Methods, devices and systems for analyte detection and analysis

Abstract

Systems and methods for analyte detection and analysis are provided. A system may include an open substrate. The open substrate may be configured to rotate or otherwise move. The open substrate may include an array of individually addressable locations, with the analyte immobilized thereon. Substrates may be spatially indexed to identify nucleic acid molecules from one or more sources and / or sequences thereof, as well as respective one or more sources. A solution comprising a plurality of probes may be directed throughout the array to couple at least one of the plurality of probes to at least one of the analytes to form a binding probe. The detector may be configured to detect a signal from the bonding probe by scanning the substrate while minimizing temperature fluctuations of the substrate or optical aberrations caused by bubbles.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of March 13, 2020, application number 202080036151.6, and invention name “Methods, devices and systems for analyte detection and analysis” (the application date of the corresponding PCT application is March 13, 2020, and the application number is PCT / US2020 / 022816).

[0002] Cross-references

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 818,549, filed on March 14, 2019, U.S. Provisional Application No. 62 / 837,684, filed on April 23, 2019, U.S. Provisional Application No. 62 / 914,293, filed on October 11, 2019, U.S. Application No. 16 / 445,798, filed on June 19, 2019, U.S. Application No. 16 / 677,067, filed on November 7, 2019, and U.S. Application No. 16 / 677,115, filed on November 7, 2019, the entire contents of each of which are incorporated herein by reference. Background Art

[0004] In the field of molecular biology and medicine (e.g., diagnosis), biological sample processing has multiple applications. For example, nucleic acid sequencing can provide information that can be used to diagnose a certain condition of a subject and can be used to customize a treatment plan in some cases. Sequencing is widely used in molecular biology applications, including vector design, gene therapy, vaccine design, industrial strain design and verification. Biological sample processing can relate to fluid systems and / or detection systems. Summary of the Invention

[0005] While biological sample processing systems and methods are common, such systems and methods can be inefficient, time consuming, and waste valuable resources such as reagents. A need is recognized herein for methods and systems for efficient sample processing and / or analysis.

[0006] The present disclosure provides methods, devices and systems for sample processing and / or analysis. The methods, devices and systems described herein may include an open substrate or its use. The open substrate may contain one or more analytes thereon. For example, one or more analytes may be coupled, attached, fixed or otherwise associated with the open substrate directly or indirectly (e.g., through an intermediate, such as an adhesive or joint). The open substrate may include an array. In some cases, the environment of the open substrate, such as the local environment surrounding the open substrate, may be controlled to promote one or more reactions, or one or more detections. The methods, devices and systems described herein may include an immersion optical system or its use. The immersion optical system may be configured to detect analytes or their activity on the open substrate. The methods, devices and systems described herein may include spatial indexing of an open substrate or its array, or its use.

[0007] In various aspects, the present disclosure provides a method for scanning a surface, the method comprising: (a) scanning a scanning field comprising a portion of the surface using a scanning system, wherein the scanning field has an orientation relative to an axis of rotation of the surface; (b) (i) rotating the surface about the axis of rotation of the surface and (ii) rotating the scanning field about the axis of rotation of the scanning field so that the scanning field substantially maintains its orientation relative to the axis of rotation of the surface before, during, or after the surface is translated relative to the scanning field.

[0008] In some embodiments, the scanning field has a substantially rectilinear shape. In some embodiments, the scanning field has a major axis, and wherein the orientation comprises a line coinciding with the major axis of the scanning field passing through the axis of rotation of the surface. In some embodiments, the scanning field traces an arc on the surface. In some embodiments, scanning the surface comprises imaging the surface. In some embodiments, the scanning field comprises an imaging field. In some embodiments, the scanning field traces a scanning path on the surface, and the scanning path comprises an imaging path. In some embodiments, the scanning system comprises an imaging system.

[0009] In some embodiments, the orientation comprises a long axis of the scan field, wherein the long axis is parallel to a radial line passing through (i) the axis of rotation of the surface and (ii) the axis of rotation of the scan field. In some embodiments, the translation of the surface relative to the scan field comprises translation in a direction that is not directly toward or away from the axis of rotation of the surface. In some embodiments, the translation of the surface relative to the scan field comprises translation along a translation path, wherein a line comprising a net displacement along the translation path does not intersect both the scan field and the axis of rotation of the surface.

[0010] In some embodiments, the scanning field rotates relative to the surface about the axis of rotation of the scanning field. In some embodiments, the axis of rotation of the scanning field is substantially perpendicular to the surface. In some embodiments, the axis of rotation of the scanning field is substantially parallel to the axis of rotation of the surface. In some embodiments, the axis of rotation of the scanning field passes through the axis of symmetry of the scanning field. In some embodiments, the scanning field is rotated by rotating the objective lens. In some embodiments, the scanning field is rotated by rotating the lens. In some embodiments, the scanning field is rotated by rotating the prism. In some embodiments, the scanning field is rotated by rotating the reflector. In some embodiments, the scanning field is rotated by rotating the camera. In some embodiments, the scanning field is rotated by rotating the diffractive optical element (DOE). In some embodiments, a motor is used to rotate the scanning field.

[0011] In some embodiments, the surface is substantially circular and wherein the scanning field translates along a chord of the surface. In some embodiments, the surface is substantially circular and wherein the axis of rotation of the scanning field translates along a chord of the surface. In some embodiments, the chord does not pass through the axis of rotation of the surface. In some embodiments, the scanning field is translated by moving the surface. In some embodiments, the scanning field is translated by moving the scanning system. In some embodiments, the scanning field traces a circle on the surface. In some embodiments, the scanning field traces a spiral on the surface. In some embodiments, the rotation of the surface and the translation of the surface are performed simultaneously. In some embodiments, the translation of the surface is linear relative to the axis of rotation of the surface. In some embodiments, the translation of the surface is not substantially circular relative to the surface. In some embodiments, the translation of the surface increases or decreases the distance between the axis of rotation of the scanning field and the axis of rotation of the surface.

[0012] In some embodiments, the scanning system includes an objective lens in optical communication with the surface. In some embodiments, the scanning system includes a camera. In some embodiments, the scanning field is in optical communication with the camera. In some embodiments, the camera is a time delay integration (TDI) camera with a linear rate. In some embodiments, the camera is a multi-line TDI camera. In some embodiments, the camera includes a sensor array and the rotation axis of the scanning field passes through the center of the sensor array. In some embodiments, the linear rate is set so that the camera captures an image when the scanning field has advanced along the surface from a first position to a second position, the second position being adjacent to the first position. In some embodiments, the linear rate is variable. In some embodiments, the linear rate is higher when the objective lens is placed farther from the rotation axis of the surface.

[0013] In some embodiments, the scanning system further comprises a tube lens. In some embodiments, the scanning system comprises two objective lenses in optical communication with the surface: the objective lens and a second objective lens. In some embodiments, the two objective lenses are located on the same side of the surface relative to a plane normal to the surface and intersecting the surface's axis of rotation. In some embodiments, the two objective lenses are located on opposite sides of the surface relative to a plane normal to the surface and intersecting the surface's axis of rotation. In some embodiments, the two objective lenses trace circular paths on the surface. In some embodiments, the circular paths are concentric. In some embodiments, the objective lens and the second objective lens trace alternating circular paths. In some embodiments, the objective lens traces a circular path closer to the axis of rotation, and the second objective lens traces a circular path further from the surface's axis of rotation. In some embodiments, the two objective lenses trace separate spiral paths on the surface. In some embodiments, the spiral paths are staggered. In some embodiments, the spiral paths are concentric and the objective lens traces a spiral path closer to the surface's axis of rotation, and the second objective lens traces a spiral path further from the surface's axis of rotation. In some embodiments, the objective lens traces a first path having a first width corresponding to a first width of a scan field, and wherein the second objective lens traces a second path having a second path width corresponding to a second width of a second scan field. In some embodiments, the first path width and the second path width overlap by no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1%.

[0014] In some embodiments, the scanning system includes four objective lenses in optical communication with the surface. In some embodiments, the four objective lenses are located on the same side of the surface relative to a plane normal to the surface and intersecting the axis of rotation of the surface. In some embodiments, the first two of the four objective lenses are located on a first side of the surface, and the last two of the four objective lenses are located on a second side of the surface opposite the first side, relative to a plane normal to the surface and intersecting the axis of rotation of the surface. In some embodiments, the scanning system includes 5, 6, 7, 8, 9, 10, 11, 12, 13 or more objective lenses in optical communication with the surface.

[0015] In some embodiments, the surface rotates at a constant angular velocity. In some embodiments, the camera is configured to capture images at a given frequency and the surface rotates relative to the objective lens at a variable angular velocity. In some embodiments, the angular velocity is varied such that, at the given frequency, the camera captures images when the scan field is at a first position and when the scan field is at a second position, the second position being adjacent to the first position.

[0016] In some embodiments, the method further comprises illuminating a portion of the surface defined by the illumination field. In some embodiments, the illumination field is illuminated using a laser. In some embodiments, the illumination field is illuminated using a light emitting diode (LED) or a lamp. In some embodiments, the power of the laser is adjusted to maintain a constant brightness on the surface and / or to not saturate the camera. In some embodiments, the illumination field at least partially overlaps with the scanning field. In some embodiments, the scanning field comprises the illumination field. In some embodiments, the illumination field has a shape substantially similar to the scanning field. In some embodiments, the illumination field is a substantially rectilinear shape. In some embodiments, the illumination field has a long axis.

[0017] In some embodiments, the scanning system further comprises a plurality of illumination fields. In some embodiments, one or more of the plurality of illumination fields has a substantially linear shape. In some embodiments, the method further comprises rotating the illumination field such that the illumination field maintains a defined orientation relative to the axis of rotation of the surface. In some embodiments, the illumination field maintains a fixed orientation relative to the scanning field. In some embodiments, the defined orientation comprises a line coinciding with a long axis of the illumination field passing through the axis of rotation of the surface. In some embodiments, the defined orientation comprises the long axis of the illumination field being parallel to a radial line passing through the axis of rotation of the surface and the axis of rotation of the illumination field. In some embodiments, the scanning field and the illumination field rotate together. In some embodiments, the long axis of the illumination field is parallel to the long axis of the scanning field. In some embodiments, the illumination field rotates about the axis of rotation of the illumination field. In some embodiments, the axis of rotation of the illumination field is substantially perpendicular to the surface. In some embodiments, the axis of rotation of the illumination field is substantially parallel to the axis of rotation of the surface. In some embodiments, the axis of rotation of the illumination field passes through an axis of symmetry of the illumination field. In some embodiments, the axis of rotation of the illumination field is the same as the axis of rotation of the scanning field. In some embodiments, the illumination field is rotated by rotating the lens. In some embodiments, the illumination field is rotated by rotating a diffractive optical element (DOE). In some embodiments, the illumination field is rotated by rotating a prism. In some embodiments, the illumination field is rotated by rotating a mirror. In some embodiments, the illumination field is rotated by rotating a laser. In some embodiments, a motor is used to rotate the illumination field.

[0018] In some embodiments, the method further includes scanning a second portion of the surface defined by the second scanning field. In some embodiments, the second scanning field is scanned using a second scanning system. In some embodiments, the second scanning system includes a second objective lens in optical communication with the surface. In some embodiments, the second objective lens is focused independently of the first objective lens. In some embodiments, the second objective lens has a fixed position relative to the first objective lens. In some embodiments, the second scanning field has an orientation relative to the axis of rotation of the surface. In some embodiments, the second scanning field is radially adjacent to the scanning field. In some embodiments, the scanning field and the second scanning field have the same orientation relative to the axis of rotation of the surface. In some embodiments, the second scanning field rotates independently of the scanning field such that the second scanning field maintains its orientation relative to the axis of rotation of the surface. In some embodiments, the second scanning field rotates in conjunction with the scanning field.

[0019] In some embodiments, the first objective lens and the second objective lens are part of a scanning module, and the scanning module translates relative to the surface along a line extending radially from the rotational axis of the surface. In some embodiments, the surface is substantially circular, and wherein at least one of the first objective lens or the second objective lens does not translate along a chord passing through the rotational axis of the surface. In some embodiments, the first objective lens and the second objective lens are located on the same side of the surface relative to a plane normal to the surface and intersecting the rotational axis of the surface, and the first objective lens and the second objective lens translate together toward or away from the rotational axis of the surface. In some embodiments, the first objective lens and the second objective lens are located on opposite sides of the surface relative to a plane normal to the surface and intersecting the rotational axis of the surface. In some embodiments, (i) the first objective lens translates toward the rotational axis of the surface when the second objective lens translates away from the rotational axis of the surface or (ii) the first objective lens translates away from the rotational axis of the surface when the second objective lens translates toward the rotational axis of the surface.

[0020] In some embodiments, the surface is substantially circular and wherein the first objective lens and the second objective lens translate along parallel chords that are on either side of a plane that is normal to the surface and intersects the surface's axis of rotation and equidistant from the surface's axis of rotation. In some embodiments, the surface is mounted on a rotation module. In some embodiments, the rotation module translates relative to the scanning system. In some embodiments, the rotation module is stationary and the scanning module is translatable. In some embodiments, the scanning module is stationary and the rotation module is translatable. In some embodiments, the rotation module is mounted on a track. In some embodiments, the scanning module is mounted on a scanning module track. In some embodiments, the scanning module track is linear. In some embodiments, multiple surfaces are mounted on multiple rotation modules and wherein the multiple rotation modules are mounted on a platform and the platform rotates so that each rotation module is in optical communication with the scanning module.

[0021] In some embodiments, after scanning the surface, the rotation module is moved to the chemical module. In some embodiments, the method further comprises translating the second rotation module so that the second surface is in optical communication with the scanning module. In some embodiments, the surface comprises an array of nucleic acid colonies. In some embodiments, the nucleic acid colonies are labeled with fluorophores. In some embodiments, the intensity of the fluorophore indicates the sequence of the nucleic acid colonies. In some embodiments, the laser excites the fluorophore at a first wavelength and the camera detects emission from the fluorophore at a second wavelength. In some embodiments, the laser illuminates the illumination field and the camera scans the scanning field.

[0022] In some embodiments, two or more of scanning, rotating the surface, rotating the scanning field, and translating occur simultaneously. In some embodiments, three or more of scanning, rotating the surface, rotating the scanning field, and translating occur simultaneously. In some embodiments, scanning, rotating the surface, rotating the scanning field, and translating occur independently. In some embodiments, the method further comprises repeating steps (a) and (b). In some embodiments, steps (a) and (b) are repeated for each base in the nucleic acid polymerization reaction, thereby sequencing the nucleic acid.

[0023] In various aspects, the present disclosure provides a scanning system comprising: a surface configured to rotate about a rotational axis of the surface; a detector in optical communication with the surface, wherein the detector has a scanning field comprising a first portion of the surface; and an illumination source configured to illuminate an illumination area comprising a second portion of the surface, wherein the illumination area and the scanning field at least partially overlap, wherein the detector is configured to maintain an orientation of the scanning field relative to the rotational axis of the surface during (i) rotation of the surface about the rotational axis and (ii) translation of the surface relative to the scanning field.

[0024] In some embodiments, the scanning field traces an arc on the surface. In some embodiments, scanning the surface includes imaging the surface. In some embodiments, the scanning field includes an imaging field. In some embodiments, the scanning field traces a scanning path along the surface, and wherein the scanning path includes an imaging path. In some embodiments, the scanning system includes an imaging system. In some embodiments, the detector includes a line scan camera. In some embodiments, the line scan camera includes a TDI line scan camera. In some embodiments, the TDI line scan camera images a first scanning field on a first camera area. In some embodiments, the TDI line scan camera images a second scanning field on a second camera area. In some embodiments, the TDI line scan camera images the first scanning field on the first camera area and images the first scanning field on the second camera area. In some embodiments, the first camera area and the second camera area detect different wavelengths. In some embodiments, the first camera area and the second camera area detect different dynamic ranges.

[0025] In some embodiments, the surface is configured to translate along a translation axis relative to the scanning field. In some embodiments, the translation axis intersects the rotation axis of the surface and the center point of the scanning field. In some embodiments, the translation axis does not intersect the rotation axis of the surface and the center point of the scanning field. In some embodiments, the orientation of the scanning field changes from a first orientation to a second orientation relative to the rotation axis of the surface when the surface translates. In some embodiments, the scanning field is configured to rotate around the rotation axis of the scanning field relative to the rotation axis of the surface to correct the orientation of the scanning field from the second orientation to the first orientation relative to the rotation axis of the surface. In some embodiments, the scanning field is configured to rotate by rotating the objective lens. In some embodiments, the scanning field is configured to rotate by rotating the lens. In some embodiments, the scanning field is configured to rotate by rotating the prism. In some embodiments, the scanning field is configured to rotate by rotating the reflector. In some embodiments, the scanning field is configured to rotate by rotating the detector. In some embodiments, the scanning field is configured to rotate by rotating the diffractive optical element (DOE).

[0026] In some embodiments, the illumination source comprises a laser or a light emitting diode (LED). In some embodiments, the illumination source comprises a substantially circular illumination profile. In some embodiments, the substantially circular illumination profile is expanded along a single axis. In some embodiments, a cylindrical lens is used to expand the substantially circular illumination profile along a single axis. In some embodiments, the scanning system further comprises a plurality of illumination sources having substantially circular illumination profiles, wherein the substantially circular illumination profile is expanded along a single axis. In some embodiments, the illumination source passes through a grating.

[0027] In some embodiments, a first portion of the surface is configured to move relative to a scanning field. In some embodiments, a first area of ​​the first portion of the surface is configured to move at a first speed relative to the scanning field, and a second area of ​​the first portion of the surface is configured to move at a second speed relative to the scanning field. In some embodiments, the first area is closer to the axis of rotation of the surface than the second area and the first speed is slower than the second speed. In some embodiments, the image of the first area is magnified on the detector by a first magnification factor, and the image of the second area is magnified on the detector by a second magnification factor. In some embodiments, the first magnification factor and the second magnification factor are different.

[0028] In some embodiments, the scanning system further comprises a lens having a lens axis positioned in a light path between the scanning field and the detector, wherein the lens axis is not perpendicular to the surface. In some embodiments, the scanning system further comprises an objective lens positioned in a light path between the scanning field and the detector. In some embodiments, the objective lens is in contact with the surface fluid. In some embodiments, the objective lens and the surface are at different temperatures.

[0029] In some embodiments, the scanning system further comprises a temperature gradient of the fluid across the contact surface and the objective lens. In some embodiments, the objective lens comprises an insulating spacer in contact with the fluid. In some embodiments, the insulating spacer comprises an air gap. In some embodiments, the objective lens is heated to reduce the temperature gradient. In some embodiments, the objective lens is cooled to increase the temperature gradient. In some embodiments, the fluid is configured to exchange during rotation.

[0030] In some embodiments, the method further comprises (i) scanning a focal region of the surface using an autofocus system to generate a focus map of the focal region and (ii) adjusting the focus of the surface relative to the scanning system based on the focus map while scanning the scanning field. In some embodiments, while scanning the focal region of the surface using the autofocus system, the surface is rotated relative to the scanning field about an axis of rotation of the surface. In some embodiments, the focal region includes the scanning field. In some embodiments, the focal region includes a field that is very close to the scanning field. In some embodiments, the focal region does not include the scanning field. In some embodiments, the focal region is scanned prior to scanning. In some embodiments, the focal region is scanned while scanning.

[0031] In some embodiments, the objective lens is configured to maintain fluid contact with the surface when the surface rotates relative to the objective lens about the axis of rotation of the surface. In some embodiments, the objective lens is configured to move in a direction generally normal to the surface to leave and re-enter fluid contact with the surface. In some embodiments, the objective lens is configured to retain fluid droplets attached to the objective lens when the objective lens leaves fluid contact with the surface. In some embodiments, the objective lens is configured to expel bubbles between the surface and the objective lens when the objective lens re-enters fluid contact with the surface. In some embodiments, the scanning system further includes an adapter attached to the objective lens and configured to facilitate bubble expulsion.

[0032] In some embodiments, the scanning system further comprises a chamber surrounding the surface and the objective lens, which is configured to maintain a higher humidity in the chamber compared to the outside of the chamber. In some embodiments, the chamber comprises a reservoir configured to collect fluid below the surface. In some embodiments, the reservoir comprises a liquid level, and wherein the reservoir is configured to maintain a substantially constant liquid level. In some embodiments, the reservoir is configured to distribute a fluid volume approximately equal to the fluid volume collected by the reservoir. In some embodiments, the top of the chamber is maintained at a first temperature, the objective lens is maintained at a second temperature, the surface is maintained at a third temperature, and the reservoir is maintained at a fourth temperature. In some embodiments, the first temperature is higher than the second temperature. In some embodiments, the third temperature is lower than the fourth temperature. In some embodiments, the second temperature is higher than the third temperature and lower than the first temperature.

[0033] In various aspects, the present disclosure provides a method for sequencing nucleic acid molecules, the method comprising: (i) providing an array of nucleic acid molecules on an uncovered surface; (ii) dispensing a layer of a solution on the uncovered surface at a rate of at least 1 nanoliter / second when measured at a temperature of 25 degrees Celsius, wherein the solution comprises a reagent comprising at least one nucleotide that is incorporated into a growing nucleic acid chain that is complementary to a nucleic acid molecule of the array of nucleic acid molecules; and (iii) detecting one or more signals, the one or more signals indicating the incorporation of the nucleotide into the growing nucleic acid chain.

[0034] In various aspects, the present disclosure provides a method for processing a plurality of nucleic acid samples, comprising: (i) providing the plurality of nucleic acid samples, wherein the plurality of nucleic acid samples comprises a first nucleic acid sample comprising a first set of nucleic acid molecules and a second nucleic acid sample comprising a second set of nucleic acid molecules, wherein each of the plurality of nucleic acid samples has an identifiable sample origin; (ii) loading the first nucleic acid sample onto a first region of a substrate as a first array of the first set of nucleic acid molecules and loading the second nucleic acid sample onto a second region of the substrate as a second array of the second set of nucleic acid molecules, wherein the first region is different from the second region; (iii) dispensing a solution throughout the substrate, wherein the solution comprises a reagent sufficient to react with the nucleic acid molecules of the first array or the second array; (iv) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array; and (v) analyzing the first nucleic acid sample and the second nucleic acid sample based at least in part on (a) the one or more signals and (b) the locations from the first region and the second region from which the one or more signals are detected, and (1) determining a first subset of the nucleic acid molecules of the first array or the second array as originating from the first nucleic acid sample and (2) determining a second subset of the nucleic acid molecules of the first array or the second array as originating from the second nucleic acid sample.

[0035] In various aspects, the present disclosure provides a method for processing a plurality of nucleic acid samples, comprising: (i) providing the plurality of nucleic acid samples, wherein the plurality of nucleic acid samples comprises a first nucleic acid sample comprising a first set of nucleic acid molecules and a second nucleic acid sample comprising a second set of nucleic acid molecules, (ii) loading the first nucleic acid sample onto a substrate to associate the first set of nucleic acid molecules with a first array of individually addressable locations; (iii) imaging the substrate to identify the first array of individually addressable locations; (iv) loading the second nucleic acid sample onto a substrate to associate the second set of nucleic acid molecules with a second array of individually addressable locations; (v) imaging the substrate to identify the second array of individually addressable locations; (vi) dispersing a solution across the substrate, wherein the solution comprising a reagent sufficient to react with the nucleic acid molecules of the first array or the second array; (vii) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array; and (viii) analyzing the first nucleic acid sample and the second nucleic acid sample based at least in part on (a) the one or more signals and (b) positions from the first array of individually addressable locations and the second array of individually addressable locations from which the one or more signals are detected, and (1) determining a first subset of the nucleic acid molecules of the first array or the second array as originating from the first nucleic acid sample and (2) determining a second subset of the nucleic acid molecules of the first array or the second array as originating from the second nucleic acid sample.

[0036] In various aspects, the present disclosure provides a method for processing a plurality of nucleic acid samples, wherein each of the plurality of nucleic acid samples comprises a fluorescent dye; (i) providing the plurality of nucleic acid samples, wherein each of the plurality of nucleic acid samples comprises a fluorescent dye; (ii) dividing the plurality of nucleic acid samples into a first set of one or more samples and a second set of one or more samples; (iii) loading the first set of one or more samples onto a first set of regions on a substrate, one sample per region in the first set of regions; (iv) imaging the substrate to identify (a) locations within the first set of regions and (b) locations within a second set of regions on the substrate, wherein the second set of regions is different from the first set of regions associated with the first set of one or more samples; (v) loading the second set of one or more samples onto the substrate (viii) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecule; and (ix) analyzing each of the plurality of nucleic acid samples based at least in part on (a) the one or more signals and (b) the positions from the first set of regions and the second set of regions from which the one or more signals are detected.

[0037] In various aspects, the present disclosure provides a method for processing a biological analyte, comprising: (i) moving a substrate through or along a reel, wherein a surface of the substrate comprises an array having the biological analyte immobilized thereon; (ii) contacting the surface of the substrate with a reservoir comprising a solution, wherein the solution comprises a plurality of probes; (iii) subjecting the biological analyte to conditions sufficient to effect a reaction between one of the plurality of probes and the biological analyte to couple the probe to the biological analyte; and (iv) detecting one or more signals from the probe coupled to the biological analyte, thereby analyzing the biological analyte, wherein the substrate is moved through or along the reel in the same direction in at least two consecutive cycles of (ii)-(iv).

