first subset of the detectors is disposed on a second side of the read head (the second side being orthogonal to the first side and orthogonal to the common plane) and the second subset of the detectors is arranged on a third side of the carriage (the third side being opposite the second side, orthogonal to the first side and orthogonal to the common plan).
[072] In addition to the previous modalities in which the excitation and emission trajectories are orthogonal, the configurations where the emission and excitation trajectories are parallel can also be useful. In this case, the excitation radiation source(s) and the detector may be present on the same side of the readhead. An exemplary sketch for a microfluorometer 800 is shown in Figure 10, where excitation radiation from excitation source 805 passes through excitation optics 806 to prism surface 807 which reflects excitation radiation to prism surface 802 which reflects the Excitation radiation through objective 801. The emission passes through objective 801, then through beam splitter 802 to projection lens 803 and then to detector 804. The emission path is parallel to most of the excitation path. The detector and excitation radiation source are located on the same side of the microfluorometer, opposite and parallel to the detection plane. A guide 810 is configured to interface with a flow cell or substrate to align the objective. A similar guide can be used on other microfluorometers presented in this document. The sketch for microfluorometer 800 is exemplary for purposes of demonstrating a parallel arrangement of excitation and emission trajectories. Other components may be included, such as those shown in other figures including, but not limited to, an autofocus module. For example, an excitation source 809 for an autofocusing module is shown and produces excitation that passes through the prism surface 807 and is reflected by the prism surface 802 to pass through the objective 801. objectives in one or more lines, as exemplified in Figures 6 and 7.
[073] As demonstrated by the exemplary modalities above, a readhead can include a plurality of objectives, with each objective being dedicated to a single microfluorometer. Thus, a microfluorometer of the present disclosure can include a variety of optical components, such as one or more detectors, excitation radiation sources, beamsplitter lenses, mirrors, or the like, that form an optical train that directs excitation radiation through a single objective and/or receiving emission radiation through a single objective. In these modalities, the objective can be configured as a macro-lens having a wide field of view. In alternative embodiments, a microfluorometer of the present disclosure can include a variety of optical components that direct excitation radiation through multiple objectives and/or receive emission radiation through multiple objectives. Therefore, an individual microfluorometer can include multiple optical trains that include multiple objectives. In modalities that include multiple objectives per microfluorometer, the objectives can optionally be configured as a microlens array. Each objective among several in a particular microfluorometer (for example, each microlens in a microlens array) can optionally be set to independent focusing, so each objective can be moved in z dimension independently of other objectives in the same microfluorometer. Alternatively or additionally, the various objectives can be set to global focus so that they can all be moved together in the z dimension.
[074] It will be understood that the various components of a readhead that are presented in this document can be mixed and matched in various ways in order to achieve a function similar to those exemplified in this document. For example, as shown in the previous paragraph, a readhead can include multiple objectives and each of these objectives can be dedicated to a single microfluorometer, or alternatively, several of these objectives can be shared by a single microfluorometer. Similarly, and as discussed previously herein, each microfluorometer may include at least one dedicated excitation source or, alternatively, two or more microfluorometers may receive excitation radiation from a shared radiation source. Therefore, there is no need for a one-to-one correspondence between the number of microfluorometers in a particular readhead and the number of components exemplified herein for any given microfluorometer modality. Preferably, one or more of the components exemplified herein as being useful in a microfluorometer may be shared by several microfluorometers in a particular readhead.
[075] A readhead of the present disclosure is particularly useful for scanning methods and apparatus, for example, due to its relatively compact size and small mass that provides low inertia. Reduced inertia allows the readhead to come to rest more quickly following movement, thus allowing high resolution images to be obtained faster than they would be in the case of a higher inertia readhead at which residual motion of the readhead reading would cause blurring and loss of resolution. The settings for achieving readhead movement will be presented in more detail below. However, it should first be noted that the low inertia advantage is not intended to be a limitation or requirement for an apparatus or method presented in this document. Preferably, a readhead of the present disclosure can be held in a static position for all or part of a detection protocol. For example, a sequencing method, such as one using the fluidic and imaging steps presented here, can be performed using a readhead that is static for at least one and perhaps all cycles of the sequencing method. sequencing.
[076] As a first example of a static readhead embodiment, a readhead may include a sufficient number of microfluorometers to detect or image a desired portion of a surface or other object. Therefore, the readhead does not need to move in x or y dimensions. For example, multiple microfluorometers can be linearly arranged to capture image frames along the entire length (or at least along the target effective length) of a flow cell channel. Similarly, using an appropriate packaging arrangement of multiple rows of microfluorometers such as that shown herein, multiple flow cell channels (present in one or more flow cells) can be imaged along their length. total (or at least along the target effective length). As shown above, image frames taken for an individual channel can, but need not, be contiguous.
[077] As a second example of a static readhead modality, a readhead can remain in a fixed position with respect to x and y dimensions while a substrate being detected by the readhead is translated in x and/or y dimension . For example, an apparatus may be provided having a translation stage which is configured to present a substrate to the readhead. The translation stage can move in a “step-and-shoot” or contiguous motion to allow scanning of the substrate by the static readhead. In particular embodiments, the substrate is a flow cell that can be attached to the translation stage. The flow cell can be translated as part of a fluidic cartridge, such as the one exemplified below, or the flow cell can be translated independently of any fluidic cartridge. Hence, the translation stage can be configured to attach a fluidic cartridge to which a flow cell is attached and move the fluidic cartridge along which the flow cell or the translation stage can be configured to move only the flow cell while the fluidic cartridge remains in a static or fixed position.
[078] According to the previous examples, the relative movement between a scan head (or microfluorometer) and a substrate can be achieved by physical movement of the scan head (or microfluorometer), physical movement of the substrate, or physical movement both. It will be appreciated that the static readheads referred to in the first and second exemplary embodiments above need not be static with respect to movement in the z dimension. Preferably, static readheads can include one or more microfluorometers having autofocus modules. Alternatively or additionally, the readheads can be moved as a whole in the z dimension, for example, to achieve a global focus at least to an approximation.
[079] Turning now to the modalities in which a read head is translated, Figures 11 and 12 show top and bottom views, respectively, of a translation stage and an example 200 for a read head. In this exemplary modality, the y stage is set to translation in the y dimension, but not in the x dimension. Therefore, a readhead transported by the y 200 translation stage will be able to make a movement in the y dimension and the readhead or individual microfluorometers on it may be able to make a movement in the z dimension (for example, through autofocusing), but the readhead will not be able to make a move in dimension x. A readhead can be attached to carriage 201 having a base area 241 positioned to support the underside of the readhead and a frame 240 configured to restrict lateral movement of the readhead. Carriage 201 further includes a flange guide 243 and a collar guide 242. An aperture 244 in the base area 241 provides a window between a readhead and a substrate to be detected by the readhead. The aforementioned components of carriage 201 can form a monolithic structure.
[080] The carriage is configured to move along a y stage frame 207 via a first axis 203, along which the collar guide 242 extends and a second axis 204 along which the flange guide 243 extends. The axes are oriented along the y axis so that the carriage 201 is directed to slide back and forth along the y dimension through the guides. The first axis 203 is held to the y-stage frame 207 by insertion at the reference plane 215 on a first side wall 250 and at the reference plane 218 on a second side wall 251. The first axis 203 is fixed to the reference plane 215 by support member 252 and secured to reference plane 218 by support member 253. Second axis 204 is held to y stage frame 207 by insertion into reference plane 214 in a first sidewall 250 and in reference plane 217 in a second sidewall 251. The first axis 204 is fixed to the reference plane 214 by the rod clamp 206 and fixed to the reference plane 217 by the rod clamp 205.
[081] The movement of the carriage 201 is triggered by the rotation of the feed thread 202 which is threaded through a feed nut 260 and which is fixed to the y stage frame 207 by insertion into a reference plane on the first side wall 250 and in a reference plane 219 on the second side wall 251. The lead screw 202 is held in position by the same support members 252 and 253 that secure the first shaft 203. The rotation of the lead thread 202 is driven by the motor 212 which is mounted to support member 252. An encoder 208 is configured to interact with motor 212 through a belt 210 that interacts with rotor 209 on encoder and rotor 211 on motor 212. A belt tensioner 220 interacts with belt 210.
[082] An aperture 230 passes through the floor 216 of the y-stage frame 207. The aperture 230 is positioned to accommodate the aperture path 244 in the base area 241 of the carriage 201 as it traverses the y-stage frame. A readhead is positioned on the carriage so that objectives are directed through aperture 244 and through aperture 230 along a path traversed by the carriage. Correspondingly, aperture 230 accommodates imaging of an elongated area along the y axis through movement of a readhead attached to the carriage.
[083] The structural and functional relationship between the y 200 translation stage and the 1000 read head is shown in Figure 13. The orientation of the 1010 objectives in relation to the scan direction of the 200 y translation stage is similar to that exemplified in Figure 7 (except that the 1000 readhead has two additional objectives). A flow cell can be oriented relative to the y 200 translation stage as shown in Figure 7.
[084] As exemplified above, a carriage can be configured to move a readhead, for example, in a scan operation. Alternatively or additionally, a carriage can be configured to prevent relative movement between the individual microfluorometers of a readhead in x and y dimensions. A carriage does not need to provide this function, for example, if the readhead includes other structural elements that prevent relative movement between individual microfluorometers. For example, a readhead can be formed from a co-molded assembly. The co-molded mount can successively be fixed to a car. However, in some modalities, the carriage can play at least an auxiliary role in preventing a relative transverse movement between the individual microfluorometers of a readhead. Additionally, it will be appreciated that a readhead that is formed from a co-molded assembly can be used for embodiments that do not employ a carriage.
[085] A y stage that is used in a method or apparatus presented in the present invention can be configured to sweep through a discontinuous or continuous motion. Discontinuous scanning, often referred to as step-and-shoot scanning, usually involves incremental movement of a microfluorometer or scan head in the y (or x) direction and detection (eg, image acquisition) between movements, while the microfluorometer or scanhead is in a temporarily static state. Continuous scanning, on the other hand, usually involves detecting or acquiring an image while the microfluorometer or scan head is in motion. In a particular modality, a continuous scan can be performed in a time delay integration (TDI) mode. Correspondingly, the signal obtained by pixel elements along the scan dimension can be collected into a common binary and read as a single value. TDI mode can provide advantages of increased signal processing rate and increased accuracy. Exemplary optical arrangements that may be included in a microfluorometer or readhead to accommodate detection in TDI mode are described, for example, in US 7,329,860, which is incorporated herein by reference.
[086] The movement of a microfluorometer or scan head in an x or y dimension, for example, to accommodate continuous or discontinuous scanning modalities, can be controlled by an encoder or other device. In the example of stage 200, motion can be controlled by encoder 208. As shown earlier in this document, scanning (whether by continuous or discontinuous techniques) can result in the acquisition of contiguous or non-contiguous frames from a substrate or another object under detection. Thus, the sum total of portions that have raster-formed images can be contiguous (but not superimposed), non-contiguous, or superimposed. The system does not need to be configured to image the entire substrate or object (eg array surface) and does not need to do so in order to allow a composite image to be produced.
[087] An electrical block diagram for a sensing apparatus is shown in Figure 14. A read printed circuit board (PCB) is present in a read head (see, for example, PCB 1701 and 1702 in Figure 8) and is connected to a main PCB which is typically contained in the housing detection apparatus. In alternative modalities, the main PCB can be located externally to the instrument. Data can be communicated between the read PCB and the main PCB via the LVDS line. For example, a flat flex cable (FFC) 36-cond and 0.5 mm slope can be used for the LVDS line. The LVDS line can be configured to communicate image data from the read PCB to the main PCB, and camera control instructions from the main PCB to the read PCB. The two PCBs are also connected by a power line, such as a 1mm slope copper clad FFC SO-cond that transmits power from a 24-volt power supply through the main PCB. FFC connections are configured to be of sufficient length and flexibility to allow the read PCB to move with the read head while the main PCB remains stationary.
[088] In the example in Figure 14, the main PCB is also connected to an external primary analysis personal computer (PC) via the SS LF USB 3.0 connectors. In some embodiments, the primary analysis computer may be located within the detection apparatus housing. However, leaving the primary analysis computer instrumentless allows interchangeable use of a variety of computers to be used for different applications, convenient maintenance of the primary analysis computer by replacement without having to interrupt detection apparatus activity, and small footprint for the detection apparatus. Any of a variety of computers can be used including, for example, a desktop computer, a laptop computer, or a server containing a processor in operational communication with accessible memory and instructions for implementing the computer-implemented methods described herein. The main PCB is also connected to a liquid crystal display (LCD) for communication with a human user. Other interfaces can also be used.
[089] In some embodiments, a user interface may include a screen (eg an LCD) to display or request information from a user and a user input device (eg a keyboard) to receive input from a user. user. In some modes, the screen and user input device are the same device. For example, the user interface might include a touchscreen configured to detect the presence of an individual touch and also identify a touch location on the screen. However, other user input devices can be used, such as a mouse, touchpad, keyboard, numeric keypad, hand scanner, voice recognition system, motion recognition system, and the like.
[090] The read PCB includes eight DS90CR217 transmitters to transfer data from individual sensors (ie, detectors) to the LVDS line, 3.3 volt switching regulator, a 5 volt switching regulator, and high-end buck drives. LED for the LED excitation radiation sources.
[091] The main PCB includes an FPGA + processor configured to accept image data from the LVDS. A DDR3 DIMM frame buffer is electronically connected to the FPGA + processor. The main PCB also includes a thermal control regulator and control circuitry for various drive motors, such as a y-axis motor, a cartridge motor, a valve motor, and a pump motor.
[092] The present disclosure further provides a method for imaging a substrate, including the steps of (a) providing a substrate including fluorescent features on a surface; (b) acquiring a plurality of wide-field images of a first portion of the surface using a plurality of microfluorometers, wherein each of the microfluorometers acquires a wide-field image from a different location on the surface, wherein the plurality of microfluorometers is attached to a car; and (c) translate the carriage in a direction parallel to the surface and repeat (b) for a second portion of the surface. The method can use any apparatus shown anywhere in this document, but need not be limited in all embodiments.
[093] The modalities of the present methods are particularly used for nucleic acid sequencing techniques. For example, sequencing by synthesis (SBS) protocols are particularly applicable. In SBS, the extension of a nucleic acid primer along a nucleic acid template is monitored to determine the nucleotide sequence in the template. The basic chemical process can be polymerization (eg, as catalyzed by a polymerase enzyme) or ligation (eg, catalyzed by a ligase enzyme). In a particular polymerase-based SBS embodiment, fluorescently labeled nucleotides are added to a primer (thus extending the primer) in a template-dependent manner so that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the model. A plurality of different models can be subjected to an SBS technique on a surface under conditions where events that occur for different models can be distinguished. For example, models can be present on the surface of an array so that the different models are spatially distinguishable from each other. Typically, models occur on resources having multiple copies of the same model (sometimes referred to as “clusters” or “colonies”). However, it is also possible to perform SBS in arrays where each resource has a single model molecule present, so that the single model molecules are solvable to each other (sometimes referred to as “single molecule arrays”).
[094] Flow cells provide a convenient substrate to house an array of nucleic acids. Flow cells are convenient for sequencing techniques because the techniques typically involve the repeated distribution of reagents in cycles. For example, to initiate a first cycle of SBS, one or more labeled nucleotides, DNA polymerase, etc., can be eluted through a flow cell that houses an array of nucleic acid templates. Features where the primer extension that causes a labeled nucleotide to be incorporated can be detected, for example, using methods or apparatus disclosed herein. Optionally, nucleotides can further include a reversible termination property that terminates primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer so that further extension cannot occur until an unlocking agent is delivered to remove the moiety. Therefore, for modalities that use a reversible termination, an unlocking agent can be delivered to the flow cell (before or after detection takes place). Washes can be carried out between the various stages of distribution. The cycle can then be repeated n times to extend the primer by n nucleotides, thus detecting a sequence of length n. Exemplary sequencing techniques are described, for example, in Bentley et al, Nature 456:53-59 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 , and US 2008/0108082 , each of which is incorporated herein by reference.