[0038] In various aspects, the present disclosure provides a system for analyzing a biological analyte, comprising: a substrate containing the biological analyte, wherein the substrate is maintained at or above a first temperature, the first temperature being higher than an ambient temperature of an environment to which the substrate is exposed; and an optical imaging objective in optical communication with the substrate and exposed to the environment, wherein the optical imaging objective is subject to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, wherein the optical imaging objective comprises a first optical element and a second optical element adjacent to the first optical element, wherein the second optical element is disposed farther from the substrate than the first optical element, wherein the first optical element is configured to be at least partially immersed in an immersion fluid in contact with the substrate, wherein the second optical element is in optical communication with the substrate via the first optical element, and wherein the first optical element is configured such that a second temperature of the second optical element is maintained at or below a predetermined threshold.

[0039] In various aspects, the present disclosure provides a method for analyzing a biological analyte, comprising: (i) providing a substrate containing the biological analyte, wherein the substrate is at a first temperature greater than an ambient temperature of an environment to which the substrate is exposed; (ii) providing an optical imaging objective in optical communication with the substrate and exposed to the environment, wherein the optical imaging objective is subject to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, wherein the optical imaging objective comprises a first optical element and a second optical element adjacent to the first optical element, wherein the second optical element is disposed farther from the substrate than the first optical element, and wherein the first optical element is at least partially immersed in an immersion fluid in contact with the substrate; (iii) controlling or maintaining a second temperature of the first optical element to regulate a magnitude or position of the temperature gradient across the optical imaging objective such that a third temperature gradient across the optical element remains below a predetermined threshold; and (iv) detecting one or more signals from the biological analyte using the optical imaging objective during movement of the substrate relative to the optical imaging objective.

[0040] In various aspects, the present disclosure provides a method for storing a substrate comprising a surface coated with nucleic acid molecules, comprising: (i) providing the substrate having a surface, the surface comprising a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples; (ii) contacting the substrate comprising the surface comprising the first set of nucleic acid molecules with a second set of nucleic acid molecules under conditions sufficient to produce a treated surface, wherein at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules hybridize to nucleic acid molecules of the second set of nucleic acid molecules, wherein the second set of nucleic acid molecules are not the sample nucleic acid molecules; and (iii) storing the substrate having the treated surface for a period of at least 1 hour.

[0041] In various aspects, the present disclosure provides a method for nucleic acid processing, comprising: (i) providing a substrate having a treated surface, the treated surface comprising a first set of nucleic acid molecules immobilized thereon, wherein at least 90% of the nucleic acid molecules of the first set of nucleic acid molecules are hybridized with nucleic acid molecules of a second set of nucleic acid molecules, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples, wherein the second set of nucleic acid molecules are not the sample nucleic acid molecules, and wherein the substrate having the treated surface has been stored for a period of at least 1 hour; and (ii) removing the nucleic acid molecules of the second set of nucleic acid molecules from the treated surface.

[0042] In various aspects, the present disclosure provides a kit comprising: a substrate comprising a treated surface, wherein the treated surface comprises a plurality of pairs of bound nucleic acid molecules, wherein each of the plurality of pairs comprises a first nucleic acid molecule of a first group of nucleic acid molecules at least partially hybridized to a second nucleic acid molecule of a second group of nucleic acid molecules, wherein the first group of nucleic acid molecules is immobilized to the surface, wherein at least 90% of the nucleic acid molecules of the first group of nucleic acid molecules are paired with nucleic acid molecules of the second group of nucleic acid molecules, wherein the nucleic acid molecules of the first group of nucleic acid molecules are configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples when the nucleic acid molecules of the first group of nucleic acid molecules are not paired with nucleic acid molecules of the second group of nucleic acid molecules.

[0043] In various aspects, the present disclosure provides a kit comprising: a substrate comprising a surface comprising a first set of nucleic acid molecules immobilized thereon, wherein the first set of nucleic acid molecules comprises one or more first nucleic acid molecules, the one or more first nucleic acid molecules being configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples; and a solution comprising a second set of nucleic acid molecules, wherein the second set of nucleic acid molecules comprises one or more second nucleic acid molecules, the one or more second nucleic acid molecules being not the sample nucleic acid molecules; wherein the second set of nucleic acid molecules is selected such that upon contacting the solution with the surface, at least 70% of the one or more first nucleic acid molecules bind to the second nucleic acid molecules of the second set of nucleic acid molecules to produce one or more pairs of bound nucleic acid molecules, wherein each of the one or more pairs comprises (i) a first nucleic acid molecule of the first set of nucleic acid molecules and a second nucleic acid molecule of the second set of nucleic acid molecules, and (ii) a substantially complementary sequence.

[0044] In various aspects, the present disclosure provides a method for storing a substrate comprising a surface coated with nucleic acid molecules, comprising: (i) providing a substrate having a surface, the surface comprising a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples, and wherein each nucleic acid molecule of the first set of nucleic acid molecules comprises a first nucleic acid sequence and a second nucleic acid sequence, the second nucleic acid sequence being substantially complementary to the first nucleic acid sequence; (ii) producing a treated surface by subjecting the surface to conditions sufficient to bind the first nucleic acid sequence of the nucleic acid molecules of the first set of nucleic acid molecules to the second nucleic acid sequence of the nucleic acid molecules to provide immobilized hairpin molecules; and (iii) storing the substrate having the treated surface for a period of at least 1 hour.

[0045] In various aspects, the present disclosure provides a method for storing a substrate comprising a surface coated with nucleic acid molecules, comprising: (i) providing a substrate having a surface comprising a first set of nucleic acid molecules immobilized thereon, wherein the nucleic acid molecules of the first set of nucleic acid molecules are configured to capture sample nucleic acid molecules derived from one or more nucleic acid samples, and wherein each nucleic acid molecule of the nucleic acid molecules of the first set of nucleic acid molecules comprises a first nucleic acid sequence; (ii) providing a second set of nucleic acid molecules, wherein each nucleic acid molecule of the second set of nucleic acid molecules comprises a second nucleic acid sequence that is substantially complementary to the first nucleic acid sequence, and wherein the second set of nucleic acid molecules is not the sample nucleic acid molecule; (iii) contacting the surface comprising the first set of nucleic acid molecules with the second set of nucleic acid molecules to produce a treated surface, wherein at least 70% of the nucleic acid molecules of the first set of nucleic acid molecules hybridize to the nucleic acid molecules of the second set of nucleic acid molecules; and (iv) storing the treated surface for at least one hour, wherein, for each nucleic acid molecule of the first set of nucleic acid molecules that hybridizes to the nucleic acid molecules of the second set of nucleic acid molecules, the first nucleic acid sequence hybridizes to the second nucleic acid sequence, and wherein the first nucleic acid sequence hybridized to the second nucleic acid sequence is at least partially denatured at approximately 40 degrees Celsius to 60 degrees Celsius.

[0046] In various aspects, the present disclosure provides a method for detecting or analyzing an analyte, comprising: (i) providing an open substrate comprising a central axis, the open substrate comprising an analyte array adjacently fixed to the open substrate, wherein at least one analyte in the analyte array is bound to a probe; and (ii) performing a nonlinear scan of the open substrate using a detector system to detect at least one signal or signal change from the bound probe, wherein the detector system comprises a line scan camera and an illumination source, wherein the illumination source is configured to produce an illumination area on the open substrate, wherein the open substrate comprises a first area and a second area, wherein the first area and the second area: (a) comprise different subsets of the analyte array, (b) are located at different radial positions of the open substrate relative to the central axis, and (c) are spatially resolved by the detector system; and wherein the bound probe is disposed in the first area of ​​the open substrate, and wherein the nonlinear scanning is performed during relative nonlinear motion between the open substrate and one or both of (1) the line scan camera and (2) the illumination area.

[0047] In various aspects, the present disclosure provides an apparatus for analyte detection or analysis, comprising: a housing configured to receive an open substrate having an array of analytes immobilized adjacent to the open substrate, wherein at least one analyte in the analyte array is bound to a probe; and a detector system, wherein the detector system comprises a line scan camera and an illumination source, wherein the illumination source is configured to produce an illumination area on the open substrate, wherein the open substrate comprises a first area and a second area, wherein the first area and the second area: (a) contain a subset of the array of immobilized analytes, (b) are located at different radial positions of the open substrate relative to the central axis, and (c) are spatially resolved by the detector system; wherein the bound probe is disposed in the first area of ​​the open substrate, and wherein the detector system is programmed to perform a nonlinear scan of the open substrate and detect at least one signal or signal change from the bound probe at the first area of ​​the open substrate, wherein the nonlinear scan is performed during relative nonlinear motion between the open substrate and one or both of (1) the line scan camera and (2) the illumination area.

[0048] In various aspects, the present disclosure provides a computer-readable medium comprising non-transitory instructions stored thereon that, when executed, cause one or more computer processors to implement a method for detecting or analyzing an analyte, the method comprising: providing an open substrate about a central axis, the open substrate comprising an analyte array abutting against the open substrate, wherein at least one analyte in the analyte array is bound to a probe; and performing a non-linear scan of the open substrate using a detector system to detect at least one signal or signal change from the bound probe, wherein the detector system comprises a line scan camera and an illumination source, wherein the illumination source is configured to produce an illumination area on the open substrate, wherein the open substrate comprises a first area and a second area, wherein the first area and the second area: (a) comprise different subsets of the analyte array, (b) are located at different radial positions of the open substrate relative to the central axis, and (c) are spatially resolved by the detector system; wherein the bound probe is disposed in the first area of ​​the open substrate, and wherein the non-linear scanning is performed during relative non-linear motion between the open substrate and one or both of (1) the line scan camera and (2) the illumination area.

[0049] In various aspects, the present disclosure provides a method for nucleic acid sample processing, comprising: (i) providing a first source comprising a first set of nucleic acid molecules and a second source comprising a second set of nucleic acid molecules, wherein the first source is different from the second source; (ii) directing the first set of nucleic acid molecules from the first source to a substrate to produce the first set of nucleic acid molecules immobilized in a first array adjacent to the substrate; (iii) imaging the substrate to identify a first set of locations on the substrate, wherein the first array is adjacent to the substrate; (iv) directing the second set of nucleic acid molecules from the second source to the substrate to produce the second set of nucleic acid molecules immobilized in a second array adjacent to the substrate, wherein the second array is different from the first array; (v) imaging the substrate to identify a second set of locations on the substrate, wherein the second array is adjacent to the substrate; and (vi) using (a) signals detected from the first array and the second array and (b) the locations from which the signals are detected to identify (1) the first set of nucleic acid molecules or sequences thereof having the first source and (2) the second set of nucleic acid molecules or sequences thereof having the second source, wherein the first set of locations and the second set of locations each comprise at least 1,000,000 locations.

[0050] In various aspects, the present disclosure provides a method for scanning a surface, comprising: (i) scanning a scanning field including a portion of the surface using a scanner, wherein the scanning field has an orientation relative to an axis of rotation of the surface; and (ii) before, during, or after the surface and the scanning field are translated relative to each other, (a) rotating the surface about the axis of rotation of the surface and (b) rotating the scanning field about the axis of rotation of the scanning field to substantially maintain the orientation of the scanning field relative to the axis of rotation of the surface.

[0051] In various aspects, the present disclosure provides a system comprising: a scanner configured to scan a scanning field comprising a portion of a surface, wherein the scanning field has an orientation relative to an axis of rotation of the surface; and a controller configured to instruct (i) the surface to rotate about the axis of rotation of the surface and (ii) the scanning field to rotate about the axis of rotation of the scanning field to substantially maintain the orientation of the scanning field relative to the axis of rotation of the surface before, during, or after the surface and the scanning field are translated relative to each other.

[0052] In various aspects, the present disclosure provides a method for analyzing biological material, comprising: (i) activating an apparatus comprising: (a) a substrate comprising a surface having the biological material, wherein the surface is at a first temperature greater than an ambient temperature, (b) an optical imaging objective in optical communication with the surface, wherein the optical imaging objective comprises a temperature gradient between the first temperature and the ambient temperature, wherein the optical imaging objective comprises (1) a first optical element at least partially immersed in an immersion fluid in contact with the surface, and (2) a second optical element in optical communication with the surface through at least the first optical element, and wherein the second optical element is maintained at or below a second temperature different from the first temperature; and (ii) collecting a signal from the surface having the biological material using the optical imaging objective.

[0053] In various aspects, the present disclosure provides a system for analyzing biological material, comprising: a platform configured to support a substrate comprising a surface having the biological material, wherein the surface is configured to be at a first temperature greater than an ambient temperature when the substrate is supported by the platform; an optical imaging objective configured to optically communicate with the surface when the substrate is supported by the platform, wherein the optical imaging objective is configured to include a temperature gradient between the first temperature and the ambient temperature, wherein the optical imaging objective comprises (1) a first optical element configured to be at least partially immersed in an immersion fluid in contact with the surface, and (2) a second optical element in optical communication with the surface through at least the first optical element, and wherein the second optical element is configured to be maintained at or below a second temperature, the second temperature being different from the first temperature; and one or more computer processors programmed individually or collectively to instruct the collection of signals from the surface having the biological material using at least the optical imaging objective.

[0054] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods above or elsewhere herein.

[0055] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto. The computer memory comprises machine executable code that, when executed by the one or more computer processors, implements any of the methods described above or elsewhere herein.

[0056] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0057] Incorporation by reference

[0058] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent such incorporated by reference publications and patents or patent applications contradict the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as the "Figures"), in which:

[0060] Figure 1 A computer control system is shown that is programmed or otherwise configured to implement the methods provided herein;

[0061] Figure 2 A flow chart illustrating an example of a method for sequencing a nucleic acid molecule;

[0062] Figure 3 A system for sequencing nucleic acid molecules is shown;

[0063] Figure 4A A system for sequencing nucleic acid molecules on a first vertical level is shown;

[0064] Figure 4B A system for sequencing nucleic acid molecules on a second vertical level is shown;

[0065] Figure 5A A first example of a system for sequencing nucleic acid molecules using an array of fluid flow channels is shown;

[0066] Figure 5B A second example of a system for sequencing nucleic acid molecules using an array of fluid flow channels is shown;

[0067] Figure 6A computerized system for sequencing nucleic acid molecules is shown;

[0068] Figure 7 Shown are systems with different environmental conditions in an open substrate system;

[0069] Figures 8A-8D The scheme for a line scan camera is shown. Figure 8A Shown are rows of pixels for a time delay and integration (TDI) line scan camera. Figure 8B A trilinear pixel scheme for a color line scan camera comprising red (R), green (G), and blue (B) pixels is shown. Figure 8C and Figure 8D A bilinear pixel scheme for a color line scan camera comprising red, green, and blue pixels is shown;

[0070] Figure 9 An optical system for scanning a substrate over a continuous area during its rotational motion is shown;

[0071] Figure 10A An optical system for imaging a substrate during its rotational motion using tailored optical distortion is shown;

[0072] Figure 10B An optical system for imaging a substrate during its rotational motion using tailored optical distortion is shown;

[0073] Figure 10C An example of custom optical distortion caused by using a cylindrical lens is shown;

[0074] Figure 11A A scheme for expanding a laser beam to provide a laser line is schematically shown;

[0075] Figure 11B A scheme for expanding a laser beam to provide a laser line is schematically shown;

[0076] Figure 11C An optical system for shaping the laser beam is shown;

[0077] Figures 12A-12C A scheme for detecting a signal emitted by a material coupled to an open substrate is shown; Figure 12A shows a scheme in which the open substrate is rotated and the detector system remains stationary during detection; Figure 12B shows a scheme in which the open substrate remains stationary and the detector system rotates during detection; Figure 12C shows a scheme in which the open substrate is rotated during delivery and dispersion of the solution onto the open substrate (left panel) and remains stationary during detection with a rotating detector system (right panel);

[0078] Figure 13A A first example of an interleaved helical imaging scan is shown;

[0079] Figure 13B A second example of an interleaved imaging scan is shown;

[0080] Figure 13C An example of a nested imaging scan is shown;

[0081] Figure 14 The configuration of a nested circular imaging scan is shown;

[0082] Figure 15 shows a cross-sectional view of an immersion optical system;

[0083] Figure 16 schematically illustrates an exemplary temperature gradient during optical imaging;

[0084] Figures 17A-17B An exemplary method for regulating substrate temperature is schematically illustrated;

[0085] Figure 17C-17D An exemplary method for regulating substrate temperature is schematically illustrated;

[0086] Figure 17E An exemplary method for regulating substrate temperature is schematically illustrated;

[0087] Figure 18 Schematic illustration of bubble formation in a fluid;

[0088] Figure 19 schematically illustrates an adapter for an optical imaging system;

[0089] Figure 20A An exemplary method for displacing bubbles is schematically illustrated, showing a substrate having a fluid dispensed thereon;

[0090] Figure 20B An exemplary method for displacing bubbles is schematically illustrated, showing an optical imaging objective in contact with a fluid;

[0091] Figure 21 schematically illustrates a method for dispensing and removing immersion fluid on a substrate;

[0092] Figures 22A-22B Schematically illustrates a method for capturing bubbles and an exemplary adapter for an optical imaging objective;

[0093] Figure 23A The architecture of the system including the stationary axis substrate and the moving fluidics and optics is shown;

[0094] Figure 23B The architecture of the system including the translation axis substrate and stationary fluidics and optics is shown;

[0095] Figure 23C The architecture of a system including multiple stationary substrates and moving fluidics and optics is shown;

[0096] Figure 23D The architecture of a system comprising multiple moving substrates on a rotating stage and stationary fluidics and optics is shown;

[0097] Figure 23E The architecture of a system including multiple stationary substrates and moving optics is shown;

[0098] Figure 23F The architecture of a system including multiple moving substrates and stationary fluidics and optics is shown;

[0099] Figure 23G The architecture of a system including multiple moving substrates and stationary fluidics and optics is shown;

[0100] Figure 23H The architecture of a system including multiple substrates moving between multiple processing bays is shown;

[0101] Figure 23I shows the architecture of a system including multiple imaging heads that scan with shared translation and rotation axes and rotate the field independently;

[0102] Figure 23J shows the architecture of a system including multiple imaging heads that scan with shared translation and rotation axes and rotate the field independently;

[0103] Figure 23K shows the architecture of a system including multiple spindles that are scanned with a shared optical detection system;

[0104] Figure 24 shows the architecture of a system including multiple rotating spindles;

[0105] Figure 25 A flow chart illustrating an example of a method for processing an analyte;

[0106] Figure 26 A first example of a system for separating analytes is shown; and

[0107] Figure 27 A second example of a system for separating analytes is shown;

[0108] Figure 28An example of a control system for compensating for velocity gradients during scanning is shown;

[0109] Figure 29A shows the motion of a substrate relative to two imaging heads located on the same side of the substrate's axis of rotation;

[0110] Figure 29B shows the motion of a substrate relative to two imaging heads located on opposite sides of the substrate's axis of rotation;

[0111] Figure 29C The motion of the substrate relative to the three imaging heads is shown;

[0112] Figure 29D The motion of the substrate relative to the four imaging heads is shown;

[0113] Figure 29E The motion of the substrate relative to the four imaging heads is shown;

[0114] Figure 29F The motion of the substrate relative to the four imaging heads is shown;

[0115] Figure 29G The motion of the substrate relative to the four imaging heads is shown;

[0116] Figure 30A shows a series of annular paths for two imaging heads located on the same side of the substrate's axis of rotation;

[0117] Figure 30B shows a series of annular paths for two imaging heads located on opposite sides of the substrate's axis of rotation;

[0118] Figure 30C shows the staggered annular paths of two imaging heads located on the same side of the substrate rotation axis;

[0119] Figure 30D shows the staggered annular paths of two imaging heads located on opposite sides of the substrate's axis of rotation;

[0120] Figure 31A shows the rotational scan direction of the imaging head due to non-radial motion of the substrate;

[0121] Figure 31B shows the rotational scan direction of the imaging head due to non-radial motion of the substrate;

[0122] Figure 32 A flow chart illustrating an example of a method for analyte detection or analysis;

[0123] Figure 33 The rotation axis and relative translation of the surface and the rotation axis of the imaging field are schematically shown;

[0124] Figure 34A Schematically illustrates an optical system for rotating the imaging field;

[0125] Figure 34B Schematically illustrates an optical system for rotating the imaging field;

[0126] Figure 34C Schematically illustrates an optical system for rotating the imaging field;

[0127] Figure 35A An example of imaging head positioning for optimal scanning efficiency is shown;

[0128] Figure 35B An example of imaging head positioning for optimal scanning efficiency is shown;

[0129] Figure 35C An example of imaging head positioning for optimal scanning efficiency is shown;

[0130] Figure 36A-Figure 36B A method for processing a biological analyte is schematically illustrated;

[0131] Figures 37A-37G Different examples of cross-sectional surface profiles of substrates are shown;

[0132] Figures 38A-38D Methods for rendering oligonucleotide-coated surfaces resistant to nucleic acid contaminants are shown;

[0133] Figure 39A-Figure 39B Two examples of spatial loading schemes are shown;

[0134] Figure 40 A multiplexed sample processing scheme is shown;

[0135] Figure 41 An exemplary optical layout is schematically shown;

[0136] Figure 42 An example of an image produced by imaging a substrate having an analyte immobilized thereon is shown;

[0137] Figure 43 Examples of data obtained from diagnostic procedures are shown;

[0138] Figure 44 shows example data of a diagnostic procedure; panels AF show spatial maps of diagnostic indicators calculated on a scanned image at different individually addressable locations;

[0139] Figure 45A Example data for flow-based sequencing are shown;

[0140] Figures 45B-45CExample data from processed images is shown;

[0141] Figure 46A A plot of aligned genomic reads is shown; Figure 46B The alignment coverage distribution plot on the reference genome is shown;

[0142] Figure 47A showing a partial cross-sectional view of a barrier system maintaining a fluid barrier;

[0143] Figure 47B Shown Figure 47A A perspective view of a chamber of a barrier system;

[0144] Figure 48 Systems and methods for spiral loading samples or reagents onto a substrate are shown;

[0145] Figure 49 An exemplary coating of a substrate having a hexagonal bead lattice is shown;

[0146] Figure 50A and Figure 50B A method for loading beads onto a substrate is shown; Figure 50A A method for loading beads onto a specific area of ​​a substrate is shown; Figure 50B A method for loading a subset of beads onto a specific area of ​​a substrate is shown;

[0147] Figure 51 A method for spiral loading samples or reagents onto an open substrate is shown. DETAILED DESCRIPTION

[0148] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions may be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0149] As used herein, the term "processing analyte" generally refers to one or more stages interacting with one or more sample substances. Processing analyte can include carrying out chemical reaction, biochemical reaction, enzymatic reaction, hybridization reaction, polymerization reaction, physical reaction, any other reaction or combination thereof in the presence of analyte or on analyte. Processing analyte can include physical and / or chemical manipulation of analyte. For example, processing analyte can include detecting chemical change or physical change, adding or subtracting material, atom or molecule, molecular confirmation, detecting the presence of fluorescent labeling, detecting Forster resonance energy transfer (FRET) interaction or inferring that there is no fluorescence. The term "analyte" can refer to molecule, cell, biological particle or organism. In some cases, molecule can be nucleic acid molecule, antibody, antigen, peptide, protein or other biomolecule obtained from or derived from biological sample. Analyte can be derived from and / or derived from biological sample, such as cell or organism. Analyte can be synthetic.

[0150] As used herein, the term "sequencing" generally refers to a process for generating or identifying the sequence of a biomolecule, such as a nucleic acid molecule. Such a sequence can be a nucleic acid sequence, which can include a sequence of nucleic acid bases. Sequencing can be, for example, single molecule sequencing or sequencing by synthesis. Sequencing can be performed using a template nucleic acid molecule immobilized on a support, such as a flow cell or one or more beads.

[0151] As used herein, the term "biological sample" generally refers to any sample from a subject or sample. A biological sample can be a fluid or tissue from a subject or sample. The fluid can be blood (e.g., whole blood), saliva, urine, or sweat. The tissue can be from an organ (e.g., liver, lung, or thyroid) or from a block of cellular material, such as a tumor. A biological sample can be a stool sample, a cell collection (e.g., a cheek swab), or a hair sample. A biological sample can be a cell-free sample or a cell sample. Examples of biological samples include nucleic acid molecules, amino acids, polypeptides, proteins, carbohydrates, fats, or viruses. In one example, a biological sample is a nucleic acid sample comprising one or more nucleic acid molecules, such as deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). Nucleic acid molecules can be cell-free or cell-free nucleic acid molecules, such as cell-free DNA or cell-free RNA. Nucleic acid molecules can be derived from a variety of sources, including humans, mammals, non-human mammals, apes, monkeys, chimpanzees, reptiles, amphibians, birds, or plant sources. Additionally, samples containing cell-free sequences can be extracted from various animal fluids including, but not limited to, blood, serum, plasma, vitreous, sputum, urine, tears, sweat, saliva, semen, mucosal secretions, mucus, spinal fluid, amniotic fluid, lymph fluid, etc. Cell-free polynucleotides can be of fetal origin (by taking fluids from a pregnant subject) or derived from the subject's own tissues.