[095] For the nucleotide delivery step of an SBS cycle, a single nucleotide type can be delivered at once, or multiple different nucleotide types (eg, A, C, T and G together) can be delivered . For a nucleotide distribution configuration where only a single nucleotide type is present at a time, the different nucleotides need not have distinct tags as they can be distinguished based on temporal separation inherent in the individualized distribution. Correspondingly, a sequencing method or apparatus may use single color detection. For example, the microfluorometer or readhead need only provide excitation at a single wavelength or a single wavelength range. Therefore, a microfluorometer or read head only needs a single excitation source and filtration in multiple excitation bands is not necessary. For a nucleotide delivery configuration where the delivery results in multiple different nucleotides being present in the flow cell at once, features that incorporate different nucleotide types can be distinguished based on different fluorescent labels that are attached to the respective nucleotide types in the mix. For example, four different nucleotides can be used, each having one of four different fluorophores. In one embodiment, the four different fluorophores can be distinguished using excitation in four different regions of the spectrum. For example, a microfluorometer or readhead can include four different excitation radiation sources. Alternatively, a readhead may include fewer than four different excitation radiation sources, but may use optical filtration of excitation radiation from a single source to produce different bands of excitation radiation in the flow cell.
[096] In some embodiments, four different nucleotides can be detected in a sample (eg, nucleic acid resource array) using fewer than four different colors. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity of one member of the pair compared to the other, or based on a change in one member of the pair. (eg, through chemical modification, photochemical modification or physical modification) which causes an evident signal to appear or disappear compared to the signal detected by the other member of the pair. As a second example, three out of four different nucleotide types may be detectable under particular conditions while a fourth nucleotide type is lacking a label that is detectable under those conditions. In an SBS embodiment of the second example, the incorporation of the first three nucleotide types in a nucleic acid can be determined based on the presence of their respective signals, and the incorporation of the fourth nucleotide type in the nucleic acid can be determined based on the absence of any sign. As a third example, a nucleotide type can be detected in two different images or in two different channels (eg a mixture of two species that have the same base but different markers can be used, or a single species that has two markers can be used or a single species that has a marker that is detected in both channels can be used), while other nucleotide types are detected in more than one of the images or channels. In this third example, comparing the two images or two channels serves to distinguish the different types of nucleotides.
[097] The three exemplary configurations in the paragraph above are not mutually exclusive and can be used in various combinations. An exemplary embodiment is an SBS method that uses reversibly blocked nucleotides (rbNTPs) that have fluorescent labels. In this format, four different nucleotide types can be distributed to an array of nucleic acid resources and will be sequenced and due to reversible blocking groups one and only one incorporation event will occur in each resource. The nucleotides distributed in the array in this example may include a first type of nucleotide that is detected in a first channel (eg, rbATP which has a tag that is detected in the first channel when excited by a first excitation wavelength), a second type of nucleotide that is detected in a second channel (eg rbCTP that has a tag that is detected in the second channel when excited by a second excitation wavelength), a third type of nucleotide that is detected in the first and second channel (eg rbTTP which has at least one label which is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type which is devoid of a label which is detected in each channel (eg rbGTP which has no extrinsic tag).
[098] Once the four nucleotide types have been contacted with the array in the example above, a detection procedure can be performed, for example, to capture two images of the array. Images can be taken in separate channels and can be taken simultaneously or sequentially. A first image obtained using the first excitation wavelength and emission in the first channel will show features that incorporate the first and/or third nucleotide type (eg, A and/or T). A second image taken using the second excitation and emission wavelength in the second channel will show features that incorporate the second and/or third nucleotide type (eg, C and/or T). Unambiguous identification of the nucleotide type embedded in each feature can be determined by comparing the two images to arrive at the following: features that appear in the first channel incorporate the first nucleotide type (eg A), features that appear only in the second channel incorporate the second type of nucleotide (eg C), features that appear in the channel incorporate the third type of nucleotide (eg T) and features that do not appear in each channel incorporate the fourth type of nucleotide (eg G). Note that the location of features that incorporate G in this example can be determined from other cycles (where at least one of the other three nucleotide types is incorporated). Exemplary apparatus and methods for distinguishing four different nucleotides using less than four color detection are described, for example, in US Patent Application Ser. 61/538,294, which is incorporated herein by reference.
[099] In a sequencing method, a microfluorometer can acquire at least two wide-field images of the same area of a surface during each cycle, where each of at least two wide-field images is acquired using excitation wavelengths or different issue. For example, during each cycle a microfluorometer can acquire two, three, or four wide-field images of the same area of a surface during each cycle, where each of the two wide-field images detects fluorescence in different regions of the spectrum. Alternatively or additionally, a microfluorometer can acquire wide-field images that detect fluorescence in no more than two, three, or four different regions of the spectrum for a given area of a surface during a given sequencing cycle. For example, a microfluorometer can excite an area of a flow cell surface with radiation in no more than two, three, or four different regions of the spectrum during a given cycle and/or a microfluorometer can acquire wide-field images of a given area. of a surface in no more than two, three, or four different regions of the spectrum during a given cycle. Different wide-field images can be taken at different times (eg sequentially) or in some modalities two or more wide-field images can be taken simultaneously.
[0100] In the context of the present disclosure "different wide-field images of an area" refers to two or more wide-field images of the same area that are acquired under different excitation and/or emission conditions. Alternatively, two or more separate wide-field images of the same area can be acquired under the same or at least similar excitation and emission conditions. For example, multiple frames can be taken from an area of a given object under a given fluorescence detection condition and frames can be co-added. Coaddition can provide the advantage of increasing signal to noise as compared to obtaining a single frame under the same conditions. A further example is that coaddition can be performed in conjunction with pulsed excitation to reduce sun exposure damage to the sample as compared to continuous excitation of the sample for an extended period of time (which may or may not obtain signal strength or signal ratio for similar noise).
[0101] In some embodiments, nucleic acids can be attached to a surface and amplified before or during sequencing. For example, amplification can be performed using bridging amplification to form clusters of nucleic acid on a surface. Useful bridge amplification methods are described, for example, in US 5,641,658; US 2002/0055100; US 7,115,400; US 2004/0096853; US 2004/0002090; US 2007/0128624; or US 2008/0009420, each of which is incorporated herein by reference. Another useful method for amplifying nucleic acids on a surface is rolling circle amplification (RCA), for example, as described in Lizardi et al, Nat. Genet. 19:225-232 (1998) and US 2007/0099208 A1, each of which is incorporated herein by reference. Emulsion PCR on microspheres can also be used, for example, as described in Dressman et al, Proc. Natl. Academic Sci. USA 100:8817-8822 (2003), WO 05/010145, US 2005/0130173 or US 2005/0064460, each of which is incorporated herein by reference.
[0102] As shown above, the sequencing modalities are an example of a repetitive process. The methods of the present disclosure are well suited for repetitive processes. Some modalities are presented below.
[0103] This disclosure provides a method of imaging a substrate, which includes the steps of (a) providing a substrate that includes fluorescent features on a surface; (b) acquiring a plurality of wide-field images of a first portion of the surface using a plurality of microfluorometers, wherein each of the microfluorometers acquires a wide-field image of a different location on the surface, wherein the plurality of microfluorometers is fixed To a car; (c) moving the car in a direction parallel to the surface and repeating (b) for a second portion of the surface; and (d) returning the carriage to a position so as to acquire a second plurality of wide-field images of the first portion of the surface. Optionally, the method may further include a step of modifying the fluorescent features on the surface after (c) and before (d), wherein the second plurality of widefield images is different from the first plurality of widefield images.
[0104] In particular embodiments, steps (a) to (c) of the above method correspond to the detection step(s) of a sequencing technique. In a related embodiment, step (d) whereby the carriage is returned corresponds to a second cycle of a sequencing technique. In this example, the modification of surface fluorescent features can include one or more biochemical steps of a sequencing technique. Exemplary sequencing techniques that can be used in the method are set forth above or otherwise known.
[0105] A fluidic cartridge is also provided which includes (a) a flow cell having an optically transparent surface, an inlet and an outlet; and (b) a housing made of a material that is optically opaque and impervious to aqueous liquids, wherein the housing contains: (i) a sample reservoir; (ii) a fluidic line between the sample reservoir and the flow cell inlet; (iii) a plurality of reagent reservoirs in fluid communication with the flow cell through the flow cell inlet, (iv) at least one valve configured to mediate fluid communication between the reservoirs and the flow cell inlet; and (v) at least one pressure source configured to move liquids from the sample reservoir or the reagent reservoirs to the flow cell through the inlet of the flow cell, wherein an optically transparent window cuts through the housing and an inlet port cuts through the housing, where the inlet port is in fluid communication with the sample reservoir, and where the optically transparent surface is positioned on the window.