[0152] As used herein, the term "subject" generally refers to an individual from whom a biological sample is obtained. A subject can be a mammal or a non-mammal. A subject can be an animal, such as a monkey, a dog, a cat, a bird, or a rodent. A subject can be a human. A subject can be a patient. A subject can be showing symptoms of a disease. A subject can be asymptomatic. A subject can be receiving treatment. A subject can not be receiving treatment. A subject can have or be suspected of having a disease, such as cancer (e.g., breast cancer, colorectal cancer, brain cancer, leukemia, lung cancer, skin cancer, liver cancer, pancreatic cancer, lymphoma, esophageal cancer, or cervical cancer) or an infectious disease. A subject can have or be suspected of having a genetic disorder, such as achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-tooth, cry-a-cat syndrome, Crohn's disease, cystic fibrosis, Dercum disease, Down syndrome, Duane syndrome, syndrome), Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson disease, phenylketonuria, Polen anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, or Wilson disease

[0153] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," and "polynucleotide" generally refer to polynucleotides, such as deoxyribonucleotides or deoxyribonucleic acid (DNA) or ribonucleotides or ribonucleic acid (RNA), or analogs thereof, which can be of various lengths. Non-limiting examples of nucleic acids include DNA, RNA, genomic DNA or synthetic DNA / RNA, or coding or non-coding regions of a gene or gene fragment, a locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, and isolated RNA of any sequence. The nucleic acid molecule can have a length of at least about 10 nucleic acid bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2kb, 3, kb, 4kb, 5kb, 10kb, 20kb, 30kb, 40kb, 50kb, 100kb, 200kb, 300kb, 400kb, 500kb, 1 megabase (Mb) or more. A nucleic acid molecule (e.g., a polynucleotide) can include a sequence of the following four natural nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (when the polynucleotide is RNA, thymine (T) is replaced by uracil (U)). A nucleic acid molecule can include one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0154] Non-standard nucleotides, nucleotide analogs and / or modified analogs may include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquenoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-glycine, Nucleosyl quercetin, 5'-methoxycarboxymethyl uracil, 5-methoxy uracil, 2-methylthio-D46-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, quercetin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, ethynyl nucleotide base, 1-propynyl nucleotide base, azido nucleotide base, phosphoroselenoate nucleic acid, etc. In some cases, the nucleotide may include modifications at its phosphate moiety, including modifications to the triphosphate moiety. In addition, non-limiting examples of modifications include phosphate chains of greater length (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties), modifications with sulfhydryl moieties (e.g., α-thiotriphosphates and β-thiotriphosphates), or modifications with selenium moieties (e.g., selenophosphate nucleic acids). Nucleic acid molecules can also be modified at the base moiety (e.g., at one or more atoms that are typically available for hydrogen bonding with complementary nucleotides and / or at one or more atoms that are typically unable to hydrogen bond with complementary nucleotides), at the sugar moiety, or at the phosphate backbone. Nucleic acid molecules can also contain amine modifying groups, such as aminoallyl dUTP (aa-dUTP) and aminohexyl acrylamide dCTP (aha-dCTP), to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide ester (NHS). Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher densities (in units of nucleotides per mm 3 Nucleotide analogs may be capable of reacting or binding with a detectable moiety for nucleotide detection.

[0155] As used herein, the term "nucleotide" generally refers to any nucleotide or nucleotide analog. Nucleotides can be naturally occurring or non-naturally occurring. Nucleotide analogs can be modified, synthetic or engineered nucleotides. Nucleotide analogs may not be naturally occurring or may include non-canonical bases. Naturally occurring nucleotides may include canonical bases. Nucleotide analogs may include modified polyphosphate chains (e.g., triphosphates coupled to a fluorophore). Nucleotide analogs may include labels. Nucleotide analogs may be terminated (e.g., reversibly terminated). Nucleotide analogs may include alternative bases.

[0156] The terms "amplifying," "amplification," and "nucleic acid amplification" are used interchangeably and generally refer to the generation of one or more copies of a nucleic acid or template. For example, "amplification" of DNA generally refers to the generation of one or more copies of a DNA molecule. Additionally, the amplification of nucleic acids can be linear, exponential, or a combination thereof. Amplification can be emulsion-based or non-emulsion-based. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction (PCR), ligase chain reaction (LCR), helicase-dependent amplification, asymmetric amplification, rolling circle amplification, recombinase polymerase reaction (RPA), and multiple displacement amplification (MDA). Where PCR is used, any form of PCR can be used, non-limiting examples of which include real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, mini-primer PCR, multiplex PCR, nested PCR, overlap extension PCR, thermal asymmetric staggered PCR, and touchdown PCR. In addition, amplification can be carried out in the reaction mixture including the various components (for example, primers, templates, nucleotides, polymerases, buffer components, cofactors, etc.) that participate in or promote amplification. In some cases, the reaction mixture includes a buffer that allows nucleotides to be incorporated independently of the environment. Non-limiting examples include magnesium ions, manganese ions and isocitrate buffers. Other examples of such buffers are described in Tabor, S. et al., CCPNAS, 1989, 86,4076-4080 and U.S. Patent No. 5,409,811 and 5,674,716, each of which is incorporated herein by reference in its entirety.

[0157] The terms "distribute" and "disperse" are used interchangeably herein. In some cases, distributing may include dispersing and / or dispersing may include distributing. Distributing generally refers to distributing, depositing, providing or supplying a reagent, solution or other object, etc. Distributing may include dispersing, which may generally refer to spreading.

[0158] Useful methods for clonal amplification from single molecules include rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), which is incorporated herein by reference), bridge PCR (Adams and Kron, Method for Performing Amplification of Nucleic Acid with Two Primers Bound to a Single Solid Support, Mosaic Technologies, Inc. (Winter Hill, Mass.); Whitehead Institute for Biomedical Research, Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000); Pemov et al., Nucl. Acids Res. 33:e11 (2005); or U.S. Pat. No. 5,641,658, each of which is incorporated herein by reference), polymerase clone passaging (Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931 (2003); Mitra et al., Anal. Biochem. 320:55-65 (2003), each of which is incorporated herein by reference) and clonal amplification using emulsion on beads (Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), each of which is incorporated herein by reference) or ligation to bead-based adapter libraries (Brenner et al., Nat. Biotechnol. 18:630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci. USA 97:1665-1670 (2000)); Reinartz et al., Brief Funct. Genomic Proteomic 1:95-104 (2002), each of which is incorporated herein by reference).

[0159] As used herein, the term "detector" generally refers to a device capable of detecting a signal, including a signal indicating the presence or absence of one or more incorporated nucleotides or fluorescent labels. The detector can detect multiple signals. A signal or multiple signals can be detected in real time during or after a biological reaction such as a sequencing reaction (e.g., sequencing during a primer extension reaction). In some cases, the detector can include optical and / or electronic components that can detect signals. The term "detector" can be used in a detection method. Non-limiting examples of detection methods include optical detection, spectral detection, electrostatic detection, electrochemical detection, acoustic detection, magnetic detection, etc. Optical detection methods include but are not limited to light absorption, ultraviolet-visible (UV-vis) light absorption, infrared light absorption, light scattering, Rayleigh scattering, Raman scattering, surface-enhanced Raman scattering, Mie scattering, fluorescence, luminescence, and phosphorescence. Spectral detection methods include but are not limited to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include but are not limited to gel-based techniques, such as gel electrophoresis. Electrochemical detection methods include but are not limited to electrochemical detection of amplification products after high performance liquid chromatography separation of the amplification products.

[0160] As used herein, the term "continuous area scanning" generally refers to the use of an optical imaging system and a detector to perform area scanning on a rotating substrate in a circular, spiral, or arc shape. The continuous area scan may scan a substrate or array along a non-linear path. Alternatively or additionally, the continuous area scan may scan a substrate or array along a linear or substantially linear path. The detector may be a continuous area scan detector. The scanning direction may be substantially θ in an (R,θ) coordinate system, where the object rotational motion is in the θ direction. Over any field of view on the object (substrate) imaged by the scanning system, the apparent velocity may vary with the radial position (R) of the field point on the object A continuous area scan detector may scan all image positions at the same rate and therefore may not operate at the correct scan rate for all imaging points in a curved (or arcuate or nonlinear) scan. Therefore, for imaging field points that move at a speed different from the scan speed, the scan may be affected by velocity blur. Continuous rotational area scanning may include an optical detection system or method that performs algorithmic correction, optical correction and / or electronic correction to substantially compensate for the tangential velocity blur, thereby reducing the scanning aberration. For example, compensation is algorithmically implemented by using an image processing algorithm that deconvolutes the differential velocity blur at various image positions corresponding to different radii on the rotating substrate to compensate for the differential velocity blur. In some cases, the camera or scanner may apply or use blur to compensate for the differential velocity blur.

[0161] In another example, compensation is accomplished by using a deformation magnification gradient. This can be used to magnify the substrate by different amounts (deformation magnification) along one axis at two or more substrate locations transverse to the scan direction. The deformation magnification gradient can modify the imaging velocity at the two or more locations to be substantially equal, thereby compensating for a difference in tangential velocity at the two locations on the substrate. This compensation can be adjustable to account for different velocity gradients across the field of view at different radii on the substrate.

[0162] The imaging field of view can be divided into two or more regions, each of which can be electronically controlled to scan at a different rate. These rates can be adjusted to the average projected object velocity within each region. The regions can be optically defined using one or more beam splitters or one or more mirrors. Two or more regions can be assigned to two or more detectors. Regions can be defined as segments of a single detector.

[0163] As used herein, the term "continuous area scan detector" generally refers to an imaging array sensor capable of continuous integration over a scan area, wherein the scan is electronically synchronized to the image of an object in relative motion. The continuous area scan detector may include a time delay integration (TDI) charge coupled device (CCD), a hybrid TDI, or a complementary metal oxide semiconductor (CMOS) pseudo TDI device. For example, the continuous area scan detector may include a TDI line scan camera.

[0164] As used herein, the term "open substrate" generally refers to a substantially planar substrate in which a single active surface is physically accessible from any point perpendicular to the substrate. Substantially planar refers to planarity on the micrometer or nanometer scale. Alternatively, substantially planar refers to planarity below the nanometer scale or greater than the micrometer scale (e.g., the millimeter scale).

[0165] As used herein, the term "anamorphic magnification" generally refers to the differential magnification between two axes of an image. The anamorphic magnification gradient may include the differential anamorphic magnification of a displacement in a first axis across a displacement in a second axis. The magnification in the second axis may be uniform or any other value that is substantially constant across the field.

[0166] As used herein, the term "field of view" generally refers to the area on a sample or substrate that is optically mapped to the active area of ​​a detector.

[0167] Analyte processing using open substrates

[0168] Existing microfluidic systems have utilized substrates that include many long, narrow channels. The typical flow cell geometry of such substrates results in a compromise between two competing requirements: 1) minimizing volume to minimize reagent usage; and 2) maximizing the effective hydraulic diameter to minimize flow time. This trade-off can be particularly important for cleaning operations, which may require a large cleaning volume and therefore take a long time to complete. The trade-off is illustrated by the Poiseuille equation, which indicates laminar flow, and is therefore inherent to microfluidic systems that utilize such flow cell geometries. Such flow cell geometries may also be susceptible to contamination. Because such flow cell geometries allow a limited, restricted number of channels to be used in a microfluidic system, such a limited number of channels can be shared between a variety of different mixtures including different analytes, reagents, agents, and / or buffers. The contents of the fluid flowing through the same channel may become contaminated.

[0169] This paper describes the device, system and method for processing analyte using open substrate or flow cell geometry, which can at least solve the above problems.The device, system and method can be used to promote any application or process of the reaction or interaction between analyte and fluid (for example, including the fluid of reagent, medicament, buffer, other analytes, etc.).Such reaction or interaction can be chemical (for example polymerase reaction) or physical (for example displacement).System and method as described herein can benefit from higher efficiency, such as benefiting from faster reagent delivery and smaller reagent volume required for each surface area.System and method as described herein can avoid the common contamination problem of the microfluidic channel flow pool supplied by multi-port valve, and multi-port valve can be the source of the legacy from one reagent to another reagent.The device, system and method can benefit from shorter completion time, less resources (for example, various reagents) use and / or reduce the system cost.Open substrate or flow cell geometry can be used to process any analyte, such as but not limited to nucleic acid molecules, protein molecules, antibodies, antigens, cells and / or organisms as described herein. The open substrate or flow cell geometry can be used for any application or process, such as, but not limited to, sequencing by synthesis, sequencing by ligation, amplification, proteomics, single cell processing, barcoding, and sample preparation as described herein.

[0170] The system and method can utilize a substrate including an array (such as a planar array) with an individually addressable position. Each position or a subset of such positions may have fixed an analyte (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.). For example, an analyte can be fixed on an individually addressable position via a support such as a bead. A plurality of analytes fixed to a substrate can be copies of a template analyte. For example, a plurality of analytes can have sequence homology. In other cases, a plurality of analytes fixed to a substrate can be different. A plurality of analytes can be analytes of the same type (e.g., a nucleic acid molecule), or can be a combination of different types of analytes (e.g., a nucleic acid molecule, a protein molecule, etc.). One or more surfaces of a substrate can be exposed to an open environment around and can be touched from the open environment around this. For example, an array can be exposed and can be touched from the open environment around this. In some cases, as described elsewhere herein, the open environment around can be controlled and / or limited in a larger controlled environment.

[0171] In some cases, reagent can be assigned to substrate to multiple positions, and / or multiple reagents can be assigned to substrate to single position. In some cases, distribution (to multiple positions and / or multiple reagents can be assigned to single position) can be realized by the relative motion of substrate and dispenser (for example, nozzle). For example, reagent can be assigned to substrate in the first position, then owing to the force (for example, centrifugal force, centripetal force, inertial force etc.) caused by substrate motion, advance to the second position that is different from the first position. In another example, reagent can be assigned to reference position, and substrate can move relative to reference position so that reagent is assigned to multiple positions of substrate. In some cases, distribution (to multiple positions and / or multiple reagents can be assigned to single position) can be realized when there is no relative motion between substrate and dispenser. For example, multiple dispensers can be used for reagent distribution to different positions, and / or multiple reagents can be assigned to single position, or its combination (for example, multiple reagents can be assigned to multiple positions). In another example, external force (for example, relating to pressure difference), for example wind, can be applied to one or more surfaces of substrate to guide reagent to the different positions on substrate. In another example, the method for distributing a reagent (for example, to multiple positions and / or multiple reagents are distributed to a single position) can include vibration. In such an example, reagent can be distributed or distributed on a single area or multiple areas (or the surface of the substrate) of the substrate. The substrate (or its surface) can then be subjected to vibration, and this can spread reagent to the different positions on the substrate (or surface). Alternatively or in combination, the method can include using machinery, electricity, physical means or other means that reagent is distributed on the substrate. For example, a solution can be distributed on the substrate, and a physical scraper (for example, a scraper) can be used to spread the material distributed or reagent is distributed to different positions and / or obtain the thickness or uniformity of expectation on the whole substrate. Advantageously, this flexible distribution can be realized when not contaminating reagent. In some cases, a reagent of a certain volume is distributed to the substrate in the first position, then advances to the second position that is different from the first position, and the reagent of this volume can advance in one or more paths so that one or more paths of travel are coated with reagent. In some cases, such one or more paths of travel can comprise the desired surface area (for example, whole surface area, partial surface area (a plurality of) etc.) of the substrate.

[0172] Reagent can be distributed on uncovered surface or substrate with the flow velocity that expects.The flow velocity of fluid distribution can be about (for example, at ambient temperature or approximately 25 degrees Celsius) 1 picoliter / minute, 10 picoliter / minute, 100 picoliter / minute, 1 nanoliter / minute, 10 nanoliter / minute, 100 nanoliter / minute, 1 microliter / minute, 10 microliter / minute, 100 microliter / minute, 1 milliliter / minute, 10 milliliter / minute, 100 milliliter / minute, up to 1 liter / minute.The flow velocity of fluid distribution can be between any one of these flow velocities.The flow velocity of fluid distribution can be at least any one in these flow velocities.Alternatively, the flow velocity of fluid distribution can be at most any one in these flow velocities.Can regulate flow velocity according to the desired property (for example, thickness) of reagent or solution layer.

[0173] The solution may contain a reagent, a sample, or any useful substance. The solution may contain a fluid having desired flow properties. For example, the fluid may have a viscosity that varies with temperature. The solution may contain a non-Newtonian fluid. The solution may contain a power-law fluid, such as a shear-thinning (thixotropic) or shear-thickening fluid. The solution may contain a Newtonian fluid.

[0174] In some cases, substrate can rotate about axis.Analyte can be fixed to substrate during rotation.Can before substrate rotation (for example, with high rotational speed rotation) or during reagent (for example nucleotide, antibody, washing reagent, enzyme etc.) be distributed on substrate, to coat array with reagent and allow analyte to interact with reagent.For example, when analyte is nucleic acid molecule and when reagent includes nucleotide, nucleic acid molecule can be incorporated into one or more nucleotide or otherwise react with one or more nucleotide (for example, transient binding).In another example, when analyte is protein molecule and when reagent includes antibody, protein molecule can be in conjunction with one or more antibodies or otherwise react with one or more antibodies.In another example, when reagent includes washing reagent, any unreacted (and / or uncombined) reagent, medicament, buffer and / or other particles can be washed from substrate (and / or analyte on substrate).

[0175] In some cases, the substrate can move in any vector or direction, as described elsewhere herein. For example, this motion can be nonlinear (e.g., rotating about an axis). In another example, this motion can be linear. In other examples, motion can be a mixture of linear and nonlinear motion. During any such motion, the analyte can be fixed on the substrate. Reagents (e.g., nucleotides, antibodies, washing reagents, enzymes, etc.) can be distributed to the substrate before or during substrate movement to facilitate coating the array with reagents and allowing the analyte to interact with the reagents.

[0176] In some cases, when substrate is rotatable, can during rotation, via tangential inertia along part radial direction (i.e. away from axis of rotation) guide unconstrained rotation reagent and realize the high speed coating of all over substrate, this phenomenon is commonly referred to as centrifugal force.High-speed rotation can relate to at least 1 revolution (rpm), at least 2rpm, at least 5rpm, at least 10rpm, at least 20rpm, at least 50rpm, at least 100rpm, at least 200rpm, at least 500rpm, at least 1,000rpm, at least 2,000rpm, at least 5,000rpm, at least 10,000rpm or higher rotational speed per minute.This pattern that reagent is guided all over substrate can be referred to as centrifugal or inertial pumping in this article. During any type of motion (for example, rotational motion, non-rotating motion, linear motion, non-linear motion, accelerated motion etc.) of substrate, inertial force can guide unconstrained reagent all over substrate along any direction.

[0177] Before, during or after distributing reagent, one or more signals (such as light signal) can be detected from the detection area on substrate to generate output.For example, output can be an intermediate result or a final result obtained from the processing of analyte. Signal can be detected in a variety of situations. Distribution, rotation (or other motion) and / or detection operation of any number can be repeated in any order (independently or simultaneously) to process analyte. In some cases, substrate can be washed (for example, via distributing washing reagent) between continuous distribution reagent. One or more detection operations can be performed within the desired time frame. For example, detection operation can be carried out in about 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds. In some cases, at least two detection operations can be carried out in 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds etc. In some cases, at least three detection operations can be carried out in 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds.

[0178] This article provides a method for processing a biological analyte, the method comprising providing a substrate comprising an array having a biological analyte fixed thereon, wherein the substrate is rotatable about a central axis. In some cases, the array can be a planar array. In some cases, the array can be an array of wells. In some cases, the substrate can be textured or patterned. The method may include guiding a solution throughout the substrate and contacting the solution with the biological analyte during rotation of the substrate. The solution may be guided in a radial direction (e.g., outward) relative to the substrate to coat the substrate and contact the biological analyte fixed to the array. In some cases, the solution may include a plurality of probes. In some cases, the solution may be a washing solution. The method may include subjecting the biological analyte to conditions sufficient to react between at least one of the plurality of probes and the biological analyte. The reaction may generate one or more signals from at least one probe coupled to the biological analyte. The method may include detecting one or more signals to analyze the biological analyte.

[0179] In other cases, provided herein is a method for processing a biological analyte, comprising providing a substrate comprising an array having immobilized biological analytes, wherein the substrate is movable relative to a reference axis. The method may include directing a solution over the substrate and contacting the solution with the biological analyte during the movement of the substrate. In some cases, the motion may be linear. In some cases, the motion may be nonlinear. In some cases, the motion may be a mixture of linear and nonlinear motion.

[0180] In other cases, a method for processing a biological analyte is provided herein, comprising providing a substrate comprising an array having a biological analyte fixed thereto. In some cases, the method may include distributing a solution to two different positions on the substrate and / or array. In some cases, the method may include, for example, distributing a plurality of solutions to a single position on the substrate and / or array using a plurality of dispensers. In some cases, the method may include distributing a plurality of solutions to a plurality of positions on the substrate and / or array. In some cases, the method may include distributing a single solution to a single position. The substrate may move relative to one or more dispensers. The substrate may be stationary relative to one or more dispensers. One or more distributing operations may be performed within a desired time frame. For example, the distributing operation may be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds. In some cases, at least two distributing operations may be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds. In some cases, at least three dispensing operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, or less than 10 seconds.

[0181] In some cases, the method for detecting a specific target gene or a specific target gene may be used to determine the target gene or a specific target gene. In some cases, the method for detecting a specific target gene or a specific target gene may be used to determine the target gene or a specific target gene. In some cases, the method for detecting a specific target gene or a specific target gene may be used to determine the target gene or a specific target gene. In some cases, the method for detecting a specific target gene or a specific target gene may be used to determine the target gene or a specific target gene. In some cases, the method for detecting a specific target gene or a specific target gene may be used to determine the target gene or a specific target gene.

[0182] One or more methods disclosed herein can eliminate the need for barcoding of analytes (e.g., nucleic acid molecules), which can be time-consuming and expensive. For example, instead of or in addition to barcoding, substrates and / or arrays can be spatially indexed to identify analytes, as described elsewhere herein. One or more methods disclosed herein can eliminate the need for unique barcoding of individual analytes (e.g., individual nucleic acid molecules).

[0183] A biological analyte can be any analyte from a sample. For example, a biological analyte can be a macromolecule, such as a nucleic acid molecule, a carbohydrate, a protein, a lipid, etc. A biological analyte can comprise a plurality of macromolecular groups, such as glycoproteins, proteoglycans, ribozymes, liposomes, etc. A biological analyte can be an antibody, an antibody fragment or an engineered variant thereof, an antigen, a cell, a peptide, a polypeptide, etc. In some cases, a biological analyte comprises a nucleic acid molecule. A nucleic acid molecule can comprise at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or more nucleotides. Alternatively or additionally, nucleic acid molecules can comprise at most about 1,000,000,000, 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 100,100,100,10 or less nucleotide. Nucleic acid molecules can have a plurality of nucleotides in the scope limited by any two of the aforementioned values. In some cases, nucleic acid molecules can also comprise the common sequence that N-mer can combine. N-mer can comprise 1,2,3,4,5 or 6 nucleotides and can combine the common sequence. In some cases, nucleic acid molecules can be amplified to produce a nucleic acid molecule colony that is attached to a substrate or is attached to a bead, and the bead can be associated with a substrate or be fixed to a substrate. In some cases, nucleic acid molecules can be attached to a bead and carry out a nucleic acid reaction, for example, amplification, to produce a clonal population of nucleic acid molecules that are attached to a bead.

[0184] The nucleic acid molecules in any given nucleic acid sample can each comprise a key sequence. The key sequence can be a synthetic sequence. In some cases, the key sequence can be at most about 6 bases in length, 5 bases in length, 4 bases in length, 3 bases in length, 2 bases in length or 1 base in length. Alternatively, the length of the key sequence can be greater than 6 bases. The key sequence can indicate the original sample. For example, the key sequence can be unique for the sample so that each sample in a plurality of samples has a unique key sequence. The individual analyte in a single sample can share the same key sequence. Alternatively, when loaded onto the substrate, each sample can have a unique key sequence between its adjacent samples. Advantageously, where two samples containing different key sequences are loaded into adjacent or otherwise proximate regions on a substrate, nucleic acid molecules originating from different samples can be readily distinguished based on the different key sequences, even in the presence of cross-contamination (e.g., peripheral nucleic acid molecules inadvertently loaded into adjacent regions due to overflowing, etc.) between regions having relatively short read sequences (e.g., much shorter than the read sequences of unique barcode sequences configured to distinguish individual molecules).

[0185] The substrate may be a solid substrate. The substrate may comprise, in whole or in part, one or more of rubber, glass, silicon, metal (such as aluminum, copper, titanium, chromium or steel), ceramic (such as titanium oxide or silicon nitride), plastic (such as polyethylene (PE), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high-impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamide, polycarbonate, polyester, polyurethane, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea formaldehyde (UF), polyetheretherketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), furan, silicone, polysulfone), any mixture of any of the foregoing materials, or any other suitable material. The substrate may be coated in whole or in part with one or more layers of metal (such as aluminum, copper, silver or gold), oxide (such as silicon oxide (SiO2), or a plurality of ... x O y, where x and y can take any possible value), a photoresist (such as SU8), a surface coating (such as aminosilane or hydrogel), polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the foregoing materials, or any other suitable coating. The substrate may be completely or partially opaque to visible light. In some cases, the substrate may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% opaque to visible light. The substrate may have an opacity within a range defined by any two of the foregoing values. The substrate may be completely or partially transparent to visible light. In some cases, the substrate can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% transparent to visible light. The substrate can have a transparency within a range defined by any two of the aforementioned values. In some cases, the irradiation power (e.g., laser power) can be adjusted based on the opacity or transparency of the substrate. One or more layers can have a thickness of at least 1 nanometer (nm), at least 2nm, at least 5nm, at least 10nm, at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1 micron (μm), at least 2μm, at least 5μm, at least 10μm, at least 20μm, at least 50μm, at least 100μm, at least 200μm, at least 500μm or at least 1 millimeter (mm). One or more layers can have a thickness within the range defined by any two of the aforementioned values. The surface of the substrate can be modified to include any adhesive or joint as described herein. The surface of the substrate can be modified to include active chemical groups, such as amine, ester, hydroxyl, epoxide, etc., or a combination thereof. In some cases, such adhesives, joints, active chemical groups, etc. can be added to the substrate as additional layers or coatings.