[0106] The external view of an exemplary fluid cartridge 10 is shown in Figure 1 and is described above. Figure 15 shows an exploded view of a fluidic cartridge 2000. The fluidic cartridge housing is formed by a 2001 housing that is compatible with a 2002 base. The housing is on the upper side of the fluidic cartridge as shown and includes a 2009 receiving area. of a flow cell 2020. The flow cell is exposed to the outside of the housing through a 2010 window. One or more 2013 ports in the housing allow sample or other reagents to be dispensed to reservoirs on the inside of the 2000 fluidic cartridge. 2002 base includes a 2012 opening that is configured to accept a 2003 reagent tray. The 2003 reagent tray can be attached to the fluidic cartridge by insertion into the 2012 opening so that the individual reagent reservoirs in the tray are in fluidic communication with fluidic lines for the delivery of reagents to the flow cell. The housing also contains 2005 and 2006 valves that interface with pumps to move reagents through fluid lines. Also contained within the housing is a 2004 waste bag that has a 2011 inlet that interfaces with the fluid lines of the 2020 flow cell.
[0107] The housing of a fluidic cartridge (eg, casing 2001 and/or base 2002 of fluidic cartridge 2000) may be made of a material that is opaque to radiation in a particular part of the spectrum. For example, the housing can be opaque to UV, VIS and/or IR radiation to protect the reagents from damage from sun exposure due to radiation at these wavelengths. For example, a material that is opaque to UV radiation is beneficial in preventing damage from sun exposure to nucleic acids among other reagents used in sequencing reactions. As another example, it may be desired to use a material that is opaque to radiation in the wavelength range absorbed by fluorophores used as markers in a sequencing reaction.
[0108] The housing of a fluidic cartridge will typically be impermeable to liquids housed in it. Thus, the casing can provide a secondary barrier in addition to the reservoirs contained therein. Exemplary materials include plastics such as polycarbonate or polystyrene, or metals such as aluminum or stainless acid. Materials that are chemically inert to the reagents housed in the fluidic cartridge are generally desired. Individual reservoirs or other fluidic components in a fluidic cartridge will have similar fluid impermeability properties and may also optionally be opaque. Materials can be rigid or flexible. For example, any one of a variety of reagent reservoirs shown here or otherwise used in a fluidic cartridge can be a flexible bag as exemplified for the 2004 waste reservoir.
[0109] As exemplified by Figure 1 and Figure 15, a fluidic cartridge can be configured to contain a variety of components within a housing. For example, in various embodiments one or more fluidic components disclosed herein may be completely contained within the housing. In fact, in particular modalities, all fluidic components of a particular modality can be completely contained in the fluidic cartridge. For example, a cartridge housing may contain one or more sample reservoirs, one or more reagent reservoirs, one or more waste reservoirs, one or more mixing reservoirs, one or more valves configured to mediate fluid communication between a reservoir. and a flow cell, one or more pressure sources configured to move liquids from a reservoir to a flow cell, or one or more fluid lines between a reservoir and a flow cell. However, it will be understood that in some embodiments at least part of some fluidic components may be present outside the carcass. For example, a surface of a flow cell through which detection will occur may be outside a cartridge housing.
[0110] In particular embodiments, a fluidic cartridge may include a receiving area that is sized to securely retain a flow cell, for example, by a compression fit. However, in other embodiments, the receiving area may be larger than the occupied area of a flow cell that is or will be present in the fluidic cartridge. Thus, the flow cell can occupy a receiving space in the housing that is sized and shaped to allow the flow cell to float relative to the housing. A configuration that accommodates buoyancy of a flow cell may be advantageous for aligning the flow cell with the optical component of a sensing apparatus after the fluidic cartridge is placed in the instrument. Alignment can be accomplished by inserting one or more alignment pins into the receiving area by the detection device, for example, in cases where the pins are pre-aligned in a microfluorometer of a read head of the detection device. Correspondingly, the receiving area may include an adjustment of at least one alignment pin or other alignment member. Exemplary configurations for aligning a floating flow cell that can be adapted to a fluidic cartridge of the present disclosure are described in US Ser. No. 13/273,666 (published as US 2012/0270305 A1), which is incorporated herein by reference. . In embodiments utilizing flow cell flotation, the fluidic connection of the flow cell to other fluidic components of the cartridge will generally be flexible. For example, flexible tubing can connect a flow cell to fixed fluidic components of a cartridge.
[0111] A fluidic cartridge of the present disclosure need not include a sensing device or other sensing components described herein. For example, a fluidic cartridge can be configured to exclude a detector, microfluorometer, or readhead such as those described here or those useful in a method presented here. In nucleic acid sequencing modalities, a detector, microfluorometer, or readhead that is used to detect nucleic acids in a flow cell (or other substrate) of a fluid cartridge may be located outside of the fluid cartridge housing. Similarly in other embodiments, a detector, microfluorometer or read head that is used to detect a particular characteristic of a substrate may be excluded from the inside of a fluid cartridge housing, which is located outside the housing. It will be understood that in at least some configurations one type of detector may be excluded from a fluidic cartridge while another type of detector may be present. For example, a fluidic cartridge may exclude a detection device used to detect nucleic acids in a flow cell, but may include a detector used to assess a characteristic of a fluid in the cartridge or assess a cartridge component. More specifically, a cartridge can include a detector for temperature, pressure, flow rate, or other characteristics of the fluids used in the cartridge. Other examples of components that may be excluded from a fluidic cartridge include, but are not limited to, optical filters, lenses, objectives, cameras (eg CCD cameras or CMOS cameras), excitation radiation sources (eg LEDs) or similar.
[0112] A fluidic map of an exemplary fluidic cartridge is shown in Figure 16. Flowcell 2020 has eight lines fluidly connected to one of eight individual fluidic lines (collectively marked 2047) that are individually actuated by inlet valve 2044. Inlet valve 2044 controls fluid flow from four sample reservoirs 2030 through 2033. Inlet valve 2044 also controls fluid flow from multiple SBS 2035 reagent reservoirs and multiple 2036 amplifying reagent reservoirs. fluid flows from the SBS 2035 reagent reservoirs are controlled by the 2043 reagent selection valve. The distribution and flow of fluids from the 2036 amplifying reagent reservoirs are controlled by the 2042 reagent selection valve which is located upstream of the 2036 reagent selection valve. reagents 2043. Correspondingly, reagent selection valve 2043 is positioned to control View the distribution and flow of reagents from both the SBS 2035 reagent reservoirs and the 2036 amplifying reagent reservoirs.
[0113] Fluid flow through the system of Figure 16 is conducted by eight separate syringe pumps 2051 to 2058. The syringe pumps are positioned to draw fluid through the fluidic system and each pump can be individually actuated by valve 2045. Logo , the flow through each channel of the flow cell can be individually controlled by a dedicated pressure source. Valve 2045 is also configured to control the flow of fluids to the refuse reservoir 2060.
[0114] Figure 16 exemplifies a fluidic system in which fluids are pulled by the action of syringe pumps downstream. It will be appreciated that a useful fluidic system may use other types of devices in place of syringe slugs to convey fluids including, for example, positive or negative pressure, peristaltic pump, diaphragm pump, piston pump, gear pump or Archimedes' screw. Additionally, these and other devices can be configured to pull fluids from a position downstream relative to a flow cell or push fluids from an upstream position.
[0115] Figure 16 also exemplifies the use of eight syringe pumps for eight channels of a flow cell. Therefore, the fluidic system includes a series of pumps that is equivalent to the number of channels in use. It will be appreciated that a fluidic system that is useful in a fluidic cartridge of the present disclosure may have fewer pumps (or other sources of pressure) than the number of channels in use. For example, multiple channels can be fluidly connected to a shared pump and a valve can be used to actuate the flow of fluid through an individual channel.
[0116] An exemplary swivel valve 400 is shown in Figure 17. The structure and function of swivel valve 400 can be understood in the context of a sequencing procedure as shown below. Of course, it will be appreciated that the valve can be used in similar ways for other applications. In a sequencing protocol where four different samples must be fluidly processed, the rotary valve rotary valve 400 can function as a four-sample injection rotary valve using a 45-degree tilt and can also function as a four-to-one pipeline for sequencing reagents . In the top view of Figure 17, swivel valve 400a is positioned to allow flow from common reagent reservoir 401 to four lines of a flow cell. More specifically, in this position, fluids can flow from common reagents 401 through port 402 to Line 1 (through port 411), to Line 2 (through port 412), to Line 3 (through port 413 ), and to Line 4 (through port 414). However, in this position fluids do not flow from sample reservoirs S1, S2, S3 or S4 because ports 421, 422, 423 and 424 are closed to flow from port 402. 400b in the position shown in the bottom view of Figure 17, thus allowing samples S1, S2, S3 and S4 to be injected because ports 411, 412, 413 and 414 are open to the flow from ports 421.422, 423 and 424, respectively. However, in this position, the flow from port 402 is closed, thus preventing the flow of reagents common to the flow cell lines.
[0117] In particular embodiments, a fluidic cartridge can be configured to allow the reuse of one or more reagents. For example, the fluidic cartridge can be configured to deliver a reagent to a flow cell, then remove the reagent from the flow cell, and then reintroduce the reagent to the flow cell. In one configuration, as exemplified in Figure 18, cartridge fluids can be configured so that a reagent reservoir is in fluid communication with a flow cell inlet port and the flow cell outlet port is also found. in fluid communication with the reagent reservoir. One or more of the reagents can be reused in a piping network of similar reagent loops as shown in Figure 18. For example, valve 522 controls the flow from wash tank 524, from IMX tank 525, from the SMX 526, from the CLM reservoir 527 and from the cleavage reservoir 528 to the flow cell 520. The pump 521 is downstream of the flow cell 520 and upstream of the valve 523. The valve 523 controls the flow from the flow cell flow 520 to the refuse reservoir 535, the IMX reservoir 525, the SMX reservoir 526, the CLM reservoir 527 and the cleavage reservoir 528.
[0118] The fluid lines connecting the previous components of Figure 18 will be described in the context of a sequencing cycle where the reagents are distributed from the reservoirs to the flow cell and the used reagents are distributed from the flow cell to the respective reservoirs. In all stages of the cycle exemplified below, the fluids are moved under the force of the pressure produced by the pump 521. In a first stage of the cycle, the flow cell is washed by opening a valve 522 to line 501 and opening the valve 523 to line 505 so that fluid flows from wash reservoir 524 to waste reservoir 535 through a path between valve 522 and valve 523 that crosses through flow cell 520. Figure 18, the path between valve 522 and valve 523 leads from valve 522 to line 502 to inlet 530 of flow cell 520, through channel 531 of flow cell 520, through outlet 532 of the cell of flow 520, through line 503, to pump 521 and then through line 504 to valve 523. In a second step of the cycle, IMX is introduced to the flow cell by opening valve 522 to line 506 and opening it. if valve 523 to line 505 so that fluid flows from the reservoir. from IMX 525 to the refuse reservoir 535 through the path between valve 522 and valve 523 that crosses through flow cell 520. In a third step of the cycle, used IMX is moved from flow cell 520 to the reservoir of IMX 525 by opening valve 522 to line 501 and valve 523 opening to line 510 so that the flushing fluid displaces the IMX from the path between valve 522 and valve 523 that crosses through the flow 520. In a fourth step of the cycle, SMX is introduced to the flow cell by opening valve 522 to line 507 and valve 523 opening to line 505 so that fluid flows from the SMX reservoir 526 to the refuse reservoir 535 through the path between valve 522 and valve 523 that crosses through flow cell 520. In a fifth step of the cycle, used SMX is moved from flow cell 520 to SMX reservoir 526 opening valve 522 to line 501 and valve 523 to line 511 opening so that the wash fluid displaces the SMX from the path between valve 522 and valve 523 that crosses through flow cell 520. Similar pairs of steps can be repeated to (1) introduce CLM reagent to the flow cell and returning the used CLM reagent to the CLM reservoir, and (2) introducing the cleavage reagent to the flow cell and returning the used cleavage reagent to the cleavage reservoir.
[0119] Another example of a fluidic configuration that provides reagent reuse is shown in Figure 19. In this example, the fluidics for a cartridge are configured so that each reagent reservoir is in fluidic communication with a single flow cell port 620 . Alternating flow allows each reagent to flow from a reservoir to flow cell 620 and from flow cell 620 back to the reservoir, where reagents ingress to flow cell 620 and reagents egress from of flow cell 620 occur through the same port as flow cell 620. Reuse for four reagents is exemplified in Figure 19, however, a fluidic system can be configured for more or fewer reagents to be reused in a similar alternate format. As shown in Figure 19, valve 622 controls the flow of fluid between flow cell 620 and each of: wash tank 624, IMX tank 625, SMX tank 626, CLM tank 627 and cleavage reservoir 628. In a first flow direction, pump 621 is configured to pull fluid from flow cell 620 through line 603 and push fluids to refuse reservoir 635 through line 605.
[0120] The fluid lines connecting the previous components of Figure 19 will be described in the context of a sequencing cycle where reagents are delivered from the reservoirs to the flow cell and used reagents are delivered from the flow cell to the respective reservoirs. In a first step of the cycle, the flow cell is flushed by opening valve 622 to line 601 so that fluid flows from flushing reservoir 624 to refuse reservoir 635. The path between valve 622 and reservoir of scrap 635 extends from valve 622 to line 602 to port 630 of flow cell 620, through channel 631 of flow cell 620, through port 632 of flow cell 620, through line 603 to pump 621 and then through line 605 to refuse reservoir 635. In a second step of the cycle, IMX is introduced to the flow cell by opening valve 622 to line 606 so that fluid flows from IMX reservoir 625 through valve 622 to line 602, to port 630 of flow cell 620, through channel 631 of flow cell 620, through port 632 of flow cell 620, and partially through line 603 (thus leaving a residual wash solution in a portion downstream of line 603 through pump 621). In a third step of the cycle, the used IMX reagent is returned from flow cell 620 to the IMX 625 reservoir by opening valve 622 to line 606 and reversing the direction of pump 621 so that the IMX reagent used is returned from flow cell 620 to the IMX 625 reservoir through port 630 of flow cell 620, through fluid line 602, through valve 622, then through fluid line 606 to IMX reservoir 625. Step three, the flow from the 621 pump to the IMX 625 reservoir occurs long enough for a portion of the IMX reagent to return to the IMX 625 reservoir, but not long enough to cause a substantial amount of the residual wash from line 603 enters IMX 625 reservoir. In a fourth step of the cycle, the flow cell is washed as described for the first step. In a fifth step of the cycle, SMX is introduced to the flow cell by opening valve 622 to line 607 so that fluid flows from the SMX reservoir 626 through valve 622 to line 602 to port 630 of the cell of flow cell 620, through channel 631 of flow cell 620, through port 632 of flow cell 620, and partially through line 603 (thus leaving a residual wash solution in a portion downstream of line 603 through the pump 621). In a sixth step of the cycle, used SMX reagent is returned from flow cell 620 to SMX reservoir 626 by opening valve 622 to line 607 and reversing the direction of pump 621 so that used SMX reagent is returned from flow cell 620 to the SMX 626 reservoir through port 630 of flow cell 620, to fluidic line 602, through valve 622, then through fluidic line 607 to SMX 626 reservoir. As per step three, the flow from pump 621 during step six causes a portion of the SMX reagent to return to the SMX 626 reservoir, but little or no residual wash solution from line 603 enters the SMX 626 reservoir. - repeating similar triplets of steps to (1) introduce a CLM reagent to flow cell 620, return the used CLM reagent to the CLM reservoir 627 and wash flow cell 620, and (2) introduce the reagent from cleavage to flow cell 620, return the rea cleavage agent used to cleavage reservoir 628 and wash flow cell 620.
[0121] The examples in Figure 18 and Figure 19 show a single reservoir for each reagent. Correspondingly, mixing of used reagents with unused reagents of the same type can occur throughout the fluidic process. In this modality, the fraction of reagent reused in the reservoir will increase with each fluidic cycle. Correspondingly, a sufficiently large volume of starting reagent can be provided to accommodate any dilution or contamination that may occur while maintaining a desired level of overall reaction quality.
[0122] As an alternative to using a single reservoir for each reagent, the fluidic system can include multiple reservoirs for each type of reagent. Each of the reservoirs can be configured for reuse. However, each reservoir can be subjected to a series of mixing events that are less than the number of cycles for the flow cell. Correspondingly, an appropriate number of reservoirs for each type of reagent can be provided to accommodate both a desired number of cycles for a flow cell and the limited number of acceptable reuse cycles for each reagent. For example, ten reservoirs can be provided for a particular reagent in order to accommodate a fluidic process having one hundred cycles and a reagent that should only be used ten times (ie, reused 9 times). In this example, once one of the ten reservoirs has been pulled out of the ten times, the system can switch to a second of the ten reservoirs. Multiple reagent reservoirs can be configured for reuse in the exemplary system shown in Figure 18, for example, by interfacing the reservoirs with valve 522 and valve 523 or by interfacing each subassembly of reservoirs with a dedicated upstream valve of valve 522 and downstream of valve 523. Using the example in Figure 19, multiple reagents can be configured for reuse by interfacing the additional reservoirs with valve 622 or by interfacing the reservoir subassembly with a dedicated valve upstream of valve 622 (in the flow cell inlet direction which is downstream of valve 622 in the flow cell outlet direction).