[0186] The substrate may have a general form of a cylinder, a cylindrical shell or disk, a rectangular prism, or any other geometric structure. The substrate may have a thickness (e.g., minimum dimension) of at least 100 μm, at least 200 μm, at least 500 μm, at least 1 mm, at least 2 mm, at least 5 mm, or at least 10 mm. The substrate may have a thickness within a range defined by any two of the aforementioned values. The substrate may have a first lateral dimension (such as a width for a substrate having the general form of a rectangular prism or a radius for a substrate having the general form of a cylinder) of at least 1 mm, at least 2 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, at least 200 mm, at least 500 mm, or at least 1,000 mm. The substrate may have a first lateral dimension within a range defined by any two of the aforementioned values. The substrate can have a second lateral dimension (such as the length for a substrate having the general form of a rectangular prism) of at least 1 mm, at least 2 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, at least 200 mm, at least 500 mm, or at least 1,000 mm. The substrate can have a second lateral dimension within a range defined by any two of the foregoing values.

[0187] The surface of the substrate may be planar. The surface of the substrate may be uncovered and may be exposed to the atmosphere. Alternatively or additionally, the surface of the substrate may be textured or patterned. For example, the substrate may include grooves, slots, slopes and / or columns. The substrate may define one or more cavities (e.g., micrometer-scale cavities or nanometer-scale cavities). The substrate may define one or more channels. The substrate may have a regular texture and / or pattern over the entire surface of the substrate. For example, the substrate may have a regular geometric structure (e.g., a wedge, a cuboid, a cylinder, a sphere, a hemisphere, etc.) above or below a reference level of the surface. Alternatively, the substrate may have an irregular texture and / or pattern over the entire surface of the substrate. For example, the substrate may have any arbitrary structure above or below a reference level of the substrate. In some cases, the texture of the substrate can include structures having a maximum dimension that is at most about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% of the total thickness of the substrate or substrate layer. In some cases, the texture and / or pattern of the substrate can define at least a portion of the individually addressable locations on the substrate. The textured and / or patterned substrate can be substantially planar.

[0188] For example, Figures 37A-37G Different examples of cross-sectional surface profiles of substrates are shown. Figure 37AA cross-sectional surface profile of a substrate having a completely flat surface is shown. Figure 37B A cross-sectional surface profile of a substrate having hemispherical grooves or recesses is shown. Figure 37C A cross-sectional surface profile of a substrate having pillars or alternatively or in combination with wells is shown. Figure 37D The cross-sectional surface profile of the substrate with the coating is shown. Figure 37E A cross-sectional surface profile of a substrate with spherical particles is shown. Figure 37F Shown Figure 37B A cross-sectional surface profile wherein a first type of adhesive is seeded in or associated with corresponding grooves. Figure 37G Shown Figure 37B A cross-sectional surface profile wherein the second type of adhesive is seeded in or associated with the corresponding grooves.

[0189] The substrate may include an array. For example, the array may be located on a side surface of the substrate. The array may be a planar array. The array may have a general shape such as a circle, annulus, rectangle, or any other shape. The array may include linear and / or nonlinear rows. The array may be evenly spaced or distributed. The array may be randomly spaced or distributed. The array may have regular spacing. The array may have irregular spacing. The array may be a textured array. The array may be a patterned array. The array may include a plurality of individually addressable locations. The individually addressable locations may be arranged in any convenient pattern. For example, the individually addressable locations may be randomly oriented on the array. The plurality of individually addressable locations may form separate radial regions around the disc-shaped substrate. The plurality of individually addressable locations may form a square, rectangle, disc, circle, ring, pentagon, hexagon, heptagon, octagon, array, or any other pattern. One or more types of individually addressable locations may be generated. One or more types of individually addressable locations may form alternating regions of different types of individually addressable locations. The independently addressable positions of one or more types can form the blocking area of ​​the independently addressable positions of different types.For example, when the independently addressable positions of two types (A and B) are needed, the independently addressable positions can be arranged as alternating ABABAB, blocked AAABBB or random arrangement, such as ABBAAB, AABBBA, BABBAA etc. The independently addressable position type can be arranged in any useful pattern, such as square, rectangle, disk, ring, pentagon, hexagon, radial pattern etc. In some cases, the independently addressable positions of two types may have different chemical, physical and / or biological properties (for example, hydrophobicity, charge, color, morphology, size, dimensions, geometric structure etc.). For example, the independently addressable position of the first type can be in conjunction with the biological analyte of the first type but not in conjunction with the biological analyte of the second type, and the independently addressable position of the second type can be in conjunction with the biological analyte of the second type but not in conjunction with the biological analyte of the first type.

[0190] Analyte to be processed can be fixed to array.Array can include one or more binding agents as described herein, such as one or more physical or chemical joints or adapters coupled to biological analytes.For example, array can include joints or adapters coupled to nucleic acid molecules.Alternatively or additionally, biological analytes can be coupled to beads, which are fixed to array.In some cases, a subset of array may not be coupled to sample or analyte.For example, in a substrate configured to rotate around a central axis, a sample may not be coupled to a plurality of individually addressable positions of an array located near the central axis.In some cases, array can be coupled to sample or analyte, but not all arrays can be processed.For example, a substrate can be coupled to a sample or analyte (for example, comprising nucleic acid molecules), but the array area close to the array boundary may not be further processed (for example, detected).

[0191] Individually addressable locations can include locations accessible to manipulated analytes or analyte groups. Manipulation can include placement, extraction, reagent distribution, inoculation, heating, cooling, or stirring. Extraction can include extracting a single analyte or analyte group. For example, extraction can include extracting at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1,000 analytes or analyte groups. Alternatively or additionally, extraction can include extracting at most 1,000, at most 500, at most 200, at most 100, at most 50, at most 20, at most 10, at most 5, or at most 2 analytes or analyte groups. Manipulation can be accomplished by, for example, local microfluidics, pipetting, light, laser, sound, magnetic, and / or electromagnetic interactions with the analyte or its surroundings.

[0192] In some cases, it is possible to index individually addressable positions, for example, spatially, so that the analyte fixed or coupled to each individually addressable position can be identified. In some embodiments, individually addressable positions are indexed by demarcating a portion of a substrate. In some embodiments, etching is used to demarcate the surface of a substrate. In some embodiments, recesses in the surface are used to demarcate the surface of a substrate. In some embodiments, dyes or inks are used to demarcate the surface of a substrate. In some embodiments, topographical markers are deposited on the surface to demarcate the surface of a substrate. In some embodiments, a sample, such as a control nucleic acid sample, can be used to demarcate the surface of a substrate. It is understood that a combination of positive demarcation and negative demarcation (which is lacking) can be used to index individually addressable positions. In some cases, a single reference point or axis (e.g., a single demarcation) can be used to index all individually addressable positions. In some embodiments, each individually addressable position is indexed. In some embodiments, a subset of individually addressable positions is indexed. In some embodiments, individually addressable positions are not indexed, and different regions of the substrate are indexed.

[0193] Individually addressable locations or individual regions comprising individually addressable locations may be indexed or otherwise distinguished. In some cases, individually addressable locations or individual regions may be distinguished solely by sample loading (e.g., without physical demarcation). In some cases, a single region may be distinguished from other regions. In some cases, a region of a single type may be distinguished from regions of other types. For example, different types of regions may include different types of analytes or different groups of samples. For example, a first type of region ("A") may include a first group of samples (or samples of the first type), and a second type of region ("B") may include a second group of samples (or samples of the second type). The substrate may include a set of multiple regions A' and a set of multiple regions B', wherein the multiple regions A' are distinguishable from the multiple regions B'. Different samples may be loaded onto different types of regions in a predetermined spatial configuration to allow such distinction.

[0194] In some cases, the key or barcode sequence on the sample can be used to distinguish and / or index spatial position, original sample or its combination. For example, the nucleic acid molecules in any given nucleic acid sample can each comprise a key sequence. The key sequence can be a synthetic sequence. The key sequence can be at most about 6 bases in length, 5 bases in length, 4 bases in length, 3 bases in length, 2 bases in length or 1 base in length. Alternatively, the length of the key sequence can be greater than 6 bases. The key sequence can indicate the original sample. For example, the key sequence can be unique for the sample so that each sample in a plurality of samples has a unique key sequence. The individual analytes of a single sample can share a common key sequence. Alternatively, when loaded onto the substrate, each sample can have a unique key sequence between its adjacent samples. Advantageously, where two samples containing different key sequences are loaded into adjacent or otherwise proximate regions on a substrate, nucleic acid molecules originating from different samples can be readily distinguished based on the different key sequences, even in the presence of cross-contamination (e.g., peripheral nucleic acid molecules inadvertently loaded into adjacent regions due to overflowing, etc.) between regions having relatively short read sequences (e.g., much shorter than the read sequences of barcode sequences configured to distinguish individual molecules).

[0195] In some cases, spatial separation of analytes can be used to augment or replace the use of key or barcode sequences. For example, Figure 40 Schemes for analyzing analytes in a single region or multiple regions (including 7, 15, and 96 regions) are shown. Figure 40As shown, the analyte may be distributed across the entire surface (top left, "1-clusters"), in discrete regions, including 7 regions (top right, "7-clusters"), 15 regions (bottom left, "15-clusters"), or 96 regions (bottom right, "96-clusters"). The analyte may be distributed in about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, or about 500 regions. The analytes can be distributed among 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 200, 200 to 300, 300 to 400, or 400 to 500 regions. In some cases, each region can contain a different analyte.

[0196] In some cases, different types of areas can be used for sample processing. An area of ​​a first type ("A") can include a first group of samples (or samples of the first type), and an area of ​​a second type ("B") can include a second group of samples (or samples of the second type). As described elsewhere herein, the areas of the first type and the areas of the second type can be arranged separately from each other in an orderly manner. In some cases, the areas of the first type and the areas of the second type can be arranged at a distance from a reference axis of the substrate. For example, the areas of the first type can be arranged at least 1 micron, 10 microns, 100 microns, 1 millimeter, 10 millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100 centimeters, or more from the reference axis of the substrate. Similarly, the areas of the second type can be arranged at a distance from the reference axis of the substrate. For example, the areas of the first type can be arranged at least 1 micron, 10 microns, 100 microns, 1 millimeter, 10 millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100 centimeters, or more from the reference axis of the substrate. Both types of regions may be positioned at least 1 micron, 10 microns, 100 microns, 1 millimeter, 10 millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100 centimeters, or more from a reference axis of the substrate.

[0197] For example, Figure 39A-Figure 39B Two examples of spatial loading schemes are shown. Figure 39A In FIG, the substrate includes two types of regions "A" and "B" which are arranged in a radially alternating manner relative to the central axis of the substrate. Figure 39BIn the present invention, the substrate includes two types of areas "A" and "B" arranged across the substrate in a triangular alternating manner. The sample positions can be determined by loading a first set of samples into area A, wherein the first set of samples includes a plurality of beads coupled to the analyte of the first set of samples, and detecting the plurality of beads and / or analytes and their positions on the substrate, and then loading a second set of samples into area B, wherein the second set of samples includes a plurality of beads coupled to the analyte of the second set of samples, and detecting the plurality of beads and / or analytes and their positions on the substrate. Each sample in the first and second sets of samples can be associated with a label (e.g., a fluorescent dye). Even if the first set of samples is primarily loaded into area A, there may be some intersection, where stray beads from the first set of samples are fixed to area B. Even if the second set of samples is primarily loaded into area B, there may be some intersection, where stray beads from the second set of samples are fixed to area A. The positions of the analytes of the first set of samples, including the crossed beads, can be determined from the first image. The positions of the analytes of the second set of samples, including the crossed beads, can be determined from the second image. Advantageously, when the same type of fluorescent dye identifies analytes from two different samples ("P" and "Q"), and "P" is deposited in region A and "Q" is deposited in region B, it is possible to identify whether the analyte is a "P" sample or a "Q" sample based on the type of region in which the fluorescent signal is detected. The different regions can be alternating. The multiple regions can form any pattern, such as a triangle, square, rectangle, disk, circle, ring, pentagon, hexagon, heptagon, octagon, array, or any other pattern. The multiple regions can form an irregular pattern. The multiple regions can be unpatterned discrete regions. The multiple regions can be staggered, interspersed, discontinuous, and / or of different sizes.

[0198] Although the examples herein describe two types of regions, there can be any number of regions (e.g., alternating regions) to achieve the alternating spatial distinctions described herein. For example, there can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 regions.

[0199] In some cases, the individually addressable locations can include different surface chemistries. Different surface chemistries can distinguish different addressable locations. Different surface chemistries can distinguish different regions. For example, a first location has a first affinity for an object (e.g., a bead containing a nucleic acid molecule immobilized thereon, e.g., an amplicon), and a second location has a second, different affinity for the object due to a different surface chemistry. The first location and the second location may or may not be located in the same region. The first location and the second location may or may not be arranged in an alternating manner on the surface. In another example, due to different surface chemical properties, a first region (e.g., including multiple individually addressable locations) has a first affinity for the object, and a second region has a second, different affinity for the object. A first location type or region type can include a first surface chemistry, and a second location type or region type can include a second surface chemistry. In some cases, a third location type or region type can include a third surface chemistry. For example, a first location type or region type can include a positively charged surface chemistry and / or a hydrophobic surface chemistry, and a second location type or region type can include a negatively charged surface chemistry and / or a hydrophilic surface chemistry, such as Figure 50A As shown in . The same object (e.g., beads containing nucleic acid molecules, e.g., amplicons, immobilized thereon) can have a higher affinity for a first location type or region type as compared to a second location type or region type. The same object may be attracted to a first location type or region type and repelled by a second location type or region type. In other examples, a first location type or region type comprising a first surface chemistry (e.g., a positively charged surface chemistry or a negatively charged surface chemistry) can interact with (e.g., have an affinity for) a first sample type (e.g., beads containing nucleic acid molecules, e.g., amplicons, immobilized thereon) and exclude a second sample type (e.g., beads lacking, e.g., nucleic acid molecules, e.g., amplicons, immobilized thereon, completely or in a substantial volume), e.g., in Figure 50B In some cases, the surface chemistry may include amines. In some cases, the surface chemistry may include silanes (e.g., tetramethylsilane). In some cases, the surface chemistry may include hexamethyldisilazane (HMDS). In some cases, the surface chemistry may include (3-aminopropyl)triethoxysilane (APTMS). In some cases, the surface chemistry may include surface primer molecules or any oligonucleotide molecules with any degree of affinity for another molecule.

[0200] An individually addressable location in a plurality of locations (eg, alternating locations) may have an area. In some cases, a location may have an area of ​​approximately 0.1 square micrometers (μm). 2 ), about 0.1 square micrometer (μm 2 ), about 0.2μm 2 , about 0.25μm2 , about 0.3μm 2 , about 0.4μm 2 , about 0.5μm 2 , about 0.6μm 2 , about 0.7μm 2 , about 0.8μm 2 , about 0.9μm 2 , about 1μm 2 , about 1.1μm 2 , about 1.2μm 2 , about 1.25μm 2 , about 1.3μm 2 , about 1.4μm 2 , about 1.5μm 2 , about 1.6μm 2 , about 1.7μm 2 , about 1.75μm 2 , about 1.8μm 2 , about 1.9μm 2 , about 2μm 2 , about 2.25μm 2 , about 2.5μm 2 , about 2.75μm 2 , about 3μm 2 , about 3.25μm 2 , about 3.5μm 2 , about 3.75μm 2 , about 4μm 2 , about 4.25μm 2 , about 4.5μm 2 , about 4.75μm 2 , about 5μm 2 , about 5.5μm 2 or about 6 μm 2 The location may have an area within the range defined by any two of the aforementioned values. The location may have an area less than about 0.1 μm 2 or greater than about 6 μm 2In some cases, the location may have an area of ​​about 0.1 micrometers (μm), about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 1.1 μm, about 1.2 μm, about 1.25 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1 In some cases, a position may have a width within a range defined by any two of the aforementioned values. A position may have a width less than about 0.1 μm or greater than about 6 μm. Positions (e.g., of the same type) may be distributed on the substrate at a spacing determined by the distance between the center of a first position and the center of a nearest or adjacent position (e.g., of the same type). The position can be about 0.1 micrometer (μm), about 0.2 μm, about 0.25 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 1.1 μm, about 1.2 μm, about 1.25 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.75 μm, about 1.8 μm, about 1. In some cases, the position can be positioned at a spacing of about 9 μm, about 2 μm, about 2.25 μm, about 2.5 μm, about 2.75 μm, about 3 μm, about 3.25 μm, about 3.5 μm, about 3.75 μm, about 4 μm, about 4.25 μm, about 4.5 μm, about 4.75 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm or about 10 μm. In some cases, the position can be positioned at a spacing within the range limited by any two of the aforementioned values. The position can be positioned at a spacing less than about 0.1 μm or greater than about 10 μm. In some cases, the spacing between any two positions of the same type can be determined as a function of the size of the loaded object (e.g., bead). For example, where the loading objects are beads having a maximum diameter, the spacing may be at least about the maximum diameter of the loading objects.

[0201] While the embodiments herein generally describe loading two samples or two groups of samples, any number of samples or sample groups may be affixed to a substrate. For example, a substrate may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 samples or sample groups affixed thereto. In some cases, at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or more samples or sample groups affixed thereto may be affixed thereto. Alternatively or additionally, the substrate can contain up to about 1,000,000,000, 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 100, 100, 10 or fewer samples or sample groups. When the samples are nucleic acid samples, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 nucleic acid samples can be immobilized on the substrate. In some cases, at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000 or more nucleic acid samples can be immobilized. Alternatively or additionally, the substrate may contain up to about 1,000,000,000, 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 100,000, 100, 100, 10, 10, or fewer nucleic acid samples. Advantageously, multiple samples can be processed simultaneously on the same substrate without the need to barcode the multiple samples (e.g., each sample has a common barcode sequence) to distinguish them.

[0202] Indexing can be carried out using detection methods and can be carried out in any convenient or useful step. The substrate indexed (e.g., demarcation) can be detected, such as optical imaging, to locate the indexed position, individually addressable position and / or biological analyte. A detection unit can be used for imaging. One or more sensors can be used for imaging. It may not be possible to use the naked eye for imaging. The substrate indexed can be imaged before loading the biological analyte. After the biological analyte is loaded into the individually addressable position, the substrate can be imaged again, such as to determine the position occupied or determine the location of the biological analyte relative to the substrate. In some cases, as described elsewhere herein, the substrate can be imaged after the iterative cycle of nucleotide addition (or other probes or other reagents). The index of the substrate and the known initial position (individually addressable position) of the biological analyte can allow analysis and identification of each individually addressable position and / or the sequence information of positioning. In addition, spatial indexing can allow identification of possible errors, such as sample contamination, sample loss, etc.

[0203] In some cases, indexing can be performed to identify, process, and / or analyze more than one type of biological analyte, as described above. For example, a first type of biological analyte that can be labeled can be loaded onto a first set of locations within a substrate. The substrate can be imaged for a first indexing step for the first type of biological analyte. A second type of biological analyte can be loaded onto a second set of locations within the substrate and imaged for a second indexing step for the second type of biological analyte. In some cases, the second type of biological analyte can be labeled in a manner that makes the second type of biological analyte distinguishable from the first type of biological analyte. Alternatively, the first type of biological analyte and the second type of biological analyte can be labeled with substantially the same detectable means (e.g., the same dye), and the first and second images can be processed to generate a difference image in which overlapping signals are attributed to the locations of the first type of biological analyte and different signals are attributed to the locations of the second type of biological analyte. Alternatively, the first type of biological analyte and the second type of biological analyte can be labeled with a cleavable (or otherwise removable) marker or label (e.g., a fluorescent label), and the label is cleaved after each imaging operation so that only the relevant analyte locations are imaged during each imaging operation. Thereafter, the substrate can be analyzed and all locations containing a first biological analyte can be attributed to the first biological analyte, and all locations containing a second biological analyte can be attributed to the second analyte. In some cases, labeling of the first and second analytes may not be necessary, and location attribution for the first or second analyte can be performed based solely on spatial location. This process can be repeated for any number or type of biological analytes.

[0204] The array can be coated with adhesive. For example, the array can be randomly coated with adhesive. Alternatively, the array can be coated with adhesive arranged in a regular pattern (for example, with a linear array, a radial array, a hexagonal array, etc.). The array can be coated with adhesive on at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the number of independently addressable positions or substrate surface area. The array can be coated with adhesive on a portion of partially independently addressable positions or substrate surface area, the portion being within the scope limited by any two of the aforementioned values. The adhesive can be integral with the array. The adhesive can be added to the array. For example, the adhesive can be added to the array as one or more coatings on the array.

[0205] Adhesive can be by non-specific interaction, such as one or more of hydrophilic interaction, hydrophobic interaction, electrostatic interaction, physical interaction (for example, adhesion to column or sedimentation in trap) etc. to fix biological analyte. Adhesive can be by specific interaction to fix biological analyte. For example, when biological analyte is nucleic acid molecule, adhesive can include the oligonucleotide adapter that is configured to be bonded to nucleic acid molecule. Alternatively or additionally, such as in order to combine other types of analytes, adhesive can include one or more of antibody, oligonucleotide, nucleic acid molecule, aptamer, affinity binding protein, lipid, carbohydrate etc. Adhesive can be by any possible interaction combination to fix biological analyte. For example, adhesive can be by the combination of physical and chemical interaction, by the combination of protein and nucleic acid interaction etc. to fix nucleic acid molecule. Array can include at least about 10,100,1000,10,000,100,000,1,000,000,10,000,000,100,000,000 or more adhesives. Alternatively or additionally, array can include at most about 100,000,000,10,000,000,1,000,000,100,000,10,000,100,100,10 or less adhesive.Array can have multiple adhesives in the scope limited by any two of the aforementioned values.In some cases, single adhesive can be in conjunction with single biological analyte (for example, nucleic acid molecules).In some cases, single adhesive can be in conjunction with multiple biological analyte (for example, multiple nucleic acid molecules).In some cases, multiple adhesives can be in conjunction with single biological analyte.Although the example herein describes the interaction of adhesive and nucleic acid molecules, adhesive can fix other molecules (such as protein), other particles, cells, viruses, other organisms etc.

[0206] In some cases, each position or a subset of such positions may have been fixed with an analyte (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.). In other cases, a portion of a plurality of individually addressable positions may have been fixed with an analyte. The plurality of analytes fixed to the substrate may be copies of the template analyte. For example, a plurality of analytes (e.g., a nucleic acid molecule) may have sequence homology. In other cases, the plurality of analytes fixed to the substrate may not be copies. A plurality of analytes may be analytes of the same type (e.g., a nucleic acid molecule), or may be a combination of different types of analytes (e.g., a nucleic acid molecule, a protein molecule, etc.).

[0207] In some cases, the array may include multiple types of adhesives. For example, the array may include different types of adhesives to combine different types of analytes. For example, the array may include a first type of adhesive (e.g., oligonucleotide) configured to combine a first type of analyte (e.g., nucleic acid molecule) and a second type of adhesive (e.g., antibody) configured to combine a second type of analyte (e.g., protein) etc. In another example, the array may include a first type of adhesive (e.g., oligonucleotide molecule of the first type) to combine a first type of nucleic acid molecule and a second type of adhesive (e.g., oligonucleotide molecule of the second type) to combine a second type of nucleic acid molecule. For example, the substrate may be configured to combine different types of analytes in certain parts or specific locations of the substrate by having different types of adhesives in certain parts or specific locations of the substrate.

[0208] The biological analyte can be affixed to the array at a given individually addressable location in a plurality of individually addressable locations. The array can have any number of individually addressable locations. For example, the array can have at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20 The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. The array may have at least one individually addressable location. Each individually addressable location may be digitally and / or physically accessible (from the plurality of individually addressable locations). For example, each individually addressable location can be located, identified and / or accessed electronically or digitally for mapping, sensing, being associated with a device (e.g., a detector, a processor, a dispenser, etc.) or otherwise processed. As described elsewhere herein, each individually addressable location can be indexed. Alternatively, the substrate can be indexed so that each individually addressable location can be identified during at least one step of the process. Alternatively or additionally, each individually addressable location can be physically located, identified and / or accessed, such as for physical manipulation or extraction of an analyte, reagent, particle or other component located at the individually addressable location.

[0209] A variety of biological analytes can be fixed to the array at a spatially discrete position. The spatial separation of biological analytes can be obtained using a mask or barrier, as described elsewhere herein. Alternatively or in combination, different fluid compositions can be used to separate biological analytes. In some cases, the fluid composition may be immiscible. For example, a first solution (e.g., oil, organic solution or other hydrophobic or lipophilic solution) may contain a first biological analyte, and a second solution (e.g., hydrophilic, aqueous, polar or ionic solution) may contain a second biological analyte. The first and second solutions may be immiscible. The substrate may be exposed to the first solution in a defined area, such as using a mask (e.g., covering or shielding other areas of the substrate). In some cases, the first biological analyte is associated with a defined area (e.g., an individually addressable location), and the first solution can be removed from the substrate. The substrate can then be exposed to the second solution. The second biological analyte can then be associated with an unoccupied site of the substrate. Alternatively, the substrate can be pre-treated so that the biological analyte can be loaded in a discrete position. In one non-limiting example, the substrate can be patterned with discrete hydrophobic and hydrophilic regions (e.g., using photolithography, soft lithography, etching, etc.) that can attract or repel a subset of biological analytes. In another non-limiting example, an inert polymer such as polyethylene glycol (PEG) can be patterned in discrete regions to prevent attachment of biological analytes to the substrate in the discrete regions.

[0210] Each individually addressable location can have a general shape or form that is circular, concave, convex, rectangular, or any other shape or form. Each individually addressable location can have a first lateral dimension (such as a radius for an individually addressable location having a general shape of a circle or a width for an individually addressable location having a general shape of a rectangle). The first lateral dimension can be at least 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or at least 10,000 nm. The first lateral dimension can be within a range defined by any two of the aforementioned values. Each individually addressable location can have a second lateral dimension (such as a length for an individually addressable location having a general shape of a rectangle). In some embodiments, the second lateral dimension can be at least 1 nanometer (nm), at least 2nm, at least 5nm, at least 10nm, at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1,000nm, at least 2,000nm, at least 5,000nm or at least 10,000nm.The second lateral dimension can be in the scope limited by any two of the aforementioned values.In some cases, each independently addressable position can have adhesive as described herein or with adhesive coupling as described herein, so that analyte is fixed with it.In some cases, only a part can have adhesive or with adhesive coupling in the independently addressable position.In some cases, independently addressable position can have multiple adhesive or with multiple adhesive coupling, so that analyte is fixed with it.

[0211] A variety of methods can be used to generate individually addressable locations. In one embodiment, the method can include using one or more barriers to generate individually addressable locations. In some embodiments, the barriers can be removed during any convenient operation. For example, the barriers can be removed before or after coupling the analyte to the individually addressable location. The barriers can be removed before or after loading the solution containing multiple probes. The barriers can be removed before or after subjecting the analyte to conditions sufficient for a reaction between the probe and the analyte. The barriers can be removed before or after detecting one or more signals from the coupled probes and analyte. The barriers can be removed before or after detecting the coupled probes and analyte. The barriers can be removed before or after repeating any of the above processes. In some cases, the barriers may not be removed.

[0212] The barrier may comprise a physical, chemical, biological, or any other type of obstacle. In some embodiments, the barrier comprises a physical barrier. In one such example, a mold may be used, wherein a portion of the mold may impede the movement of the fluid to a designated area. The mold may be produced using a variety of methods, such as injection molding, machining, heat treatment, fiber spinning, bonding and gluing, casting, rolling, forging, 3D printing, and the like. In some embodiments, the barrier may be configured to dissolve in any convenient step. The barrier may be configured to dissolve, evaporate, or sublime. In some cases, the barrier may be melted and removed. In some cases, removal of the barrier or a portion of the barrier may be achieved using an air knife. In some cases, the barrier comprises a chemical barrier. In some cases, the barrier comprises a polymer. The barrier may comprise polyethylene glycol (PEG). In some cases, the barrier may comprise a solution. The solution may be viscous. The solution may have a viscosity that varies with temperature. The solution may be a non-Newtonian fluid. The solution may be a power-law fluid, such as a shear-thinning (e.g., thixotropic) or shear-thickening fluid. The solution may be a Newtonian fluid. In some embodiments, the barrier comprises a fluid that is immiscible with the loading solution. In some cases, the barrier is a hydrophobic region on the substrate.

[0213] A mask may additionally or alternatively be used to prevent coupling of the sample and / or biological analyte to areas of the substrate. Alternatively, or in combination, a subset of individually addressable locations containing biological analytes may be shielded, for example, to prevent coupling of a probe to the biological analyte. The mask may comprise a barrier, such as a physical, chemical, or biological barrier. The mask may comprise a membrane with removed portions. In some cases, the mask may interface with the substrate prior to the introduction of the biological analyte. In this case, the introduction of the biological analyte may allow the biological analyte to couple to exposed areas of the mask-substrate interface, while unexposed areas may remain free of the biological analyte. In any convenient process, the substrate may be unmasked. Any combination of masks may be used. For example, a first mask may be used to load a first biological analyte into a desired area. Subsequently, the first mask may be removed, and a second mask may be used to load a second biological analyte into the desired area. The first and second areas may have overlapping regions or may remain spatially distinct. The barrier and mask may be used in combination or separately.

[0214] The analyte that is bound to the addressable position alone can include but is not limited to molecule, cell, organism, nucleic acid molecule, nucleic acid colony, bead, cluster, polymerase clone, DNA nano ball or its any combination (for example, be attached with one or more nucleic acid molecules, for example, the bead of the clonal population of one or more nucleic acid molecules).The analyte of combination can be fixed to array in regular, patterned, periodic, random or pseudo-random configuration or any other spatial arrangement.In some embodiments, analyte is combined with bead (multiple), and bead can then be associated with substrate or substrate area (for example, addressable position alone) or be fixed thereto.In some embodiments, analyte comprises one bead or multiple beads.In some cases, bead or multiple beads can comprise another analyte (for example, nucleic acid molecule) or the clonal population of other analytes (for example, nucleic acid molecule amplified on bead).Such other analytes can be attached or otherwise coupled to bead.For example, analyte can comprise multiple beads, and each bead has the clonal population of nucleic acid molecule attached thereto. In some cases, the beads are magnetic, and the application of a magnetic field or the use of a magnet can be used to guide the analyte or beads containing the analyte to an individually addressable location. In some cases, a fluid can be used to guide the analyte to an individually addressable location. The fluid can be a ferrofluid, and a magnet can be used to guide the fluid to an individually addressable location. The individually addressable location can alternatively or in combination include materials that are sensitive to stimuli, such as thermal, chemical, or electrical or magnetic stimuli. For example, the individually addressable location can include a photosensitive polymer or reagent that is activated when exposed to electromagnetic radiation. In some cases, caged molecules can be used to reveal a binding (e.g., biotin) portion on the substrate. Subsequent exposure to light of a specific wavelength can result in the uncaging of the binding portion. Then, beads containing the analyte, such as beads with streptavidin, can be associated with the uncaged binding portion. In some cases, a subset of the individually addressable locations may not include beads. In this case, blank beads can be added to the substrate. The blank beads can then occupy the area not occupied by the analyte. In some cases, the blank beads have a higher binding affinity or avidity for the individually addressable positions than the beads containing the analyte. In some cases, the unoccupied positions may be destroyed. In some cases, the unoccupied positions may be treated to remove any unbound analyte, such as by suction, washing, air blowing, etc. In some cases, a device may be used to load a sample containing a biological analyte onto a substrate, such as a microfluidic device, a closed flow cell, etc. The loaded biological analyte may then be associated with or fixed to the substrate or the individually addressable positions of the substrate. In this case, the device may be removed after the sample is loaded.

[0215] Bioanalytes can be combined with any number of beads. Different bioanalytes can be combined with any number of beads. Beads can be unique (i.e., different from each other). Any number of unique beads can be used. For example, at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000 or more different beads can be used. Alternatively or additionally, at most about 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 100,000, 100, 10 or less different beads can be used. Many different beads can be within the range limited by any two of the aforementioned values. Using the characteristics of beads, such as color, reflectivity, anisotropy, brightness, fluorescence, etc., beads can be distinguished from each other.

[0216] The sample can be diluted to control the approximate occupancy of the individually addressable locations. The sample can be diluted to at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:10,000, 1:100,000, 1:100,000,000, 1:100,000,000. Alternatively, the sample can be diluted up to a dilution of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:10000, 1:100000, 1:10000000, 1:100000000. Dilutions between any of these dilution values ​​can also be used.

[0217] In some cases, the sample can include beads. The beads can be dispersed on the surface in any pattern or randomly. The beads can be dispersed on one or more regions of the surface (e.g., regions with a specific surface chemistry). In some cases, the beads can be dispersed on the surface or surface region in a hexagonal lattice, such as Figure 49, which shows a zoomed-out image of a portion of the surface in the right panel and a magnified image of a portion of the surface portion in the left panel. In some cases, a sample comprising beads may be dispersed on a surface comprising different locations / regions distinguished by surface chemistry (e.g., Figure 50A and Figure 50B ). For example, a sample comprising beads can be distributed on a surface comprising positively charged locations / regions and / or hydrophobic locations / regions. The beads can have a high affinity for a first location type or region type (e.g., positively charged). The beads can have a low affinity for a second location type or region type (e.g., hydrophobic). The locations can include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 beads per location. In some embodiments, the beads can be substantially centered within the individually addressable locations. A location can have a width that is up to about 0.5 times, up to about 0.6 times, up to about 0.7 times, up to about 0.8 times, up to about 0.9 times, up to about 1 times, up to about 1.1 times, up to about 1.2 times, up to about 1.3 times, up to about 1.4 times, up to about 1.5 times, up to about 1.6 times, up to about 1.7 times, up to about 1.8 times, up to about 1.9 times, up to about 2 times, up to about 2.1 times, up to about 2.2 times, up to about 2.3 times, up to about 2.4 times, up to about 2.5 times, up to about 2.6 times, up to about 2.7 times, up to about 2.8 times, up to about 2.9 times, or up to about 3 times the diameter of the bead (e.g., maximum diameter). In some embodiments, regions can be separated by a spacing determined by the distance between the center of a first location and the center of the nearest or adjacent location of the same type. The positions can be separated by a spacing of at least about 1 times, at least about 1.2 times, at least about 1.4 times, at least about 1.6 times, at least about 1.8 times, at least about 2 times, at least about 2.2 times, at least about 2.4 times, at least about 2.6 times, at least about 2.8 times, at least about 3 times, at least about 3.2 times, at least about 3.4 times, at least about 3.6 times, at least about 3.8 times, at least about 4 times, at least about 4.2 times, at least about 4.4 times, at least about 4.6 times, at least about 4.8 times, or at least about 5 times the diameter (e.g., maximum diameter) of the bead. In some cases, one or more of position size, position spacing, bead affinity, position surface chemistry can be adjusted to reduce the deviation of the bead contact point from the center of the region.

[0218] A surface comprising a plurality of individually addressable locations can be loaded with beads. The beads can be loaded onto the surface at an occupancy rate determined by the number of locations of a given location type that include at least one bead relative to the total number of locations of the same location type. A surface comprising a plurality of locations can have an occupancy rate of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99.5%, or up to about 100%. For example, a surface can have at least about 90% of the locations of a given location type loaded with at least one bead. Beads can be landed on the surface with a landing efficiency determined by the number of surface-bound beads relative to the total number of beads distributed on the surface. The beads can be distributed onto the surface with a landing efficiency of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, up to about 100%. In some embodiments, one or more of the temperature, incubation time, surfactant, or salt concentration of the solution containing the beads can be adjusted to increase bead occupancy. In some embodiments, one or more of the temperature, incubation time, surfactant, or salt concentration of the solution containing the beads can be adjusted to increase bead loading efficiency.

[0219] In some cases, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the available surface area of ​​the substrate can be configured to receive beads. In cases where less than 100% of the available surface area is loaded with beads (e.g., having beads immobilized thereon), the negative space (e.g., locations where there are no beads) can be used as a reference to identify and / or index different individually addressable locations of the positive space (e.g., locations where there are beads). In one example, a single individually addressable location acting in the negative space is sufficient to index the entire substrate. In such an example, during imaging, such as during sequencing, a single individually addressable location will always remain "dark," in contrast to other individually addressable locations in the positive space that will light up (e.g., fluoresce) at different points in time, such that the single individually addressable location that is always "dark" can serve as a reference for all other individually addressable locations. In other examples, multiple individually addressable locations acting in the negative space can facilitate indexing of the substrate. Alternatively or additionally, reference beads that are always "bright" (e.g., always fluoresce regardless of the point in time) can be used as a reference for identifying and / or indexing different individually addressable locations of the positive space. In this case, different individually addressable locations can be identified and / or indexed even if 100% or substantially 100% of the available surface area is loaded with beads, including reference beads.

[0220] The sample comprising beads can be dispensed onto the surface. In some cases, the beads can be dispensed onto the surface in a substantially spiral pattern. For example, Figure 48 The system shown and / or Figure 51 The method shown dispenses beads in a spiral pattern. In some cases, the beads can be dispersed in a spiral pattern that moves radially inward toward the axis of rotation of the surface. In some cases, the beads can be dispersed in a spiral pattern that moves radially outward from the axis of rotation of the surface. Figure 48As shown, a sample (e.g., a sample containing beads) can be dispensed from a dispensing probe 4801 (e.g., a nozzle) to a substrate 4803 (e.g., a wafer) to form a layer 4805. The dispensing probe can be positioned at a fixed height ("Z") above the substrate. In the example shown, the beads are retained in layer 4805 by electrostatic retention and can be fixed to the substrate. A set of beads can each contain a population of amplification products (e.g., nucleic acid molecules) immobilized thereon, which accumulate into a negative charge on the beads and have an affinity for positive charges. The substrate contains alternating surface chemistry between distinguishable locations, wherein a first location type contains APTMS that carries a positive charge and has an affinity for the negative charge of amplification beads (e.g., beads containing amplification products immobilized thereon, and different from negative beads that do not contain amplification products), and a second location type contains HMDS that has a lower affinity and / or repels amplified beads. In the layer 4805 comprising the dispensed sample, the amplified beads can successfully land on a first location of a first location type (as in 4807). In the example shown, the location size is 1 micron, the spacing between different locations of the same location type (e.g., the first location type) is 2 microns, and the layer has a depth of 15 microns. To obtain a substantially spiral pattern, the substrate and / or the dispensing probe can have an angular velocity and / or a linear velocity relative to each other.

[0221] The samples can be distributed as shown in the figure. Figure 51 On the open surface shown. In some cases, the substrate can rotate relative to the distribution probe. In some cases, the distribution probe can move radially relative to the substrate relative to the rotation axis of the substrate. In some cases, the substrate can move linearly relative to the distribution probe. In some cases, the substrate can rotate relative to the distribution probe and move linearly relative to the distribution probe, thereby distributing the sample in a spiral pattern. In some cases, when the distribution probe moves radially relative to the substrate relative to the rotation axis of the substrate, the substrate can rotate relative to the distribution probe, thereby distributing the sample in a spiral pattern. The substrate and the distribution probe can move relative to each other in any configuration to achieve a basic spiral pattern. The substrate can be no more than 60rpm, no more than 50rpm, no more than 40rpm, no more than 30rpm, no more than 25rpm, no more than 20rpm, no more than 15rpm, no more than 14rpm, no more than 13rpm, no more than 12rpm, no more than 11rpm, no more than 10rpm, no more than 9rpm, no more than 8rpm, no more than 7rpm, no more than 6rpm, no more than 5rpm, no more than 4rpm, no more than 3rpm, no more than 2rpm or no more than 10rpm of rotation frequency rotation. In some cases, the rotation frequency can be within the range defined by any two of the aforementioned values. In some cases, the substrate may be rotated at a rotation frequency of about 5 rpm.

[0222] The spiral dispensing pattern may have a path width determined by the width of the area coated by the fluid dispensed during a single rotation of the surface. The spiral dispensing pattern may have a path spacing determined by the distance between the center of the fluid dispensing path at a first position and the center of the fluid dispensing path at a second position after the substrate rotates once. In some cases, the path width may be greater than the path spacing. For example, the fluid dispensed along the path during a substrate rotation may overlap with the fluid dispensed along the path during a previous substrate rotation. In some cases, the path spacing may be greater than the path width. For example, the fluid dispensed along the path during a substrate rotation may be separated from the fluid dispensed along the path during a previous substrate rotation. In some cases, the path width may be similar to the path spacing. For example, the fluid dispensed along the path during a substrate rotation is substantially not separated from the fluid dispensed along the path during a previous substrate rotation, and the fluid dispensed along the path during a substrate rotation may substantially not overlap with the fluid dispensed along the path during a previous substrate rotation.

[0223] The substrate can be configured to rotate about an axis. In some cases, the systems, devices, and apparatus described herein may further include a rotation unit configured to rotate the substrate. The rotation unit may include a motor and / or a rotor to rotate the substrate. Such a motor and / or rotor may be mechanically connected to the substrate directly or indirectly via an intermediate component (e.g., a gear, a platform, an actuator, a disk, a pulley, etc.). The rotation unit may be automated. Alternatively or additionally, the rotation unit may receive manual input. The axis of rotation may be an axis passing through the center of the substrate (e.g., an attached substrate). Figure 33 As shown). The axis can be an eccentric axis. For example, the substrate can be fixed to the chuck (such as a vacuum chuck) of the spin coating equipment. The substrate can be configured to rotate at a rotation speed of at least 1 revolution per minute (rpm), at least 2rpm, at least 5rpm, at least 10rpm, at least 20rpm, at least 50rpm, at least 100rpm, at least 200rpm, at least 500rpm, at least 1,000rpm, at least 2,000rpm, at least 5,000rpm or at least 10,000rpm. The substrate can be configured to rotate at a rotation speed within the range defined by any two of the aforementioned values. The substrate can be configured to rotate at different rotation speeds during different operations described herein. The substrate can be configured to rotate at a rotation speed that varies according to a time-dependent function (such as a ramp function, a sine function, a pulse function or other function or a combination of functions). The time-varying function can be periodic or aperiodic.

[0224] The substrate can be configured to move with any vector relative to a reference point. In some cases, the systems, devices and equipment described herein may also include a motion unit configured to move the substrate. The motion unit may include any mechanical components, such as a motor, a rotor, an actuator, a linear stage, a drum, a roller, a pulley, etc., to move the substrate. These components can be directly or indirectly mechanically connected to the substrate through an intermediate component (e.g., a gear, a platform, an actuator, a disc, a pulley, etc.). The motion unit can be automated. Alternatively or additionally, the motion unit can receive manual input. The substrate can be configured to move at any speed. In some cases, the substrate can be configured to move at different speeds during different operations described herein. The substrate can be configured to move at a speed that varies according to a time-dependent function (e.g., a ramp function, a sine function, a pulse function or other function or a combination of functions). The time-varying function can be periodic or aperiodic.

[0225] Solution can be provided to the substrate before or during substrate rotation (or other motion) to guide the solution centrifugally (or otherwise inertially) throughout the array. In some cases, solution can be provided to the planar array during substrate rotation in a pulsed manner, thereby generating a circular wave in which the solution moves radially outward. In some cases, solution can be provided to the planar array during substrate other motions in a pulsed manner, thereby generating a solution wave moving in a specific direction. Pulses can have periodic or non-periodic (e.g., arbitrary) intervals. A series of pulses can include a series of waves that produce surface-reagent exchanges. Surface-reagent exchanges can include washing, wherein each subsequent pulse includes a surface reagent that reduces concentration. The solution can have a temperature different from that of the substrate, thereby providing a source or reservoir of thermal energy to the substrate or the analyte on the substrate. Thermal energy can provide temperature changes for the substrate or analyte. Temperature changes may be temporary. Temperature changes can enable, prevent, enhance, or inhibit chemical reactions (such as those to be performed on the analyte). For example, chemical reactions can include denaturation, hybridization, or annealing of nucleic acid molecules. The chemical reaction may include a step in a polymerase chain reaction (PCR), bridge amplification, or other nucleic acid amplification reaction.The temperature change may modulate, increase, or decrease, the signal detected from the analyte.

[0226] Array can be in communication with at least one sample inlet fluid (of fluid passage).Array can be in communication with sample inlet fluid via non-solid gap, for example, air gap.In some cases, array can be in communication with at least one sample outlet fluid additionally.Array can be in communication with sample outlet fluid via air gap.Sample inlet can be configured to guide solution to array.Sample outlet can be configured to receive solution from array.One or more distribution nozzles can be used to guide solution to array.For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 distribution nozzles can be used to guide solution to array.Can use a plurality of nozzles in the scope of any two limits by aforementioned value to guide solution to array.In some cases, can distribute different reagents (for example, dissimilar nucleotide solutions, different probes, washing solutions etc.) via different nozzles, such as to prevent pollution.Each nozzle can be connected to special fluid line or fluid valve, and this can further prevent pollution. Can distribute a kind of reagent type via one or more nozzles.One or more nozzles can be directed to the center of substrate or near the center of substrate.Perhaps, one or more nozzles can be directed to the position except the center of substrate or near the position except the center of substrate on substrate.Alternately or in combination, one or more nozzles can be directed to the center closer to substrate than one or more other nozzles.For example, one or more nozzles for distributing washing reagent can be directed to the center closer to substrate than one or more nozzles for distributing active agent.One or more nozzles can be arranged at the radius of different distances from the center of substrate.Two or more nozzles can be combined to operate so that fluid is more effectively delivered to substrate.One or more nozzles can be configured to deliver fluid to substrate in the form of jet, spray (or other dispersed fluid) and / or droplet.Before being delivered to substrate, one or more nozzles can be operated to atomize fluid.For example, fluid can be carried as aerosol particles.

[0227] The solution may be dispensed onto the substrate while the substrate is stationary; then, after dispensing the solution, the substrate may be subjected to rotation (or other motion). Alternatively, the substrate may be subjected to rotation (or other motion) before dispensing the solution; then, the solution may be dispensed onto the substrate while the substrate is rotating (or otherwise moving).

[0228] The rotation of the substrate can generate a centrifugal force (or an inertial force directed away from the axis) on the solution, causing the solution to flow radially outward on the array. In this way, the rotation of the substrate can guide the solution throughout the array. Continuous rotation of the substrate over a period of time can distribute a fluid film of almost constant thickness throughout the array. The rotation speed of the substrate can be selected to obtain a desired thickness of the solution film on the substrate. The film thickness can be related to the rotation speed by equation (1):

[0229]

[0230] Here, h(t) is the thickness of the fluid film at time t, μ is the viscosity of the fluid, ω is the rotational speed, and C is a constant.

[0231] Alternatively or in combination, the viscosity of the solution can be selected to obtain the desired thickness of the solution film on the substrate. For example, the rotational speed of the substrate or the viscosity of the solution can be selected to obtain a film thickness of at least 10 nanometers (nm), at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1 micron (μm), at least 2μm, at least 5μm, at least 10μm, at least 20μm, at least 50μm, at least 100μm, at least 200μm, at least 500μm or at least 1mm. The rotational speed of the substrate and / or the viscosity of the solution can be selected to obtain a film thickness within the range limited by any two of the aforementioned values. The viscosity of the solution can be controlled by controlling the temperature of the solution. The thickness of the film can be measured or monitored. The measurement of the thickness of the film or monitoring can be incorporated into a feedback system to better control the film thickness. The thickness of the film can be measured or monitored by a variety of techniques. For example, the thickness of the film can be measured or monitored by thin film spectrometers such as fiber spectrometers.

[0232] In some cases, one or more factors such as the rotational speed of the substrate, the acceleration of the substrate (e.g., rate of change of velocity), the viscosity of the solution, the angle of distribution of the solution (e.g., contact angle of the reagent stream), the radial coordinate of the solution distribution (e.g., on the center, off-center, etc.), the substrate temperature, the solution temperature, and other factors can be adjusted and / or optimized in other ways to obtain the desired wettability and / or film thickness on the substrate on the substrate to promote uniform coating of the substrate. In some cases, a surfactant can be added to the solution, or a surfactant can be added to the surface to promote uniform coating or promote sample loading efficiency. This optimization can prevent the solution from leaving the substrate along a relatively concentrated stream or travel path so that the fluid contacts the substrate only at a portion of the surface area (rather than the entire surface area) - in this case, a significantly larger volume of reagent may have to be distributed to achieve uniform and complete coating of the substrate. This optimization can also prevent the solution from scattering or otherwise reflecting or rebounding off the substrate when contacting and interfering with the surface fluid. Alternatively or in combination, mechanical, electrical, physical or other mechanisms can be used to adjust the thickness of the solution. For example, the solution can be dispensed onto the substrate and then leveled using, for example, a physical scraper such as a doctor blade to achieve a desired thickness uniformity across the substrate.