[0123] Another useful configuration for reusing a given reagent is to use a supplemental reservoir that is separate from a reagent reservoir. Taking as an example the configuration in Figure 18, lines 510, 511, 512 and 513 can flow to the respective supplemented reservoirs so that the reagents are not directed back to the reagent reservoirs 525, 526, 527 and 528 after being placed in contact with the flow cell. Used reagent can then be delivered from respective supplemental reservoirs to the flow cell through different ports in valve 522 or through a separate valve. Returning to the fluidic system example in Figure 19, supplemental reservoirs can be added to the system and ports can be added to valve 622 to route used reagents to supplemental reservoirs. Correspondingly, actuation of valve 622 can be used to direct used reagents to supplemental reservoirs rather than reagent reservoirs 625, 626, 627 and 628 after the reagents have been brought into contact with the flow cell. For embodiments that include a supplemental reservoir including, but not limited to, those exemplified in Figures 18 and 19, spent reagents (of a particular type) from several cycles may be mixed into these supplemental reservoirs prior to reuse. Alternatively, used reagents can be reused sequentially without mixing in supplemental reservoirs. Whether the used reagents are mixed or not, once the reagents have been reused a predetermined number of times or otherwise a desirable number of times, the used reagents can be sent to a scrap reservoir and the supplemental reservoir reused for subsequent cycles with subsequent aliquot(s) of used reagent.
[0124] The configurations shown in Figures 18 and 19 are exemplary. Other configurations are also possible to achieve a reuse of one or more of the reagents used in a particular process. It will be understood that in some reagent reuse configurations, fluidic configurations for reagent reuse will only be used for a subset of reagents used in a particular process. For example, a first subset of the reagents might be robust enough to be reused, while a second subset might be prone to contamination, degradation, or other unwanted effects after a single use. Correspondingly, the fluidic system can be configured to reuse the first subset of reagents, while the fluidics for the second set of reagents will be configured for single use.
[0125] A particular reagent can be reused any number of times desired to suit a particular process. For example, one or more of the reagents exemplified herein, described in a cited reference, or otherwise known for use in a process disclosed herein may be reused at least 2, 3, 4, 5, 10, 25, 50 or more often. In fact, any one of a variety of desired reagents can be reused at least as many times.
[0126] Fluid configurations and methods for reagent reuse, although exemplified for a nucleic acid sequencing process, can be applied to other processes, in particular, processes that involve repeated cycles of reagent distribution. Exemplary processes include the sequencing of polymers such as polypeptides, polysaccharides or synthetic polymers and also include the synthesis of these polymers.
[0127] Figures 18 and 19 and other examples provided herein in relation to methods and apparatus for reagent reuse have been described in the context of a single channel for a flow cell. It will be appreciated that similar methods and apparatus can be applied to a flow cell having multiple channels. Correspondingly, a fluidic cartridge of the present disclosure may include a flow cell having multiple channels and may further include a fluidic system configured to provide reagent reuse for all or a subset of channels. For example, individual channels may be connected to a fluidic system configured as shown in Figure 18 or Figure 19 or as described elsewhere in this document.
[0128] A fluidic cartridge of the present disclosure may include an input and output (I/O) connection to allow communication between the fluidic cartridge and a sensing apparatus that receives the fluidic cartridge. The I/O connection can be used to coordinate the fluidic operations taking place in the fluidic cartridge with the sensing operations taking place in the detection apparatus. For example, in a nucleic acid sequencing procedure, the fluidic delivery of sequencing reagents to a flow cell can be coordinated with the detection of the flow cell by the detection apparatus in one or more cycles of the sequencing procedure. In the embodiment of Figure 14, the I/O connector can allow communication between the fluidic cartridge and the main PCB.
[0129] As will be apparent from the exemplary nucleic acid sequencing modalities presented herein, reservoirs in a fluid cartridge of the present disclosure may contain reagents useful for a nucleic acid sequencing procedure. For example, reagents useful for a synthetic sequencing technique can be present including, for example, a polymerase, a fluorescently labeled nucleotide, or a wash solution. Several different fluorescently labeled nucleotides can be present, either as a mixture in a single pool or each alone in a separate pool. Labeled nucleotides may have reversible terminator moieties for use in a reversible terminator sequencing, in which case a reservoir containing an unlocking agent may also be present. Other nucleic acid sequencing reagents that can be included in a fluidic cartridge include those previously set forth which include, but are not limited to those described in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 , or US 2008/0108082 , each of which is incorporated herein by reference. In particular, nucleic acid sequencing reagents available from Illumina, such as those provided in SBS TruSeq® kits, can be included in a fluidic cartridge.
[0130] The reservoirs of a fluidic cartridge may also include a nucleic acid sample that must be sequenced. Multiple samples can be present in your own reservoir. In some embodiments, multiple samples can be mixed in a single reservoir, for example, in cases where the samples were previously tagged with known nucleic acid tag sequences and then mixed.
[0131] A fluidic cartridge may also include reservoirs that contain reagents used for amplifying nucleic acids. For example, reagents used for a bridge amplification (also called a cluster amplification) can be included, such as those described in US 5,641,658; US 2002/0055100; US 7,115,400; US 2004/0096853; US 2004/0002090; US 2007/0128624; or US 2008/0009420, each of which is incorporated herein by reference. In particular, bridge amplification reagents available from Illumina, such as those provided in TruSeq® RNA or DNA amplification kits, can be included in a fluidic cartridge. Reagents useful for rolling circle amplification (RCA) may also be present in a fluid cartridge including, for example, those described in Lizardi et al, Nat. Genet. 19:225-232 (1998) or US 2007/0099208 A1, each of which is incorporated herein by reference. Emulsion PCR reagents can also be used, for example those described in Dressman et al, Proc. Natl. Academic Set USA 100:8817-8822 (2003), WO 05/010145, or US 2005/0130173 or US 2005/0064460, each of which is incorporated herein by reference.
[0132] A fluidic cartridge of the present disclosure may include two or more subcomponent parts that contain different reagents. Subcomponent parts can be configured for convenient combination in a fluidic cartridge, for example, manually and without the use of tools. For example, the subcomponent parts can be combined into a fluidic cartridge using press fit, mating complementary male and female fits, insertion into appropriately sized reception ports, fastening, or the like. If desired, connections that require the use of tools can be used, for example, the use of a screwdriver to connect the screws, or the use of a wrench to turn a bolt and/or a nut.
[0133] The subcomponent parts that make up a fluidic cartridge may contain reagents that have been previously transported and/or stored under different conditions. For example, a first subcomponent may include reagents that are stored at freezing temperatures (eg, below 0°C, -20°C or -70°C) while a second subcomponent may include reagents that are stored at a higher temperature ( for example, room temperature or above 20°C, 0°C, -20°C or -70°C). Correspondingly, at least some of the reactants in the reservoirs of one subcomponent may be in frozen solid phase, while all the reactants in the reservoirs of another subcomponent are in liquid form. Two or more subcomponent parts that have been stored at different temperatures can be combined in a fluidic cartridge before or after the temperatures equilibrate to room temperature (or other common temperature).
[0134] Reagents useful for fluidic processes in addition to nucleic acid sequencing processes can be provided in the reservoirs of a fluidic cartridge. For example, a fluidic cartridge can contain reagents useful for sequencing other polymers, such as polypeptides, polysaccharides or synthetic polymers. Alternatively or in addition, reagents useful for synthesizing such polymers may also be present.
[0135] However, turning to the modalities relating to the sequencing of nucleic acid, the present disclosure also provides a sequencing method that includes the steps of (a) providing a fluidic cartridge having (i) a flow cell having an optically transparent surface, (ii) a nucleic acid sample, (iii) a plurality of reagents for a sequencing reaction, and (iv) a fluidic system for delivering the reagents to the flow cell; (b) providing a detection apparatus having (i) a plurality of microfluorometers, each of the microfluorometers comprising an objective configured for detecting wide-field images in an image plane in x and y dimensions, and (ii) a sampling stage ; (c) delivering the fluidic cartridge to the sampling stage, where the optically transparent surface is placed on the imaging plane; and (d) perform fluidic operations of a nucleic acid sequencing procedure in the fluid cartridge and detection operations of the nucleic acid sequencing procedure in the detection apparatus, in which (i) the reagents are distributed to the flow cell by the fluidic system , and (ii) the nucleic acid resources are detected by the plurality of microfluorometers.
[0136] One can use any of a variety of detection devices and/or fluidic cartridges described in the previous method. A particular advantage of the apparatus presented here is the modularity that allows for convenient sequencing of different samples using a single detection apparatus. As shown previously, sufficient sample(s), reagents, and fluidic hardware for a complete sequencing procedure can be contained in a fluidic cartridge that can be delivered to a detection device for a sequencing procedure. Once the sequencing procedure is complete, the fluidic cartridge can be removed so that the detection apparatus is ready for another sequencing cycle. By separating the detection device and the fluidic system into separate modules, the present system allows several different samples to be sequenced, avoiding the risk of cross-contamination between samples that occur in existing systems where the detection device and the fluidic system are permanently integrated. Additionally, for modalities where sensing components are relatively expensive and technically difficult to assemble, the modularity presented here provides cost savings by allowing the sensing apparatus to be kept for repeated use while fluidic components are typically lower priced and easily accessible. assembly are replaced or discarded by an action that can be as simple as pressing an eject button.
[0137] Correspondingly, a sequencing method may include the steps of (a) providing a fluid cartridge having (i) a flow cell having an optically transparent surface, (ii) a nucleic acid sample, (iii) a plurality of reagents for a sequencing reaction, and (iv) a fluidic system for delivering the reagents to the flow cell; (b) providing a detection apparatus having (i) a plurality of microfluorometers, each of the microfluorometers comprising an objective configured for detecting wide-field images in an image plane in x and y dimensions, and (ii) a sampling stage ; (c) delivering the fluidic cartridge to the sampling stage, where the optically transparent surface is placed on the imaging plane; (d) perform the fluidic operations of a nucleic acid sequencing procedure in the fluid cartridge and detection operations of the nucleic acid sequencing procedure in the detection apparatus, in which (i) the reagents are distributed to the flow cell by the fluidic system , and (ii) the nucleic acid resources are detected by the plurality of microfluorometers; (e) removing the fluidic cartridge from the sampling stage; (f) delivering a second fluid cartridge to the sampling stage; and (g) performing fluidic operations of a nucleic acid sequencing procedure in the second fluid cartridge and detecting operations of the nucleic acid sequencing procedure in the detection apparatus.
[0138] In general, a second fluid cartridge will include a second nucleic acid sample that is different from the nucleic acid sample in the first fluid cartridge. However, if desired, two fluid cartridges may include duplicate samples, for example, to provide statistical analysis or other technical comparisons. A sequencing system or method of the present disclosure can be used repeatedly for a series of fluid cartridges. For example, it is contemplated that at least 2, 5, 10, 50, 100, or 1000 or more fluidic cartridges can be used.
[0139] In particular embodiments, a flow cell that contains a plurality of channels can be fluidly manipulated and optically detected in a staggered manner. More specifically, fluidic manipulations can be performed on a first subset of channels in the flow cell while optical detection occurs for a second subset of channels. For example, in one configuration, at least four linear channels can be arranged parallel to each other in the flow cell (eg channels 1 to 4 can be arranged in sequential rows). Fluid manipulations can be performed on alternate channels (eg, channels 1 and 3) while detection occurs for the other channels (eg, channels 2 and 4). This particular configuration can be accommodated by using a readhead that fixes several microfluorometers in a spaced configuration so that the objectives are directed to alternating channels of the flow cell. In this case, the readhead can have a series of microfluorometers that is half the number of channels in the flow cell. Additionally, valves can be actuated to direct the flow of reagents for a sequencing cycle to alternate channels while the channels being detected are held in a detect state. In this example, a first set of alternate channels may undergo fluidic steps of a first sequencing cycle and a second set of alternate channels may undergo detection steps of a second sequencing cycle. Once the fluidic steps of the first cycle are complete and the sensing steps of the second cycle are complete, the readhead can be jumped (eg along dimension x) to the first set of alternating channels and valves can be actuated to deliver sequencing reagents to the second set of channels. Then, the detection steps for the first cycle can be completed (in the first set of channels) and the fluidic steps for a third cycle can take place (in the second set of channels). The steps can be repeated in this way several times until a desired number of cycles have been performed or until the sequencing procedure is complete.
[0140] An advantage of the staggered fluidic steps and detection presented above is to provide a faster overall sequencing cycle. In the previous example, a faster sequencing cycle will result from the staggered setup (compared to fluidic manipulation of all channels in parallel followed by detection of all channels in parallel) if the time required for fluidic manipulation is approximately equal to the time required for detection. Of course, in modalities where the timing for detection step is not the same as the timing for fluidic steps, the staggered configuration can be changed to alternate channels to a more appropriate pattern in order to accommodate parallel scanning of one subset of channels while another subset of channels is subjected to fluidic steps.
[0141] According to the various modalities presented above, it provides a detection device having a relatively compact form factor. In some embodiments, a sensing apparatus can have a footprint of about 0.09 m2 (1 square foot) and can occupy a volume of about 0.03 m3 (1 cubic foot). Smaller areas and/or volumes are possible. Slightly larger occupied areas and/or volumes are also useful. As exemplified herein, an apparatus may have a relatively small footprint and occupy a relatively small volume of space when in a fully functional state, for example, after accepting a fluidic cartridge internally. Several devices have been exemplified in the context of their use as autonomous units capable of performing any of a variety of desired procedures. However, these examples are not intended to be limiting and, in fact, the compact form factor of the modalities presented above allows for multiple devices to be arranged in a small space. For example, multiple appliances can be stacked and/or placed in a cabinet or rack for convenient placement. The cabinet or rack may include one or more shelves that define one or more reception spaces, and each reception space may be configured to accommodate one or more detection devices.
[0142] Correspondingly, several detection apparatus of the present disclosure can be used together in a larger system, thus, each detection apparatus effectively functions as a module or node of the system. For example, multiple detection devices can be physically co-located in a rack and can be electronically networked. The electronic network, whether for devices that are co-located or for devices that are located in distributed locations, can allow for global data analysis and/or global control of instrument function. For nucleic acid sequencing modalities, several different detection devices can function as a sequencing system, for example, to sequence the same sample (or subfractions of the same sample) in parallel. A nucleic acid sequencing system can include a control computer that provides instructions to each individual detection apparatus. As such, any of the detection devices in the nucleic acid sequencing system can adopt instructions from a control computer that is physically external to that detection device. Nucleic acid sequence data from the various detection devices can be analyzed in the control computer and/or in a separate analysis computer. Therefore, a central computer can be used for global analysis of nucleic acid sequence data from several different detection devices in a networked system.
[0143] Feedback mechanisms can be used in the control of various detection devices that form modules in a larger system. For example, quality control feedback loops can be used to observe parameters that are determinative or diagnostic of quality of nucleic acid sequence data, and appropriate responsive actions can be taken. Exemplary feedback loops that can be readily adapted for use in a modular sequencing system of the present disclosure are described, for example, in US 7,835,871, which is incorporated herein by reference. A control computer can be programmed to include feedback loops based on parameters and responses to control output quality (e.g., sequence data quality) to a detection apparatus network.
[0144] Nucleic acid sequence data that is obtained from one or more detection devices that function as modules or nodes in a system can be analyzed in real time. Sequence data can be evaluated against a parameter, for example, by comparing the acquired nucleic acid sequence in real time to a standard sequence. Based on these comparison results, a decision can be made as to whether or not to proceed with a sequencing procedure in one or more of the detection devices. For example, environmental samples or pathological samples can be sequenced using multiple modules in a sequencing system and data output from the modules can be compared to known sequences for suspected contaminants or pathogens. Once enough data has been collected to determine the presence or absence of a particular contaminant or pathogen, sequencing can be stopped in one or more of the modules. Exemplary protocols for real-time analysis that can be adapted to a network system of the present disclosure are described in US 2011/0246084 A1, which is incorporated herein by reference. The data analysis and decision procedures exemplified above can be done completely automatically without human intervention. For example, the procedures can be performed on a control computer or another computer that is part of a networked system presented in this document.