[0233] In some cases, the rotation of the substrate can be slow enough so as not to produce significant centrifugal forces (or inertial forces directed away from the axis) on the solution. Advantageously, one or more reagents dispensed onto the substrate can be kept substantially local to the landing position, e.g., without significant and / or prior travel outward relative to the axis of rotation, e.g., to invade another reaction space on the open substrate. The substrate can be rotated at a rotational frequency of no more than 60 rpm, no more than 50 rpm, no more than 40 rpm, no more than 30 rpm, no more than 25 rpm, no more than 20 rpm, no more than 15 rpm, no more than 14 rpm, no more than 13 rpm, no more than 12 rpm, no more than 11 rpm, no more than 10 rpm, no more than 9 rpm, no more than 8 rpm, no more than 7 rpm, no more than 6 rpm, no more than 5 rpm, no more than 4 rpm, no more than 3 rpm, no more than 2 rpm, or no more than 1 rpm. In some cases, the rotational frequency can be within the range defined by any two of the aforementioned values. In some cases, the substrate can be rotated at a rotational frequency of about 5 rpm. In some cases, the solution can be dispensed on the surface in a spiral pattern. For example, the solution can be dispensed on the surface using Figure 48 or Figure 51 The system shown dispenses in a spiral pattern. Figure 48As shown, a solution (e.g., a sample or a wash solution) can be dispensed from a dispensing probe (e.g., a nozzle). In some embodiments, a solution can be dispensed from multiple dispensing probes. For example, a first reagent in a solution can be dispensed from a first dispensing probe, a second reagent in a solution can be dispensed from a second dispensing probe, and a third reagent in a solution can be dispensed from a third dispensing probe. Reagents dispensed from different dispensing probes can combine on a substrate to form a homogeneous solution. The dispensing probe can be positioned at a fixed height above a substrate (e.g., a wafer). Reagents can be dispensed onto an open surface, such as Figure 51 As shown. In some cases, the substrate can rotate relative to the distribution probe. In some cases, the distribution probe can move radially relative to the substrate relative to the rotation axis of the substrate. In some cases, the substrate can move linearly relative to the distribution probe. In some cases, the substrate can rotate relative to the distribution probe and move linearly relative to the distribution probe, thereby dispensing the sample in a spiral pattern. In some cases, when the distribution probe moves radially relative to the substrate relative to the rotation axis of the substrate, the substrate can rotate relative to the distribution probe, thereby dispensing the sample in a spiral pattern. The rotation speed of the substrate, the flow rate of the solution or the viscosity of the solution can be selected to obtain a film thickness of at least 10 nanometers (nm), at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1 micron (μm), at least 2μm, at least 5μm, at least 10μm, at least 20μm, at least 50μm, at least 100μm, at least 200μm, at least 500μm or at least 1mm.

[0234] In some cases, the solution can be heated before being distributed on the substrate. The solution can be in a temperature higher than the ambient temperature. Before distribution, the solution can be heated to about 25 ° C, about 26 ° C, about 27 ° C, about 28 ° C, about 29 ° C, about 30 ° C, about 31 ° C, about 32 ° C, about 33 ° C, about 34 ° C, about 35 ° C, about 36 ° C, about 37 ° C, about 38 ° C, about 39 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C, about 60 ° C, about 65 ° C, about 70 ° C, about 75 ° C, about 80 ° C, about 85 ° C, about 90 ° C, about 95 ° C. In some cases, the solution can be heated to a temperature within the range limited by any two of the aforementioned values.

[0235] In some cases, the substrate can be rotated at a variable angular velocity. The angular velocity of the substrate can be varied so that the linear velocity of the substrate relative to the dispensing probe remains substantially constant as the radial distance between the dispensing probe and the substrate's axis of rotation changes. For example, the angular velocity of the substrate can be reduced as the dispensing probe distributes the fluid in a spiral path that travels outward relative to the substrate's axis of rotation. In another example, the angular velocity of the substrate can be increased as the dispensing probe distributes the fluid in a spiral path that travels inward relative to the substrate's axis of rotation. In some cases, the dispensing probe can distribute the fluid at a variable flow rate. The flow rate of the dispensing probe can be varied so as to substantially maintain the amount of fluid distributed per unit area of ​​the substrate. For example, the flow rate of the dispensing probe can be increased as the dispensing probe distributes the fluid in a spiral path that travels outward relative to the substrate's axis of rotation. In another example, the flow rate of the dispensing probe can be reduced as the dispensing probe distributes the fluid in a spiral path that travels inward relative to the substrate's axis of rotation.

[0236] One or more solutions distributed on the surface can react on the surface. For example, the first solution distributed on the surface (for example, comprising a reactant) can react with the second solution (for example, comprising an enzyme) distributed on the surface at the top of the first solution. One or more solutions distributed on the surface can inactivate or quench a chemical reaction. For example, a quenching solution (for example, comprising EDTA or acid) can be added to the substrate at the top of the reaction to quench the reaction. The solution (for example, a solution comprising a reactant, a solution comprising an enzyme or a quenching solution) can be distributed on the surface with a pattern (for example, a spiral pattern). In some embodiments, the quenching solution is distributed on the surface with the pattern identical with the solution comprising the reactant, so that each point on the surface to which the solution is distributed keeps a substantially constant reaction time. In some embodiments, the quenching solution is distributed on the surface with the pattern identical with the solution comprising the enzyme, so that each point on the surface to which the solution is distributed keeps a substantially constant reaction time. For example, the solution comprising the reactant can be distributed on the surface with a spiral path, which inwardly points to the axis of rotation of the substrate. The solution comprising the reactant can be distributed at a constant rate, and the substrate can be rotated at a variable rate so that the volume distributed per unit area is substantially constant. The solution that comprises enzyme can be distributed along the spiral path identical with the solution that comprises reagent.The solution that comprises enzyme can be distributed at a constant rate, and substrate can be rotated at a variable rate so that the time between the solution that comprises reactant and the solution that comprises enzyme in distribution is substantially the same at any point along the spiral path.Quenching solution can be distributed along the spiral path identical with the solution that comprises enzyme.Quenching solution can be distributed at a constant rate, and substrate can be rotated at a variable rate so that the time between the solution that comprises enzyme and quenching solution in distribution is substantially the same at any point along the spiral path.Alternatively or additionally, similarly, one or more solutions that are distributed on the surface can activate or catalyze chemical reaction.For example, activation solution (for example, comprising catalyst) can be added to substrate at the top of reaction (for example, with identical distribution pattern) to activate or catalyze reaction.

[0237] In some embodiments, one or more solutions can be distributed to the substrate to ensure that the reaction time of the substrate area contacted with the one or more solutions is basically similar. In some embodiments, the solution can be spin-coated on the surface by distributing the solution near the axis of rotation of the rotating substrate or so that the centrifugal force of the rotating substrate promotes the solution to walk away from the axis of rotation. Spin coating can be very suitable for distributing one or more solutions, and this solution triggers or quenches the reaction that occurs on the slower time scale relative to the distribution time. In some embodiments, one or more solutions can be directly delivered to the reaction site, and these one or more solutions can not be displaced in a large number from the delivery point. The method for solution being directly delivered to the reaction site can comprise the aerosol delivery of solution, use applicator coating solution, curtain coating solution, slot die coating, distributing solution from translational distribution probe, distributing solution from distribution probe array, immersing substrate in solution or contacting substrate with the sheet material that comprises solution.

[0238] Aerosol delivery can include delivering the solution to the substrate in the form of an aerosol by using a pressure nozzle or an ultrasonic nozzle to direct the solution to the substrate. Applying the solution using an applicator can include contacting the substrate with an applicator containing the solution and translating the applicator relative to the substrate. For example, applying the solution using an applicator can include painting the substrate. The solution can be applied in a pattern by translating the applicator, rotating the substrate, translating the substrate, or a combination thereof. The pattern can be a spiral pattern. The pattern can be a circular pattern. Curtain coating can include distributing the solution from a distribution probe onto the substrate in a continuous stream (e.g., a curtain or a flat plate) and translating the distribution probe relative to the substrate. The solution can be curtain coated in a pattern by translating the distribution probe, rotating the substrate, translating the substrate, or a combination thereof. The pattern can be a spiral pattern. The pattern can be a circular pattern. Slot die coating can include distributing the solution from a distribution probe located near the substrate so that the solution forms a meniscus between the substrate and the distribution probe, and translating the distribution probe relative to the substrate. The solution can be slot die coated in a pattern by translating the distribution probe, rotating the substrate, translating the substrate, or a combination thereof. The pattern can be a spiral pattern. The pattern may be a circular pattern. Dispensing the solution from a translational dispensing probe may include translating the dispensing probe relative to the substrate in a pattern (e.g., a spiral pattern, a circular pattern, a linear pattern, a striped pattern, a cross-hatch pattern, or a diagonal pattern). Dispensing the solution from an array of dispensing probes may include dispensing the solution from an array of nozzles (e.g., a showerhead) located above the substrate so that the solution is dispensed substantially simultaneously over an area of ​​the substrate. Immersing the substrate in the solution may include immersing the substrate in a reservoir containing the solution. In some embodiments, the reservoir may be a shallow reservoir to reduce the volume of solution required to coat the substrate. Contacting the substrate with a sheet containing the solution may include contacting the substrate with a sheet of material (e.g., a porous sheet or a fibrous sheet) permeated with the solution. The solution may be transferred to the substrate. In some embodiments, the sheet of material may be a disposable sheet. In some embodiments, the sheet of material may be a reusable sheet. In some embodiments, a disposable sheet may be used. Figure 51 The method shown is to dispense the solution onto the substrate. Figure 51 As shown, a jet of solution can be dispensed from a nozzle onto a rotating substrate. The nozzle can be radially translated relative to the rotating substrate to dispense the solution onto the substrate in a spiral pattern.

[0239] One or more solutions or reagents can be delivered to the substrate by any delivery method disclosed herein. In some embodiments, two or more solutions or reagents are delivered to the substrate using the same delivery method. In some embodiments, two or more solutions are delivered to the substrate so that the time between contacting the solution or reagent and the subsequent solution or reagent is substantially similar for each region of the substrate in contact with the one or more solutions or reagents. In some embodiments, the solution or reagent can be delivered in a single mixture form. In some embodiments, the solution or reagent can be distributed in two or more component solutions. For example, each component of the two or more component solutions can be distributed from different nozzles. Different nozzles can distribute the two or more component solutions to the substantially same region of the substrate at the same time, thereby forming a uniform solution on the substrate. In some embodiments, the distribution of each component in the two or more components can be separated in time. The distribution of each component can be carried out using the same method. For example, the first component and the second component are distributed to the substrate using the same method at substantially the same rate and substantially the same pattern so that the time between contacting the first component and the second component is substantially similar for each region of the substrate in contact with the first component and the second component. In some embodiments, the first solution can start reacting (for example, a solution comprising magnesium) on the substrate. In some embodiments, the second solution can terminate the reaction on the substrate (e.g., a solution comprising ethylenediaminetetraacetic acid (EDTA)). In some embodiments, the time between starting the reaction and stopping the reaction can be substantially the same in each area of ​​the substrate in contact with the first solution and the second solution. The first solution can form a substantially uniform film when delivered to the substrate. The second solution can comprise a fast-diffusing component that can diffuse rapidly when in contact with the first solution. In some embodiments, the fast-diffusing component can start the reaction, or the fast-diffusing component can stop the reaction.

[0240] In some embodiments, direct delivery of solutions or reagents can be combined with spin coating. For example, a first solution can be directly delivered in a spiral pattern using any of the direct delivery methods disclosed herein. The spiral pattern can be oriented inwardly toward the axis of rotation of the substrate. The first solution can initiate the reaction. A second solution can be delivered in the same pattern as the first solution. The second solution can stop the reaction. The second solution can wash away the first solution. The first and second solutions can be distributed so that the reaction occurs for a substantially fixed amount of time in each spatial region of the substrate.

[0241] In some embodiments, the incubation time can be in the range of two limits by any two of the aforementioned values. In some cases, incubation can continue for more than 90 minutes. In some cases, the fluid layer can maintain a film thickness of at least 10 nanometers (nm), at least 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 mm during the incubation period. One or more of the temperature of the chamber, the humidity of the chamber, the rotation of the substrate, or the composition of the fluid can be adjusted so that the fluid layer is maintained during the incubation period. In some cases, the substrate can be rotated at a rotation frequency of no more than 60 rpm, no more than 50 rpm, no more than 40 rpm, no more than 30 rpm, no more than 25 rpm, no more than 20 rpm, no more than 15 rpm, no more than 14 rpm, no more than 13 rpm, no more than 12 rpm, no more than 11 rpm, no more than 10 rpm, no more than 9 rpm, no more than 8 rpm, no more than 7 rpm, no more than 6 rpm, no more than 5 rpm, no more than 4 rpm, no more than 3 rpm, no more than 2 rpm, or no more than 1 rpm. In some cases, the rotation frequency can be within a range defined by any two of the aforementioned values. In some cases, the substrate can be rotated at a rotation frequency of about 5 rpm.

[0242] The substrate or its surface may include other features that help the solution or reagent remain on the substrate or the thickness uniformity of the solution or reagent on the substrate. In some cases, the surface may include a raised edge (e.g., a rim) that can be used to hold the solution on the surface. The surface may include a rim near the outer edge of the surface, thereby reducing the amount of solution flowing through the outer edge.

[0243] The solution can be a reaction mixture including multiple components. For example, the solution can include multiple probes configured to interact with the analyte. For example, the probe can have binding specificity to the analyte. In another example, the probe may not have binding specificity to the analyte. The probe can be configured to be permanently coupled to the analyte. The probe can be configured to be transiently coupled to the analyte. For example, a nucleotide probe can be permanently incorporated into a growing chain hybridized with a nucleic acid molecule analyte. Alternatively, a nucleotide probe can be transiently coupled to the nucleic acid molecule analyte. The transiently coupled probe can then be removed from the analyte. The transiently coupled probe removed from the analyte may or may not leave residues (such as chemical residues) on the analyte. The type of probe in the solution may depend on the type of analyte. The probe can include a functional group or part configured to perform a specific function. For example, the probe can include a label (such as a dye). The probe can be configured to generate a detectable signal (such as a light signal) when coupled to the analyte or otherwise interacting, such as via a label. In some cases, the probe can be configured to generate a detectable signal when activated (such as, applying a stimulus). In another example, the nucleotide probe can include a reversible terminator (e.g., a blocking group) configured to terminate the polymerase reaction (until deblocking). The solution can include other components (e.g., enzymes, catalysts, buffers, saline solutions, chelating agents, reducing agents, other reagents, etc.) to assist, accelerate, or decelerate the reaction between the probe and the analyte. In some cases, the solution can be a washing solution. In some cases, after the reaction or interaction between the reagent (e.g., probe) in the reaction mixture solution and the analyte fixed on the array, the washing solution is directed to the substrate so that the washing solution is contacted with the array. The washing solution can wash away any free reagent from the previous reaction mixture solution.

[0244] When the probe in the solution reacts with the analyte, a detectable signal, such as an optical signal (e.g., a fluorescent signal), can be generated. For example, the signal can be derived from the probe and / or the analyte. The detectable signal can indicate the reaction or interaction between the probe and the analyte. The detectable signal can be a non-optical signal. For example, the detectable signal can be an electrical signal. The detectable signal can be detected by one or more sensors. For example, in the optical detection scheme described elsewhere herein, the optical signal can be detected via one or more optical detectors. The signal can be detected during substrate rotation. The signal can be detected after rotation termination. The signal can be detected when the analyte contacts the solution fluid. The signal can be detected after the washing solution. In some cases, after detection, the signal can be eliminated, such as by cutting a label from the probe and / or the analyte, and / or modifying the probe and / or the analyte. Such cutting and / or modification can be subject to one or more stimuli, such as exposure to chemicals, enzymes, light (e.g., ultraviolet rays) or temperature changes (e.g., heat). In some cases, by deactivating or changing the mode (e.g., detection wavelength) of one or more sensors, or terminating or reversing the excitation of the signal, the signal may become undetectable. In some cases, detection of a signal may include capturing an image or generating a digital output (eg, between different images).

[0245] The operation of one or more signals of the reaction between the probe in solution and the analyte in array directed to solution can be repeated once or many times.Such operation can be repeated in an iterative manner.For example, the same analyte fixed on a given position in the array can interact with multiple solutions in multiple repetitive cycles.For each iteration, other signals detected can provide the increment or final data of relevant analyte during processing.For example, when analyte is a nucleic acid molecule and processing is order-checking, other signals detected in each iteration can indicate the base in the nucleotide sequence of this nucleic acid molecule. The operation can be repeated for at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, or at least 1,000,000,000 cycles to process the analyte. In some cases, a different solution may be directed to the substrate for each cycle. For example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, or at least 1,000,000,000 solutions can be directed to the substrate.

[0246] In some cases, washing solution can be directed to the substrate between each cycle (or at least once during each cycle). For example, after each type of reaction mixture solution is directed to the substrate, washing solution can be directed to the substrate. Washing solution can be different. Washing solution can be the same. Washing solution can be distributed in pulses during rotation, thereby generating annular waves as described herein. Washing solution can be distributed in a continuous stream during rotation, while the stream moves radially relative to the axis of rotation of the substrate, thereby distributing washing solution in a spiral pattern. In some cases, washing solution can be distributed in a spiral pattern that advances outwards relative to the axis of rotation of the substrate. In some cases, washing solution can be distributed in a spiral pattern that advances inwards relative to the axis of rotation of the substrate. For example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, or at least 1,000,000,000 wash solutions can be directed to the substrate.

[0247] In some cases, a portion or all of the solution may be recovered after the solution has contacted the substrate. Recycling may include collecting, filtering, and reusing a subset or all of the solution. Filtration may be molecular filtration.

[0248] Nucleic acid sequencing using rotation arrays

[0249] In some cases, sequencing methods can adopt synthesis scheme sequencing, wherein nucleic acid molecules are sequenced base by base by primer extension reaction. For example, the method for sequencing nucleic acid molecules can include providing a substrate including an array, to which nucleic acid molecules are fixed. The array can be a planar array. The substrate can be configured to rotate about an axis. The method can include guiding a solution including a plurality of nucleotides throughout the planar array before or during the rotation of the substrate. The rotation of the substrate can promote the coating of the solution on the substrate surface. The nucleic acid molecules can be subjected to a primer extension reaction under conditions sufficient to incorporate at least one nucleotide in the plurality of nucleotides into or specifically bind to a growing chain complementary to the nucleic acid molecule. A signal indicating the incorporation or binding of at least one nucleotide can be detected, thereby sequencing the nucleic acid molecules.

[0250] In some cases, the method can include, prior to providing the substrate to which the nucleic acid molecules are already immobilized, immobilizing the nucleic acid molecules to the substrate. For example, a solution comprising a plurality of nucleic acid molecules (including the nucleic acid molecules) can be introduced to the substrate before, during, or after the substrate is rotated, and the substrate can be subjected to conditions sufficient to immobilize at least a subset of the plurality of nucleic acid molecules as an array on the substrate.

[0251] Figure 2 A flow chart of an example of a method 200 for sequencing nucleic acid molecules is shown. In a first operation 210, the method may include providing a substrate as described elsewhere herein. The substrate may include an array of multiple individually addressable locations. The array may be a planar array. The array may be a textured array. The array may be a patterned array. For example, the array may define individually addressable locations with wells and / or pillars. Multiple nucleic acid molecules that may or may not be copies of the same nucleic acid molecule may be affixed to the array. Each of the multiple nucleic acid molecules may be affixed to the array at a given individually addressable location in the multiple individually addressable locations.

[0252] The substrate can be configured to rotate about an axis. The axis can be an axis passing through the center or substantially the center of the substrate. The axis can be an eccentric axis. For example, the substrate can be fixed to a chuck (such as a vacuum chuck) of a spin coating device. The substrate can be configured to rotate at a rotation speed of at least 1 revolution per minute (rpm), at least 2rpm, at least 5rpm, at least 10rpm, at least 20rpm, at least 50rpm, at least 100rpm, at least 200rpm, at least 500rpm, at least 1,000rpm, at least 2,000rpm, at least 5,000rpm or at least 10,000rpm. The substrate can be configured to rotate at a rotation speed within a range defined by any two of the aforementioned values. The substrate can be configured to rotate at different rotation speeds during different operations described herein. The substrate can be configured to rotate at a rotation speed that varies according to a time-dependent function (such as a ramp function, a sine function, a pulse function or other function or a combination of functions). The time-varying function can be periodic or aperiodic.

[0253] In second operation 220, described method can include before or during substrate rotation, solution is guided throughout array.Solution can be centrifugally guided throughout array.In some cases, solution can be guided to array during substrate rotation with pulsed formula, thereby generate the annular wave that solution moves radially outward.In some cases, solution can be guided to array with continuous stream during substrate rotation, and this stream moves radially relative to the axis of rotation of substrate simultaneously, thereby solution is guided to array with spiral pattern.In some cases, substrate can be configured to rotate at a speed of no more than 60rpm, no more than 50rpm, no more than 40rpm, no more than 30rpm, no more than 25rpm, no more than 20rpm, no more than 15rpm, no more than 14rpm, no more than 13rpm, no more than 12rpm, no more than 11rpm, no more than 10rpm, no more than 9rpm, no more than 8rpm, no more than 7rpm, no more than 6rpm, no more than 5rpm, no more than 4rpm, no more than 3rpm, no more than 2rpm or no more than 10rpm.In some cases, rotation frequency can be in the range limited by any two of the aforementioned values. The solution can have a temperature different from that of the substrate, thereby providing a source or reservoir of thermal energy to the substrate or the nucleic acid molecules on the substrate. Thermal energy can provide temperature variation for the substrate or the nucleic acid molecules. The temperature variation can be temporary. The temperature variation can enable, disable, enhance or inhibit a chemical reaction, such as a chemical reaction to be performed on a nucleic acid molecule. For example, the chemical reaction can include denaturation, hybridization or annealing of multiple nucleic acid molecules. The chemical reaction can be included in a step in a polymerase chain reaction (PCR), bridge amplification or other nucleic acid amplification reactions. The temperature variation can regulate, increase or decrease the signal detected from the nucleic acid molecule (or from a probe in the solution).

[0254] In some cases, the solution may contain beads. The beads may be coated with the nucleic acid molecules to be sequenced. The solution containing beads may be dispensed onto the substrate using the methods described herein. For example, the solution containing beads may be dispensed onto the substrate in a spiral pattern, such as Figure 48 or Figure 51 In some cases, the beads may preferentially interact with a first region type (e.g., a positively charged region) of the substrate, as shown in FIG. Figure 50A In some cases, beads may not interact with the second region type (e.g., a hydrophobic region) of the substrate. In some cases, beads coated with nucleic acid molecules may interact with the first region type (e.g., a positively charged region) of the substrate, and beads not coated with nucleic acid molecules may not interact with the first region type of the substrate, such as Figure 50B shown.

[0255] In some cases, solution can include a probe configured to interact with nucleic acid molecules. For example, in some cases, such as for performing sequencing by synthesis, solution can include multiple nucleotides (in the form of single bases). Multiple nucleotides can include nucleotide analogs, naturally occurring nucleotides and / or non-naturally occurring nucleotides, collectively referred to as "nucleotides" in this article. Multiple nucleotides can be or may not be bases of the same type (for example, A, T, G, C, etc.). For example, solution can include or may not include only one type of base. Solution can include at least 1 type of base or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 types of bases. For example, solution can include any possible mixture of A, T, C and G. In some cases, solution can include multiple natural nucleotides and non-natural nucleotides. Multiple natural nucleotides and non-natural nucleotides can be or may not be bases of the same type (for example, A, T, G, C). In some cases, solution can include oligo probes (for example, oligonucleotide primers). For example, in some cases, for example, in order to order-check by synthesis, solution can include multiple nucleic acid molecules, for example primers, which include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotide bases. Multiple nucleic acid molecules can include nucleotide analogs, naturally occurring nucleotides and / or non-naturally occurring nucleotides, collectively referred to as "nucleotides" in this article. Multiple nucleotides can be or may not be bases of the same type (for example, A, T, G, C, etc.). For example, solution can include or not include only one type of base. Solution can include at least one type of base or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 types of bases. For example, solution can include any possible mixture of A, T, C and G. In some cases, solution can include multiple natural nucleotides and non-natural nucleotides. The plurality of natural nucleotides and non-natural nucleotides may or may not be the same type of base (eg, A, T, G, C).

[0256] One or more nucleotides in a plurality of nucleotides can be terminated (for example, reversibly terminated).For example, nucleotides can include reversible terminators, or can reversibly terminate the part of primer extension. The nucleotides including reversible terminators can be accepted by polymerases and incorporated into the growing nucleic acid sequence in a manner similar to the nucleotides that irreversibly terminate. Polymerases can be any naturally occurring (i.e., natural or wild type) or engineered variants (for example, DNA polymerase, Taq polymerase, etc.) of polymerases. After the nucleotide analogs including reversible terminators are incorporated into the nucleic acid chain, the reversible terminators can be removed to allow further extension of the nucleic acid chain. Reversible terminators can include blocking groups or capping groups attached to the 3'-oxygen atom of the sugar moiety (for example, pentose) of nucleotides or nucleotide analogs. Such parts are referred to as 3'-O-sealed reversible terminators. The example of 3'-O-sealed reversible terminators includes, for example, 3'-ONH2 reversible terminators, 3'-O-allyl reversible terminators, and 3'-O-aziomethyl (aziomethyl) reversible terminators. Alternatively, the reversible terminator can include a blocking group in a linker (e.g., a cleavable linker) and / or a dye moiety of a nucleotide analog. 3'-unblocked reversible terminators can be attached to bases and fluorescent groups (e.g., labels as described herein) of nucleotide analogs. Examples of 3'-unblocked reversible terminators include, for example, "virtual terminators" developed by Helicos BioSciences Corp. and "lightning terminators" developed by Michael L.Metzker et al. The cutting of reversible terminators can be achieved by, for example, irradiating a nucleic acid molecule comprising a reversible terminator. In some cases, a plurality of nucleotides may not include terminated nucleotides.

[0257] One or more nucleotides in the plurality of nucleotides can be labeled with dyes, fluorophores, or quantum dots. For example, the solution can include labeled nucleotides. In another example, the solution can include unlabeled nucleotides. In another example, the solution can include a mixture of labeled nucleotides and unlabeled nucleotides. Non-limiting examples of dyes include SYBR Green, SYBR Blue, DAPI, propidium iodide, Hoeste, SYBR Gold, ethidium bromide, acridine, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine and acridine, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and ethidium homodimer-2, ethidium monoazide and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBRDX, SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, SYTO-45 (blue), SYTO-13, SYTO-16, SYTO-24, SYTO-21, SYTO -23, SYTO-12, SYTO-11, SYTO-20, SYTO-22, SYTO-15, SYTO-14, SYTO-25 (green), SYTO-81, SYTO-80, SYTO-82, SYT O-83, SYTO-84, SYTO-85 (orange), SYTO-64, SYTO-17, SYTO-59, SYTO-61, SYTO-62, SYTO-60, SYTO-63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red Red), Phar-Red, allophycocyanin (APC), Sybr Green I, SybrGreen II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue (Cascade Blue) blue), dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes (such as those containing europium and terbium), carboxytetrachlorofluorescein, 5-carboxyfluorescein and / or 6-carboxyfluorescein (FAM), VIC, 5-iodoacetamidofluorescein or 6-iodoacetamidofluorescein, 5-{[2-5-(acetylmercapto)-succinyl]amino}fluorescein and 5-{[3-5-(acetylmercapto)-succinyl]amino}fluorescein ]amino}fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5-carboxyrhodamine and / or 6-carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonic acid-4-amino-naphthalimide, phycobiliprotein, Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 532, Atto 565, Atto 594, Atto 633, Atto 647, Atto 647N, Atto 665, Atto 680 and Atto 700 dyes, AlexaFluor 350, AlexaFluor 405.AlexaFluor430, AlexaFluor 488, AlexaFluor 532, AlexaFluor 546, AlexaFluor 555, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 635, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, and AlexaFluor 790 dyes, DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, and DyLight 800 dyes, or other fluorophores, Black Hole quencher dyes (Biosearch Technologies), such as BH1-0, BHQ-1, BHQ-3, BHQ-10; QSY dye fluorescence quenchers (from Molecular Probes / Invitrogen), such as QSY7, QSY9, QSY21, QSY35 and other quenchers such as Dabcyl and Dabsyl; Cy5Q and Cy7Q and dark cyanine dyes (GE Healthcare); Dy-quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTO fluorescence quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q, 612Q. In some cases, the label can be a label with a linker. For example, the label can have a disulfide linker attached to the label. The non-limiting examples of such labels include Cy5-azide, Cy-2-azide, Cy-3-azide, Cy-3.5-azide, Cy5.5-azide and Cy-7-azide. In some cases, the linker can be a cleavable linker. In some cases, the label can be a type that is not self-quenching and does not show proximity quenching. The non-limiting examples of label types that are not self-quenching and do not show proximity quenching include bimane derivatives, such as bromobimane. Alternatively, the label can be a type that is self-quenching or shows proximity quenching. The non-limiting examples of such labels include Cy5-azide, Cy-2-azide, Cy-3-azide, Cy-3.5-azide, Cy5.5-azide and Cy-7-azide. In some cases, the blocking group of the reversible terminator can include a dye.

[0258] Can use one or more nozzles that solution is directed to array.In some cases, can distribute different reagents (for example, dissimilar nucleotide solutions, washing solutions etc.) via different nozzles, such as to prevent pollution.Each nozzle can be connected to special fluid line or fluid valve, and this can further prevent pollution.Can distribute a kind of type reagent via one or more nozzles.One or more nozzles can be directed to the center of substrate or near the center of substrate.Perhaps, one or more nozzles can be directed to the position except substrate center on substrate or near the position except substrate center.Can combine two or more nozzles to operate, so that fluid is more effectively delivered to substrate.

[0259] The solution can be dispensed onto the substrate while the substrate is stationary; then, after dispensing the solution, the substrate can be subjected to rotation. Alternatively, the substrate can be subjected to rotation before dispensing the solution; then, the solution can be dispensed onto the substrate while the substrate is rotating. The rotation of the substrate can generate centrifugal forces (or inertial forces away from the axis) on the solution, causing the solution to flow radially outward across the array.

[0260] In a third operation 230, the method can include subjecting the nucleic acid molecule to a primer extension reaction. The primer extension reaction can be performed under conditions sufficient to incorporate at least one nucleotide from the plurality of nucleotides into a growing chain complementary to the nucleic acid molecule. The incorporated nucleotide can be labeled or unlabeled.

[0261] In some cases, operation 230 may also include modifying at least one nucleotide. Modified nucleotides may include labeled nucleotides. For example, nucleotides may be labeled with dyes, fluorophores, or quantum dots. Nucleotides may be cleavably labeled. In some cases, modified nucleotides may include labels that activate (e.g., stimulate) nucleotides.

[0262] In the fourth operation 240, the method may include detecting a signal indicating the incorporation of at least one nucleotide. The signal may be an optical signal. The signal may be a fluorescent signal. The signal may be detected during substrate rotation. The signal may be detected after rotation is terminated. The signal may be detected when the nucleic acid molecule to be sequenced contacts a solution fluid. The signal may be detected after the nucleic acid molecule contacts a solution fluid. Operation 240 may also include modifying a label of at least one nucleotide. For example, operation 240 may also include cutting a label of the nucleotide (e.g., after detection). The nucleotide may be cut by one or more stimuli, such as exposure to chemicals, enzymes, light (e.g., ultraviolet light), or heat. Once the label is cut, the signal indicating the incorporated nucleotide may not be detected by one or more detectors.

[0263] Method 200 can also include repeating operations 220, 230 and / or 240 one or more times to identify one or more additional signals indicating the incorporation of one or more additional nucleotides, thereby sequencing the nucleic acid molecule. Method 200 can include repeating operations 220, 230 and / or 240 in an iterative manner. For each iteration, the additional signal can indicate the incorporation of additional nucleotides. The additional nucleotides can be the same nucleotides as those detected in the previous iteration. The additional nucleotides can be nucleotides different from those detected in the previous iteration. In some cases, at least one nucleotide can be modified (e.g., labeled and / or cut) between each iteration of operations 220, 230 or 240. For example, the method can include repeating operations 220, 230, and / or 240 at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 20 ... At least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, or at least 1,000,000,000 times. The method can include repeating operations 220, 230, and / or 240 multiple times, the multiple times being within the range defined by any two of the aforementioned values. Thus, method 200 can result in the sequencing of nucleic acid molecules of any size.

[0264] The method can include guiding different solutions to the array in a circular manner during substrate rotation. For example, the method can include guiding the first solution comprising the first type of nucleotide (for example, in the form of multiple first type nucleotides) to the array, subsequently guiding the second solution comprising the second type of nucleotide, subsequently guiding the third type of nucleotide, subsequently guiding the fourth type of nucleotide etc. to the array. In another example, different solutions can comprise different combinations of nucleotide types. For example, the first solution can include the nucleotides (for example, A) of the first standard type and the nucleotides (for example, C) of the second standard type, and the second solution can include the nucleotides (for example, A) of the first standard type and the nucleotides (for example, T) of the third standard type, and the third solution can include the nucleotides (for example, G) of the first standard type, the nucleotides of the second standard type, the nucleotides of the third standard type and the nucleotides of the fourth standard type. In another example, the first solution can include the nucleotides of the mark, and the second solution can include unlabeled nucleotides, and the third solution can include a mixture of marked and unlabeled nucleotides. The method can comprise directing at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, or at least 1,000,000,000 solutions to the array. The method may include directing a plurality of solutions within a range defined by any two of the aforementioned values ​​to the array. The solutions may be different. The solutions may be the same.

[0265] The method may comprise directing at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000,000, at least 50,000,000, at least 100,000,000, at least 200,000,000, at least 500,000,000, or at least 1,000,000,000 wash solutions to the substrate. For example, after each type of nucleic acid is introduced to the substrate, a wash solution can be introduced to the substrate. The wash solutions can be different. The wash solutions can be the same. The wash solution can be dispensed in pulses during rotation, thereby generating a ring wave as described herein. The wash solution can be dispensed in a continuous stream during rotation, while the stream moves radially relative to the axis of rotation of the substrate, thereby dispensing the wash solution in a spiral pattern.

[0266] The method may further comprise recovering a subset or all of the solution after the solution has contacted the substrate. Recycling may comprise collecting, filtering and reusing a subset or all of the solution. Filtration may be molecular filtration.

[0267] Operations 220 and 230 may occur at a first location, and operation 240 may occur at a second location. The first location and the second location may each include a device as described herein (e.g., with respect to Figure 23H ) of the first processing room and the second processing room. The first location and the second location can each include as described herein (e.g., Figure 24 ) and a first rotating spindle and a second rotating spindle. The first rotating spindle can be external to or internal to the second rotating spindle. The first rotating spindle and the second rotating spindle can be configured to rotate at different angular velocities. Alternatively, operation 220 can occur at the first location, and operations 230 and 240 can occur at the second location.

[0268] The method may further include transferring the substrate between the first position and the second position. Operations 220 and 230 may occur while the substrate is rotating at a first angular velocity, and operation 240 may occur while the substrate is rotating at a second angular velocity. The first angular velocity may be less than the second angular velocity. The first angular velocity may be between approximately 0 rpm and approximately 100 rpm. The second angular velocity may be between approximately 100 rpm and approximately 1,000 rpm. Alternatively, operation 220 may occur while the substrate is rotating at the first angular velocity, and operations 230 and 240 may occur while the substrate is rotating at the second angular velocity.

[0269] Many variations, changes, and adaptations based on the method 200 provided herein are possible. For example, the order of operations of method 200 can be changed, some operations can be removed, some operations can be repeated, and additional operations can be added as appropriate. Some operations can be performed continuously. Some operations can be performed in parallel. Some operations can be performed once. Some operations can be performed more than once. Some operations can include sub-operations. Some operations can be automated. Some operations can be manual. Some operations can be performed separately, such as at different locations or during different steps and / or processes. For example, directing a solution containing multiple probes to a substrate can occur separately from the reaction and detection processes.

[0270] For example, in some cases, in the third operation 230, nucleic acid molecules can be subjected to conditions that allow the nucleotides in a plurality of nucleotides to be instantaneously bound to nucleic acid molecules, rather than conditions that promote primer extension reaction. The nucleotides of instantaneous binding can be marked. The nucleotides of instantaneous binding can be removed such as after detection (for example, referring to operation 240). Then, the second solution can be directed to the substrate, this time under the conditions that promote primer extension reaction, so that the nucleotides of the second solution are incorporated into (for example, incorporated into the growing chain of hybridization with nucleic acid molecules). The nucleotides incorporated can be unlabeled. After washing and when not detecting, another solution of labeled nucleotides can be directed to the substrate, such as for another instantaneous binding cycle.

[0271] In some cases, such as in order to connect sequencing, solution can include different probes.For example, solution can include multiple oligonucleotide molecules.For example, oligonucleotide molecules can have the length of about 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases or more bases.Oligonucleotide molecules can be marked with dyestuff (for example, fluorescent dye) as described elsewhere herein.In some cases, such as in order to detect the tumor-necrosis factor glycoproteins in nucleic acid molecules, such as homopolymer tumor-necrosis factor glycoproteins, dinucleotide tumor-necrosis factor glycoproteins and trinucleotide tumor-necrosis factor glycoproteins, solution can include the targeting probe (for example, homopolymer probe) that is configured to be combined with tumor-necrosis factor glycoproteins.Solution can include a type of probe (for example, nucleotide).Solution can include different types of probes (for example, nucleotide, oligonucleotide molecules etc.).Solution can include different types of probes (for example, oligonucleotide molecules, antibody etc.), for interacting with different types of analytes (for example, nucleic acid molecules, protein etc.). Different solutions comprising different types of probes can be introduced to the substrate any number of times, with or without detection between successive cycles (e.g., detection can be performed between some successive cycles and not between other cycles), to sequence or otherwise process nucleic acid molecules, depending on the type of processing.

[0272] Figure 3 A system 300 for sequencing nucleic acid molecules or processing analytes is shown. The system can be configured to implement method 200 or 1400. Although the system (e.g., 300, 400, 500a, 500b, etc.) is described for processing nucleic acid molecules, as described herein, the system can be used to process any other type of biological analyte.

[0273] The system may include a substrate 310. The substrate may include any substrate described herein, such as Figure 2 Any substrate described herein. The substrate may include an array. The substrate may be open. The array may include one or more locations 320 configured to secure one or more nucleic acid molecules or analytes. The array may include any array described herein, such as any array described herein with respect to method 200. For example, the array may include multiple individually addressable locations. The array may include a linker (e.g., any binding agent described herein) coupled to the nucleic acid molecules to be sequenced. Alternatively or in combination, the nucleic acid molecules to be sequenced may be coupled to beads; the beads may be affixed to the array. The array may be textured. The array may be a patterned array. The array may be planar.

[0274] The substrate can be configured to rotate about an axis 305. The axis can be an axis passing through the center of the substrate. The axis can be an eccentric axis. The substrate can be configured to rotate at any rotational speed described herein, such as any rotational speed described herein with respect to methods 200 or 1400.

[0275] The substrate can be configured to undergo a change in relative position about the first longitudinal axis 315 or the second longitudinal axis 325. For example, the substrate can be translatable along the first longitudinal axis and / or the second longitudinal axis (e.g., Figure 3). Alternatively, the substrate may be stationary along the first longitudinal axis and / or the second longitudinal axis. Alternatively or in combination, the substrate may be translatable along the axis (as shown in FIG4 ). Alternatively or in combination, the substrate may be stationary along the axis. The relative position of the substrate may be configured to alternate between positions. The relative position of the substrate may be configured to alternate between positions relative to one or more longitudinal axes. The relative position of the substrate may be configured to alternate between positions relative to any fluid channel described herein. For example, the relative position of the substrate may be configured to alternate between a first position and a second position. The relative position of the substrate may be configured to alternate between at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positions. The relative position of the substrate may be configured to alternate between a plurality of positions within a range defined by any two of the aforementioned values. The first longitudinal axis or the second longitudinal axis may be substantially perpendicular to the axis. The first longitudinal axis or the second longitudinal axis may be substantially parallel to the axis. The first longitudinal axis or the second longitudinal axis may coincide with the axis.

[0276] The system may include a first fluid channel 330. The first fluid channel may include a first fluid outlet port 335. The first fluid outlet port may be configured to distribute a first fluid to the array. The first fluid outlet port may be configured to distribute any fluid described herein, such as any solution described herein. The first fluid outlet port may be external to the substrate. The first fluid outlet port may not contact the substrate. The first fluid outlet port may be a nozzle. The first fluid outlet port may have an axis substantially coincident with the axis. The first fluid outlet port may have an axis substantially parallel to the axis.

[0277] The system may include a second fluid channel 340. The second fluid channel may include a second fluid outlet port 345. The second fluid outlet port may be configured to distribute a second fluid to the array. The second fluid outlet port may be configured to distribute any fluid described herein, such as any solution described herein. The second fluid outlet port may be external to the substrate. The second fluid outlet port may not contact the substrate. The second fluid outlet port may be a nozzle. The second fluid outlet port may have an axis substantially coincident with the axis. The second fluid outlet port may have an axis substantially parallel to the axis.

[0278] The first fluid and the second fluid can comprise dissimilar reagents.For example, the first fluid can comprise the nucleotide of the first type, such as any nucleotide as described herein, or a nucleotide mixture.The second fluid can comprise the nucleotide of the second type, such as any nucleotide as described herein, or a nucleotide mixture.Perhaps, the first fluid and the second fluid can comprise the reagent of the same type (for example, distributing the fluid of the same type to increase the coating speed by a plurality of fluid outlet ports (for example, nozzle)).Alternately or in combination, the first fluid or the second fluid can comprise washing reagent.The first fluid channel 330 and the second fluid channel 340 can be isolated by fluid.Advantageously, when the first fluid and the second fluid comprise dissimilar reagents, during distribution, each different reagent can keep not being polluted by other reagents.

[0279] The first fluid outlet port can be configured to distribute the first fluid during the rotation of the substrate. The second fluid outlet port can be configured to distribute the second fluid during the rotation of the substrate. The first fluid outlet port and the second fluid outlet port can be configured to distribute at non-overlapping times. Alternatively, the first fluid outlet port and the second fluid outlet port can be configured to distribute at overlapping times (such as when the first fluid and the second fluid include the same type of reagent). When the first outlet port and the second outlet port distribute, the substrate can be configured to rotate at different speeds or different numbers of turns. Alternatively, when the first outlet port and the second outlet port distribute, the substrate can be configured to rotate at the same speed and number of turns. During rotation, the array can be configured to guide the first fluid away from the axis in a substantially radial direction. The first fluid outlet port can be configured to direct the first fluid to the array during at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, or at least 1,000,000 full rotations of the substrate. The first fluid outlet port can be configured to direct the first fluid to the array during a number of full rotations within a range defined by any two of the aforementioned values.

[0280] System can include the third fluid channel 350 that comprises the third fluid outlet port 355 that is configured to distribute the third fluid.System can include the fourth fluid channel 360 that comprises the fourth fluid outlet port 365 that is configured to distribute the fourth fluid.The third fluid channel and the fourth fluid channel can be similar to the first fluid channel and the second fluid channel as described herein.The third fluid and the fourth fluid can be the fluid that is identical or different with the first fluid and / or the second fluid.In some cases, can adopt at least 1,2,3,4,5,6,7,8,9,10,15,20 or more kinds of fluids (or reagents).For example, can adopt 5-10 kinds of fluids (or reagents).

[0281] Although Figure 3 In the embodiment of the present invention, the substrate is shown to be in a position change. Alternatively or additionally, one or more of the first, second, third, and fourth fluid channels can also be configured to undergo a position change. For example, any of the first, second, third, or fourth fluid channels can be translatable along the first and / or second longitudinal axis. Alternatively, any of the first, second, third, or fourth fluid channels can be stationary along the first and / or second longitudinal axis. Alternatively or additionally, any of the first, second, third, or fourth fluid channels can be translatable along the axis. Alternatively or additionally, any of the first, second, third, or fourth fluid channels can be stationary along the axis.

[0282] The relative position of one or more of the first, second, third and fourth fluid channels can be configured to alternate between positions relative to one or more longitudinal axes or the axis. For example, the relative position of any one of the first, second, third or fourth fluid channels can be configured to alternate between the first position and the second position (for example, by moving such a channel, by moving a substrate or by moving a channel and a substrate). The relative position of any one of the first, second, third or fourth fluid channels can be configured to alternate between at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more positions. The relative position of any one of the first, second, third or fourth fluid channels can be configured to alternate between a plurality of positions within the range defined by any two of the aforementioned values. The first longitudinal axis or the second longitudinal axis can be substantially perpendicular to the axis. The first longitudinal axis or the second longitudinal axis can be substantially parallel to the axis. The first longitudinal axis or the second longitudinal axis can coincide with the axis.

[0283] In some cases, the system may include one or more fluid channels (not shown) for receiving fluid from the substrate. Figure 3 ). Figure 4A-4B , the fifth fluid channel 430 may include a first fluid inlet port 435. The first fluid inlet port may be located at a first level of the axis (as shown in FIG4 ). In some cases, the first fluid inlet port may (e.g., circularly) surround the periphery of the substrate 310. When the substrate is in a first position such as about the axis, the first fluid inlet port may be downstream of the substrate 310 and in fluid communication with the substrate 310. The fifth fluid channel may be in fluid communication with the first fluid channel 330. For example, the first fluid inlet port may be configured to receive a solution that arrives at the substrate from the first fluid outlet port and subsequently leaves the substrate (e.g., due to inertial forces during substrate rotation). For example, the first fluid inlet port may be configured to receive a solution in a recycling process (such as the recycling process described herein with respect to method 200 or 1400). In some cases, the solution received by the fifth fluid channel via the first fluid inlet port may be fed back (e.g., after filtration) to the first fluid channel to be distributed to the substrate via the first fluid outlet port. The fifth fluid channel and the first fluid channel may define at least a portion of the first circulating fluid flow path. The first circulating fluid flow path can include a filter, such as the filter described herein with respect to method 200 or 1400. The filter can be a molecular filter. In other cases, the solution received by the fifth fluid channel can be fed back (e.g., after filtering) to a different fluid channel (in addition to the first fluid channel) for distribution via a different fluid outlet port.

[0284] The system may include a sixth fluid channel 440. The sixth fluid channel may include a second fluid inlet port 445. The second fluid inlet port may be located at a second level of the axis (as shown in FIG. 4 ). In some cases, the second fluid inlet port may surround the periphery of the substrate 310. When the substrate is in a second position, such as about the axis, the second fluid inlet port may be downstream of the substrate 310 and in fluid communication with the substrate 310. The sixth fluid channel may be in fluid communication with the second fluid channel 340. For example, the second fluid inlet port may be configured to receive a solution that arrives at the substrate from the second fluid outlet port and subsequently leaves the substrate. For example, the second fluid inlet port may be configured to receive a solution during a recovery process (such as the recovery process described herein with respect to method 200 or 1400). In some cases, the solution received by the sixth fluid channel via the second fluid inlet port may be fed back (e.g., after filtration) to the second fluid channel to be distributed to the substrate via the second fluid outlet port. The sixth fluid channel and the second fluid channel may define at least a portion of a second circulating fluid flow path. The second circulating fluid flow path may include a filter, such as the filter described herein with respect to method 200 or 1400. The filter may be a molecular filter.

[0285] The system may include a shield (not shown) that blocks fluid communication between the substrate and the second fluid inlet port when the substrate is in the first position and blocks fluid communication between the substrate and the first fluid inlet port when the substrate is in the second position.

[0286] The system may also include one or more detectors 370. The detector may be an optical detector, such as one or more photodetectors, one or more photodiodes, one or more avalanche photodiodes, one or more photomultipliers, one or more photodiode arrays, one or more avalanche photodiode arrays, one or more cameras, one or more charge coupled device (CCD) cameras, or one or more complementary metal oxide semiconductor (CMOS) cameras. The camera may be a TDI or other continuous area scanning detector as described herein. Including, for example, a TDI line scan camera. The detector may be a fluorescence detector. The detector may be in sensing communication with the array. For example, the detector may be configured to detect a signal from the array. The signal may be an optical signal. The signal may be a fluorescent signal. The detector may be configured to detect a signal from the substrate during substrate rotation. The detector may be configured to detect a signal from the substrate when the substrate is not rotating. The detector may be configured to detect a signal from the substrate after substrate rotation is terminated. Figure 3 An example area 375 on the substrate optically mapped to the detector is shown.

[0287] The system may include one or more sources (not shown) configured to deliver electromagnetic radiation to the substrate. Figure 3). The source may include one or more light sources (e.g., illumination sources). The source may include one or more incoherent light sources or coherent light sources. The source may include one or more narrow bandwidth light sources or broadband light sources. The source can be configured to emit optical radiation having a bandwidth of at most 1 Hertz (Hz), at most 2 Hz, at most 5 Hz, at most 10 Hz, at most 20 Hz, at most 50 Hz, at most 100 Hz, at most 200 Hz, at most 500 Hz, at most 1 kilohertz (kHz), at most 2 kHz, at most 5 kHz, at most 10 kHz, at most 20 kHz, at most 50 kHz, at most 100 kHz, at most 200 kHz, at most 500 kHz, at most 1 megahertz (MHz), at most 2 MHz, at most 5 MHz, at most 10 MHz, at most 20 MHz, at most 50 MHz, at most 100 MHz, at most 200 MHz, at most 500 MHz, at most 1 gigahertz (GHz), at most 2 GHz, at most 5 GHz, at most 10 GHz, at most 20 GHz, at most 50 GHz, at most 100 GHz, or at most broadband within a range defined by any two of the foregoing values. The source may include one or more light emitting diodes (LEDs). The source may include one or more lasers. The source may include one or more single-mode laser sources. The source may include one or more multi-mode laser sources. The source may include one or more laser diodes. The laser may be a continuous wave laser or a pulsed laser. The light beam emitted by the laser may be a Gaussian beam or a near-Gaussian beam, which may be manipulated using one or more optical elements (e.g., mirrors, lenses, prisms, wave plates, etc.). For example, the light beam may be collimated. In some cases, the light beam may be manipulated to provide a laser line (e.g., using one or more Powell lenses or cylindrical lenses). Figure 11A An example of using a cylindrical lens to provide beam shaping of a laser line is shown. A collimated beam of radius r0 is incident on a cylindrical plano-concave lens of focal length f. The beam will expand by a half-angle θ equal to r0 / f. At a distance z from the lens, the thickness of the laser line is about 2r0 and the length L is about 2(r0 / f)(z+f). In some embodiments, the beam thickness can expand along a single axis (e.g., the y-axis) while the beam thickness remains substantially constant along a second axis (e.g., the x-axis), as shown in FIG. Figure 11B The expansion along a single axis can be achieved using a cylindrical lens (e.g. a plano-concave cylindrical lens with a focal length f along the expansion axis). The beam shaping lens can be part of a line shaper element, such as Figure 11C As shown. The line shaper element may include one or more optical elements configured to expand the light beam along a single axis. The line shaper element may also include one or more optical elements to collimate the expanded light beam, such as a second cylindrical lens. In some embodiments, the second cylindrical lens is a plano-convex cylindrical lens. The expanded light beam may result in a laser line, such as Figure 11BThe laser line can impinge directly on the substrate or can be projected onto the substrate so that it is approximately perpendicular to a central axis about which the open substrate can be rotated.