[0145] Alternatively or in addition to being electronically networked, several detection devices that are physically co-located in a rack can be networked considering the distribution of samples and/or reagents. For example, cartridges can be delivered to appropriate detection devices using an autoloader or robotic device. Specifically, fluidic cartridges can be automatically removed from a storage location to appropriate detection devices. Automated distribution can be under instructions from a control computer or another computer that is networked to the sequencing system. Additionally, in some embodiments, not all reagents used in a nucleic acid sequencing process need to be contained in fluid cartridges that are used in a sequencing system. Preferably, several detection devices can be in fluid communication with one or more reservoirs containing bulky reagents. In this case, reagents can be delivered to various detection devices from a central fluid storage location, for example, using a central fluid delivery system. Dispensing of reagents can take place under the instructions of a control computer or another computer that is networked to a central delivery system or that is networked to an individual detection apparatus in the sequencing system.
[0146] Various embodiments of the present invention have been presented herein in the context of nucleic acid sequencing or using nucleic acid sequencing applications as an example. However, the apparatus and methods presented here are not limited to nucleic acid sequencing applications. Other applications are useful including, but not limited to, other types of nucleic acid assays, such as those using optically detected markers. Two examples are expression analyzes performed on nucleic acid arrays and genotyping analyzes performed on nucleic acid arrays. In any case, a microfluorometer, read head or detection device presented here can be used to detect the arrays. Additionally, the arrangements can be included in a fluidic cartridge and fluidly manipulated by an appropriate modification of the fluidic cartridge and methods disclosed herein. Exemplary array-based methods that may be modified for use with the apparatus and methods of the present disclosure include, for example, those described in US 2003/0108900, US 2003/0215821 or US 2005/0181394, each of which is incorporated herein by reference title.
[0147] Other solid phase assays that are performed in arrays or on multi-well substrates, such as enzyme-linked immunosorbent assays (ELISAs), can also be used in the methods and apparatus presented here. Formats that use fluorescent markers are particularly useful as the markers can be detected using microfluorometers, readheads, or detection devices discussed above. Additionally, reagents used in ELISAs or other solid phase assays can be processed in a fluidic cartridge similar to those presented herein.
[0148] The methods and apparatus presented here can also be useful to monitor the synthesis of molecules that are optically detectable or molecules that are prepared using optically detectable reagents, intermediates or by-products. Polymeric molecules that undergo cyclic reactions are particularly applicable. For example, nucleic acid or polypeptide synthesis utilize optically detectable blocking groups or intermediates that can be detected using a microfluorometer, readheads, or detection devices presented herein. The fluidic steps involved in synthetic protocols can be performed in a fluidic cartridge similar to those presented here.
[0149] Another useful application of the methods and apparatus presented here is the formation of microscopic images of objects, such as biological samples. Particularly well suited samples are tissues or cells. Samples can be presented on a substrate and detected as exemplified herein for nucleic acid arrays. Imaging the fluorescent properties of objects, such as biological samples, is particularly applicable to the methods and apparatus presented here. Microfluorometers can be used for these applications and optionally fluidic manipulations, for example, to introduce fluorescently labeled reagents, such as fluorescent tags to target molecules, can be performed.
[0150] Throughout this application, reference has been made to various publications, patents and patent applications. The disclosures of these publications in their entirety are incorporated by reference into this application in order to more fully describe the state of the art to which the present invention belongs.
[0151] In this document, the term "comprises" is intended to be unlimited, including not only the elements cited, but also covering any additional elements.
[0152] As the usage in question, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection, but does not necessarily refer to each item in the collection, except where the context clearly indicate otherwise.
[0153] Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the invention. Correspondingly, the invention is limited only by the claims.

权利要求:
Claims (17)
[0001]
1. Detection apparatus, CHARACTERIZED in that it comprises (a) a cart comprising a plurality of microfluorometers, each of the microfluorometers comprising an objective configured for detection of wide-field images, wherein the plurality of microfluorometers is positioned to simultaneously acquiring a plurality of widefield images on a common plane, and each of the widefield images coming from a different area of the common plane; (b) a translation stage configured to move the car in at least one direction parallel to the common plane; and (c) a sampling stage configured to maintain a substrate in the common plane; and wherein each of the microfluorometers further comprises a dedicated autofocusing module, and wherein the autofocusing module for a first microfluorometer of the device is configured to integrate data from an autofocusing module to a second microfluorometer of the device, where the autofocus module changes the focus of the first microfluorometer based on the focal position of the first microfluorometer and the focal position of the second microfluorometer.
[0002]
2. Detection apparatus, according to claim 1, CHARACTERIZED by the fact that the autofocusing module comprises a detector and an actuator, in which the actuator is configured to change the focus of the microfluorometer in relation to the common plane, and in which the detector is configured to direct actuator movement.
[0003]
3. Detection apparatus, according to claim 2, CHARACTERIZED by the fact that the detector is further configured to obtain the wide-field image.
[0004]
4. Detection apparatus according to claim 1 or 2, CHARACTERIZED by the fact that each of the microfluorometers further comprises a beam splitter and a detector, wherein the beam splitter is positioned to direct excitation radiation from a source of excitation radiation to the objective and direct emission radiation from the objective to the detector.
[0005]
5. Detection device, according to claim 1 or 2, CHARACTERIZED by the fact that each of the microfluorometers further comprises the excitation radiation source.
[0006]
6. Detection apparatus, according to claim 5, CHARACTERIZED by the fact that the excitation radiation source directs the excitation radiation to the objective of an individual microfluorometer in the plurality of microfluorometers, wherein each microfluorometer comprises a radiation source of separate excitement.
[0007]
7. Detection apparatus, according to claim 5 or 6, CHARACTERIZED by the fact that each of the microfluorometers further comprises at least two sources of excitation radiation.
[0008]
8. Detection device according to claim 1, 2, 5, or 6 CHARACTERIZED by the fact that the objective of each of the microfluorometers has a numerical aperture between 0.2 and 0.5.
[0009]
9. Detection apparatus according to claim 1, 2, 5, 6 or 8, CHARACTERIZED by the fact that each of the microfluorometers is configured to detect at a resolution sufficient to distinguish resources that are less than 50 microns away .
[0010]
10. Detection device according to claim 1, 2, 5, 6, 8 or 9, CHARACTERIZED by the fact that the wide-field image for each of the microfluorometers has an area of at least 1 mm2.
[0011]
11. Detection device according to claim 1, 2, 5, 6, 8, 9 or 10, CHARACTERIZED by the fact that the carriage prevents lateral movement between the microfluorometers.
[0012]
12. Detection device, according to claim 11, CHARACTERIZED by the fact that the microfluorometers are co-molded with the car.
[0013]
13. Detection device according to claim 1, 2, 5, 6, 8, 9, 10, 11 or 12, CHARACTERIZED by the fact that the car comprises at least 4 microfluorometers, in which the objectives of at least four microfluorometers are arranged in at least two rows.
[0014]
14. Method for imaging a substrate CHARACTERIZED in that it comprises: (a) providing a substrate comprising fluorescent features on a surface; (b) acquiring a plurality of wide-field images of a first portion of the surface using a plurality of microfluorometers, wherein each of the microfluorometers acquires a wide-field image from a different location on the surface, wherein the plurality of microfluorometers is attached to a car; and (c) translating the carriage in a direction parallel to the surface and repeating (b) for a second portion of the surface, further comprising individually focusing each microfluorometer on the plurality of microfluorometers using an autofocusing technique, each of the microfluorometers comprising a module self-focusing; wherein the autofocusing module used for a first microfluorometer of the plurality of microfluorometers integrates data from an autofocusing module used for a second microfluorometer of the plurality of microfluorometers, wherein the focus of the first microfluorometer is adjusted based on the focal position of the first microfluorometer and at the focal position of the second microfluorometer.
[0015]
15. Method according to claim 14, CHARACTERIZED by the fact that the plurality of wide-field images is acquired simultaneously in (b).
[0016]
16. The method of claim 14 or 15, CHARACTERIZED in that the method further comprises (d) returning the carriage to a position to acquire a second plurality of wide-field images of the first portion of the surface.
[0017]
17. Method according to claim 16, CHARACTERIZED by the fact that it further comprises modifying the surface fluorescent features after (c) and before (d), wherein the second plurality of wide field images is different from the first plurality of wide-field imaging, in which the fluorescent resources comprise nucleic acids and the modification comprises altering nucleic acids in a sequencing by synthesis technique.

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法律状态:
2018-12-04| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-11-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-10| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-03-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-25| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/02/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261619784P| true| 2012-04-03|2012-04-03|

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US61/619,784|2012-04-03|

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