[0288] The source (e.g., light source or irradiation source) of the system can be configured to emit light of one or more wavelengths in the ultraviolet (about 100 nm to about 400 nm), visible (about 400 nm to about 700 nm), or infrared (about 700 nm to about 10,000 nm) regions of the electromagnetic spectrum, or any combination thereof. For example, the source can emit radiation of one or more wavelengths in the range of 600 nm to 700 nm. The source can emit radiation having an optical power of at least 0.05 watts (W), at least 0.1 W, at least 0.2 W, at least 0.5 W, at least 1 W, at least 2 W, at least 5 W, at least 10 W, or a range defined by any two of the foregoing values, either individually or in combination. The source can be configured to interact with molecules on the substrate to generate a detectable optical signal that can be detected by a photodetector. For example, the source can be configured to generate light absorption, light reflectance, scattering, phosphorescence, fluorescence, or any other optical signal described herein.

[0289] The system can include a seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid channel. Each fluid channel can include a fluid outlet port or a fluid inlet port in fluid communication with the substrate. For example, the ninth, tenth, thirteenth, fourteenth, seventeenth, or eighteenth fluid channel can include a fluid outlet port. The seventh, eighth, eleventh, twelfth, fifteenth, sixteenth, nineteenth, or twentieth fluid channel can include a fluid inlet port. Alternatively, the system can include more than twenty fluid channels including fluid outlet ports or fluid inlet ports.

[0290] In another embodiment, the system can include a fifth, sixth, seventh, eighth, ninth or tenth fluid outlet port. The fifth, sixth, seventh, eighth, ninth or tenth fluid outlet port can be configured to distribute the fifth, sixth, seventh, eighth, ninth or tenth fluid to the array. The fifth, sixth, seventh, eighth, ninth or tenth fluid outlet port can be configured to distribute any fluid as described herein, such as any solution as described herein. The fifth, sixth, seventh, eighth, ninth or tenth fluid outlet port can be similar to the first, second, third or fourth fluid outlet port as described herein. Alternatively, the system can include more than ten fluid outlet ports.

[0291] The fluid channels can be fluidically isolated from each other. For example, the fluid channels can be fluidically isolated upstream of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port can be external to the substrate. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port can not contact the substrate. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port can be a nozzle.

[0292] The system can include a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet port. When the substrate is in the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth position (e.g., about the axis), the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet port can be in fluid communication with the substrate, respectively. Alternatively, the system can include more than ten fluid inlet ports.

[0293] The ninth, tenth, thirteenth, fourteenth, seventeenth, or eighteenth fluid channel can be in fluid communication with the seventh, eighth, eleventh, twelfth, fifteenth, or sixteenth fluid channel, respectively; each pair of fluid channels can define at least a portion of a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth circulating fluid flow path, respectively. Each circulating fluid flow path can be configured similarly to the first or second circulating fluid flow paths described herein, wherein the fluid inlet port of the circulating fluid flow path is configured to receive a solution from the fluid outlet port of the circulating fluid flow path to the substrate. Each circulating fluid flow path can be configured to receive a solution during a recovery process as described herein. Each circulating fluid flow path can include a filter as described herein.

[0294] The fifth, sixth, seventh, eighth, ninth or tenth fluid can comprise different types of reagents. For example, the fifth, sixth, seventh, eighth, ninth or tenth fluid can comprise the fifth, sixth, seventh, eighth, ninth or tenth type of nucleotides, such as any nucleotide described herein, respectively. Alternatively or in combination, the fifth, sixth, seventh, eighth, ninth or tenth fluid can comprise a washing reagent.

[0295] The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port can be configured to dispense the fifth, sixth, seventh, eighth, ninth, or tenth fluid, respectively, during substrate rotation. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port can be configured to dispense at overlapping or non-overlapping times.

[0296] Figure 4AA system 400 for sequencing nucleic acid molecules on a first vertical level is shown. The system can be substantially similar to the system 300 described herein, or can differ from the system 300 in the arrangement of one or more of its elements. The system 400 can include a substrate 310 as described herein. The system 400 can utilize vertical movement parallel to the axis 305 to expose (e.g., connect fluids to) the substrate 310 to different fluid channels. The system can include a first fluid channel 330 and a first fluid outlet port 335 as described herein. The system can include a second fluid channel 340 and a second fluid outlet port 345 as described herein. The system can include a third fluid channel 350 and a third fluid outlet port 355 as described herein. The system can include a fourth fluid channel 360 and a fourth fluid outlet port 365 as described herein. The system can include a detector 370 as described herein. The detector can be in optical communication with the region shown. The system can include any light source described herein (not shown). Figure 4A (shown in the middle).

[0297] The fifth fluid channel 430 and the first fluid inlet port 435 may be arranged at a first level along the vertical axis, such as Figure 4A and Figure 4B As shown. The sixth fluid channel 440 and the second fluid inlet port 445 can be arranged at a second level along the vertical axis. In this way, the system can be considered to include a first fluid flow path and a second fluid flow path, wherein each fluid flow path is located at a different vertical level. The substrate 310 can be vertically moved between the first level and the second level, from the first level to the second level, and from the second level to the first level. Alternatively, the substrate can be vertically fixed, but the level can be vertically moved relative to the substrate 310. As another alternative, the substrate and the level can be vertically moved.

[0298] System 400 can comprise a plurality of levels.These levels can be relative to vertical orientation.System can comprise at least 2,3,4,5,6,7,8,9,10,20,30,40,50,100 or more levels.Each level can comprise one or more sub-levels (for example, the incremental level between any two levels).Each level can be used to distribute and / or reclaim different fluids (or reagents).Some levels can be used to distribute identical fluids (or reagents).

[0299] When in the first vertical level, the substrate can be in fluid communication with the fifth fluid channel and the first fluid inlet port, but not in fluid communication with the sixth fluid channel and the second fluid inlet port. The substrate can be separated from the sixth fluid channel and the second fluid inlet port by a shield (not shown) as described herein. When the substrate is in the first vertical level, the first fluid or first solution described herein can be dispensed to the substrate. For example, when the substrate is in the first vertical level, the first fluid inlet port can receive any excess first solution thrown off from the substrate. In another example, when the substrate is in the first vertical level, the first fluid inlet port can receive some washing solution thrown off from the substrate together with some of the first fluid (for example, dispensed from a fluid outlet port different from the first fluid). The substrate can then be moved to the second vertical level by vertically moving the substrate. Alternatively, the fifth fluid channel or the sixth fluid channel can be moved vertically. Alternatively or additionally, the substrate and one or more fluid channels can move relative to one another (for example, along an axis).

[0300] Figure 4B A system 400 for sequencing nucleic acid molecules on a second vertical level is shown. When in the second vertical level, the substrate can be in fluid communication with the sixth fluid channel and the second fluid inlet port, but not in fluid communication with the fifth fluid channel and the first fluid inlet port. The substrate can be isolated from the fifth fluid channel and the first fluid inlet port by a shield (not shown) as described herein. When the substrate is in the second vertical level, the second fluid or the second solution as described herein can be dispensed to the substrate. Alternatively, the first solution can be removed when the substrate is in the second vertical position. In some cases, the first solution can be reclaimed when the substrate is in the second vertical position. Then, by vertically moving the substrate, the substrate can be moved back to the first vertical level or another vertical level as described herein. Alternatively, the fifth fluid channel or the sixth fluid channel can be moved vertically. Alternatively or additionally, the substrate and one or more fluid channels can move relative to one another (e.g., along an axis).

[0301] In some embodiments, the present invention provides the fluid inlet port of the present invention.The third, fourth, fifth, sixth, seventh, eighth, ninth or tenth fluid inlet port can be positioned at the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth vertical level respectively.Can by vertically moving substrate or by vertically moving first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth or twentieth fluid flow channel to move substrate to the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth vertical level.In any horizontal place in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more vertical levels, any fluid solution as herein described can be assigned to substrate.In any horizontal place in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more vertical levels, any fluid solution as herein described can be removed from substrate. Any of the fluid solutions described herein can be recovered from the substrate at any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more vertical levels.

[0302] Figure 5A A first example of a system 500a for sequencing nucleic acid molecules using an array of fluid flow channels is shown. The system can be substantially similar to system 300 or system 400 described herein, and can differ from system 300 or 400 in the arrangement of one or more of its elements. System 500a can utilize a geometric arrangement of multiple fluid flow channels to expose a substrate to different fluids. System 500a can include substrate 310 as described herein. The system can include a first fluid channel 330 and a first fluid outlet port 335 as described herein. The system can include a second fluid channel 340 and a second fluid outlet port 345 as described herein. The system can include a fifth fluid channel 430 and a first fluid inlet port 435 as described herein (not shown). Figure 5A The system may include the sixth fluid channel 440 and the second fluid inlet port 445 described herein (not shown). Figure 5A The system may include a detector 370 as described herein (not shown). Figure 5A The system may include any of the illumination sources described herein (not shown). Figure 5A shown in ).

[0303] The first fluid channel and the first fluid outlet port may be arranged at a first location, such as Figure 5A The second fluid channel and the second fluid outlet port may be arranged at a second position. The system may be configured to dispense a first fluid from the first fluid outlet port and a second fluid from the second fluid outlet port.

[0304] The system can include any of the third, fourth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid channels described herein. The system can include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports described herein. The system can include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet ports described herein.

[0305] The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port can be located at a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth position, respectively. The system can be configured to dispense a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid from the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port, respectively.

[0306] Any two or more of the first, second, third, fourth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, or more fluid channels may form an array of fluid flow channels. The array of fluid flow channels may be movable. Alternatively, the array of fluid flow channels may be in a fixed position relative to the substrate. Each fluid flow channel in the array of fluid flow channels may be positioned such that the longitudinal axis of the fluid flow channel forms an angle with the rotational axis of the substrate. The angle may have a value of at least 0 degrees, at least 5 degrees, at least 10 degrees, at least 15 degrees, at least 20 degrees, at least 25 degrees, at least 30 degrees, at least 35 degrees, at least 40 degrees, at least 45 degrees, at least 50 degrees, at least 55 degrees, at least 60 degrees, at least 65 degrees, at least 70 degrees, at least 75 degrees, at least 80 degrees, at least 85 degrees, or at least 90 degrees. The angle may have a value within a range defined by any two of the aforementioned values. Each fluid channel in the array of fluid channels may form a similar angle with the substrate. Alternatively, one or more fluid channels may form a different angle with the substrate.

[0307] Figure 5B A second example of a system 500b for sequencing nucleic acid molecules using an array of fluid flow channels is shown.

[0308] The system can be substantially similar to system 300 or system 400 described herein, and can differ from system 300 or system 400 in the arrangement of one or more elements thereof. System 500b can utilize multiple fluid flow channels configured to move relative to a substrate to expose the substrate to different fluids. System 500b can include substrate 310 as described herein. The system can include a first fluid channel 330 and a first fluid outlet port 335 as described herein. The system can include a second fluid channel 340 and a second fluid outlet port 345 as described herein. The system can include a fifth fluid channel 430 and a first fluid inlet port 435 as described herein (not shown). Figure 5B The system may include the sixth fluid channel 440 and the second fluid inlet port 445 described herein (not shown). Figure 5B The system may include a detector 370 as described herein (not shown). Figure 5B The system may include any light source described herein (not shown in Figure 5B shown in ).

[0309] The first fluid channel and the first fluid outlet port may be attached to a fluid dispenser 510. The fluid dispenser may be a movable fluid dispenser, such as one comprising a movable rack arm, such as Figure 5B As shown. Alternatively, the fluid dispenser can be fixed or stationary. The fluid dispenser can be configured to move to a first position to dispense a first fluid from a first fluid outlet port. A second fluid channel and a second fluid outlet port can also be attached to the fluid dispenser. The fluid dispenser can be configured to move to a second position to dispense a second fluid from a second fluid outlet port.

[0310] The system can include any of the third, fourth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth fluid channels described herein. The system can include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet ports described herein. The system can include any of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluid inlet ports described herein.

[0311] The third, fourth, fifth, sixth, seventh, eighth, ninth or tenth fluid outlet port can be attached to the fluid dispenser. The fluid dispenser can be configured to move to a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth position to dispense the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth fluid from the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth fluid outlet port, respectively. Alternatively, the fluid dispenser can remain stationary and the substrate 310 can be moved to different positions to receive different fluids.

[0312] Figure 6 Shown is a computerized system 600 for sequencing nucleic acid molecules.System can include substrate 310, such as this paper about the substrate described in method 200 or 1400 or system 300.System can also include fluid flow unit 610.Fluid flow unit can include any element relevant to fluid flow as herein described, such as this paper about any one or all of element 330,335,340,345,350,355,360,365,430,435,440,445 and 370 described in system 300,400,500a or 500b.Fluid flow unit can be configured to before or during substrate rotation will comprise a solution of a plurality of nucleotides as herein described be directed to the array of substrate.Fluid flow unit can be configured to before or during substrate rotation will wash solution as herein described be directed to the array of substrate.In some cases, fluid flow unit can include pump, compressor and / or actuator, to guide fluid flow from first position to second position. With respect to method 1400, the fluid flow system can be configured to direct any solution to substrate 310. With respect to method 1400, the fluid flow system can be configured to collect any solution from substrate 310. The system can also include a detector 370, such as any detector described herein with respect to systems 300 or 400. The detector can be in sensing communication with the array of substrates.

[0313] The system may also include one or more computer processors 620. The one or more processors may be programmed individually or collectively to implement any of the methods described herein. For example, the one or more processors may be programmed individually or collectively to implement any or all operations of the methods of the present disclosure (such as method 200 or method 1400). In particular, the one or more processors may be programmed individually or collectively to: (i) direct a fluid flow unit to direct a solution comprising a plurality of nucleotides throughout the array during or before rotation of the substrate; (ii) subject the nucleic acid molecules to a primer extension reaction under conditions sufficient to incorporate at least one nucleotide from the plurality of nucleotides into a growing chain complementary to the nucleic acid molecule; and (iii) detect a signal indicating the incorporation of at least one nucleotide using a detector, thereby sequencing the nucleic acid molecules.

[0314] Although the rotation system has been described for sequencing applications, this rotation scheme can also be used for other applications (e.g., pre-sequencing applications, sample preparation, etc.), such as template seeding and surface amplification processes. For example, the reagents dispensed during or before the substrate rotation can be customized for other applications. Although the reagents dispensed to the substrate in the rotation system have been described for nucleotides, any reagents (such as probes, adapters, enzymes, and labeling reagents) that can react with nucleic acid molecules (or any other molecules or cells) fixed to the substrate can be dispensed to the substrate before, during, or after rotation to achieve high-speed coating of the substrate with the dispensed reagents.

[0315] The systems described herein (such as any of systems 300, 400, 500a, or 500b, or any other system described herein) or any element thereof may be environmentally controlled.For example, the system may be maintained at a specified temperature or humidity. The system (or any element thereof) can be maintained at a temperature of at least 20 degrees Celsius (°C), at least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, at least 100°C, at most 100°C, at most 95°C, at most 90°C, at most 85°C, at most 80°C, at most 75°C, at most 70°C, at most 65°C, at most 60°C, at most 55°C, at most 50°C, at most 45°C, at most 40°C, at most 35°C, at most 30°C, at most 25°C, at most 20°C, or a temperature within a range defined by any two of the foregoing values.

[0316] Different components of the system can be maintained at different temperatures or within different temperature ranges, such as the temperatures or temperature ranges described herein. The temperature of the system components can be set above the dew point to prevent condensation. The temperature of the system components can be set below the dew point to collect condensed water.

[0317] Figure 7Shown is a system with different environmental conditions in an open substrate system. The open substrate system can include a substrate 3502 and a container 3504 for enclosing the substrate. The substrate 3502 can be any substrate as described herein. The container 3504 can define the surrounding environment of the substrate 3502. In some cases, the surrounding environment can be restricted and / or closed. In some cases, the surrounding environment may be sealed (e.g., airtight seal, friction seal, pneumatic seal, etc.). In some cases, a pressure difference (e.g., pneumatic pressure, mechanical pressure, etc.) can be used to seal the surrounding environment. The open substrate system can include at least two non-overlapping areas, a first area 3522 and a second area 3524, which have different environmental conditions. In some cases, the surface of the substrate 3502, for example, the first area 3522 comprising the surface of one or more analytes as described herein, can be maintained at a first set of temperature and a first set of humidity. In some cases, the second area 3524 of the top portion (or otherwise referred to as a lid or cover herein) of the contact or approaching container 3504 can be maintained at a second set of temperature and a second set of humidity. A first set of temperatures and a first set of humidities can be controlled to prevent or minimize evaporation of one or more reagents on the substrate surface. For example, the first set of temperatures and humidities can be configured to prevent evaporation of less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the volume of the solution layer dispensed on the uncovered surface. A second set of temperatures and humidities can also be controlled to enhance or limit condensation. For example, the first set of temperatures can be the lowest temperature within the surrounding environment of the open substrate system. For example, the second set of temperatures can be the highest temperature within the surrounding environment of the open substrate system. In some cases, the environmental conditions of different regions can be achieved by controlling the temperature of the housing. In some cases, the environmental conditions of different regions can be achieved by controlling the temperature of selected portions or the entire container. In some cases, the environmental conditions of different regions can be achieved by controlling the temperature of selected portions or the entire substrate. In some cases, the environmental conditions of different regions can be achieved by controlling the temperature of the reagents dispensed to the substrate. Any combination of these can be used to control the environmental conditions of different regions. Heat transfer can be achieved by any method, including, for example, conduction, convection, and radiation. For example, the first region 3522 can be maintained at a cooler temperature by controlling the temperature of the substrate 3502, and the second region 3524 can be maintained at a warmer temperature by controlling the temperature of the top portion of the container 3504 via conduction.

[0318] The system may also include a reservoir ( Figure 735). The reservoir may be configured to hold a fluid. The reservoir may be configured to collect fluid, precipitation, or condensate from other surfaces, such as from the substrate 3522 or the top portion of the container 3504. Fluid may be removed from the reservoir. In some cases, fluid may be removed from the reservoir by volume. For example, fluid may be removed from the reservoir by volume to balance the amount of fluid added to the system. In some cases, fluid is continuously added to the system and fluid is continuously removed from the reservoir. The amount of fluid added may be equal to the amount of fluid removed. In some cases, the volume of fluid in the reservoir remains constant. The volume of fluid in the reservoir may be determined based on the relative humidity of the system. The relative humidity of the system may depend on the volume of fluid in the reservoir, the amount of fluid in the system, the temperature of the system, or any combination thereof.

[0319] The open substrate systems of the present disclosure may include a barrier system configured to maintain a fluid barrier. Figure 47A A partial cross-sectional view of a barrier system 4700 holding a fluid barrier 4713 is shown. Figure 47B A perspective view of chamber 4715 of barrier system 4700 is shown. Barrier system 4700 and / or its respective components may correspond to Figure 7 The systems shown have different environmental conditions, and / or their respective components. For example, Figures 15-24 The substrate 310 shown in FIG can be positioned within the barrier system 4700 .

[0320] Barrier system 4700 includes a sample environment 4705 defined by plate 4703, chamber 4715, and fluid barrier 4713. Chamber 4715 and plate 4703 may be separated by a physical gap. Sample environment 4705 may be isolated (and / or insulated) from external environment 4707.

[0321] The fluid barrier 4713 can act as a transition area between the sample environment 4705 and the external environment 4707. Figures 15-24 4705. The substrate 310 shown can be positioned within the sample environment 4705. The fluid barrier 4713 can include fluid (e.g., air) from the sample environment 4705, the external environment 4707, or both. The fluid barrier 4713 can be a low pressure area. The fluid barrier 4713 can have a lower pressure than the sample environment, the external environment, or both. The fluid barrier 4713 can be maintained by a fluid flow unit, such as a pressure changing device 4711. The fluid barrier 4713 can include fluid in coherent motion or entangled motion.

[0322] The pressure changing device 4711 can be integrated with the chamber 4715. For example, Figure 47A and Figure 47BAs shown, the pressure changing device can be integrated into the wall of the chamber 4715 as a fluid channel 4720. For example, suction can be applied through the fluid channel 4720 to draw fluid from the external environment 4707 or the sample environment 4705, or both, to create a partial vacuum curtain (e.g., in coherent motion, entrained motion, etc.), thereby forming the fluid barrier 4713. Otherwise, the fluid may be subjected to a negative pressure. The fluid discharge can be discharged at the other end of the fluid channel. Alternatively or additionally, the device may not be integrated with the chamber 4715. The fluid flow unit and / or pressure changing device 4711 can be operated by one or more compressors (e.g., to generate negative pressure), pumps (e.g., to generate positive pressure), suction devices, and / or other devices to provide a lower pressure in the transition region. The chamber 4715 may include one or more fluid channels 4720 for implementing the fluid barrier of the present disclosure.

[0323] Although Figure 47A and Figure 47B In the figure, two pressure change devices 4711 are shown, but it should be understood that any number of such devices can be provided. For example, there can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more such devices. Alternatively or additionally, there can be at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 such devices. In some cases, one or more pressure change devices 4711 can be implemented as an annular fluid channel around the sample environment area, or other fluid channels along the periphery or boundary of the sample environment area. In some cases, one or more additional fluid flow channels (for example, 4733) can be provided near the chamber bottom to draw excess fluid (for example, liquid, gas) from the sample environment area.

[0324] Advantageously, the fluid barrier 4713 can provide a low friction or zero friction seal between the sample environment 4705 and the external environment 4707. In some cases, the fluid flow rate through the fluid barrier 4713 can be at least about 5 liters / minute (L / min), 5.5 L / min, 6 L / min, 6.5 L / min, 7 L / min, 7.5 L / min, 8 L / min, 8.5 L / min, 9 L / min, 9.5 L / min, 10 L / min, 10.5 L / min, 11 L, 11.5 L / min, 12 L / min, 12.5 L / min, 13 L / min, 13.5 L / min, 14 L / min, 14.5 L / min, 15 L / min, or more. Alternatively or additionally, the fluid flow rate can be at most about 15 L / min, 14.5 L / min, 14 L / min, 13.5 L / min, 13 L / min, 12.5 L / min, 12 L / min, 11.5 L / min, 11 L / min, 10.5 L / min, 10 L / min, 9.5 L / min, 9 L / min, 8.5 L / min, 8 L / min, 7.5 L / min, 7 L / min, 6.5 L / min, 6 L / min, 5.5 L / min, 5 L / min or less. It should be understood that the fluid flow rate can vary with different parameters (e.g., minimum distance between plate and chamber, pressure, temperature, etc.). In some examples, for a gap of about 500 microns between plate 4703 and chamber 4715, the fluid flow rate can be about 10 L / min or about 13 milliliters / minute (mL / min) / millimeter (mm) along a circumferential velocity of about 0.42 meters / second (m / s). Barrier systems, methods, and apparatus that can be used with the open substrate systems of the present disclosure are described in U.S. Patent No. 10,512,911 and International Patent Application No. PCT / US19 / 64916, filed December 6, 2019, the entire contents of each of which are incorporated herein by reference.

[0325] The system can be temperature controlled. In some cases, the components of the system can be maintained at different temperatures. The temperature difference between the various components of the system can control the accumulation of condensation or precipitation on the various components of the system. The top portion of the container 3504 can be maintained in contact with the substrate 3502, the objective lens (e.g., Figure 15As shown), or a temperature different from the reservoir. Alternatively or additionally, the substrate can be maintained at a temperature different from the top portion of the container, the objective lens, or the reservoir. Alternatively or additionally, the reservoir can be maintained at a temperature different from the top portion of the container, the objective lens, or the substrate. Alternatively or additionally, the objective lens can be maintained at a temperature different from the top portion of the container, the reservoir, or the substrate. In some cases, the top portion of the container is maintained at a higher temperature than at least one other element in the system to prevent condensation from accumulating on the top surface of the container. In an exemplary configuration, the top portion of the container is maintained at the highest temperature, the substrate is maintained at the lowest temperature, and the reservoir and objective lens are maintained at an intermediate temperature, thereby preventing condensation from forming on the top portion of the container or preventing condensation from forming or dripping onto the objective lens. In another example, the objective lens is maintained at the highest temperature, the top portion of the container is maintained at an intermediate temperature, and the substrate and reservoir are maintained at a temperature lower than the top portion of the container, thereby preventing condensation from forming on the top portion of the container or preventing condensation from forming or dripping onto the objective lens. In some cases, the objective lens can be completely or partially surrounded by a seal. The seal can be configured to prevent moisture from the container surrounding the substrate (e.g., as shown). Figure 7 as shown) to other optical components in the system (e.g., as Figure 41 The seal may comprise a flexible material. The flexible seal may be configured to allow relative movement of various components of the system while maintaining a seal. In some embodiments, the flexible seal may be able to stretch, expand, or contract. For example, the flexible seal may be configured to allow independent movement of two or more imaging heads, as described with respect to FIG. Figure 29F-29G As described above. Alternatively or additionally, the seal may comprise a waterproof material. For example, the seal may be rubber, silicone, latex, plastic, Teflon, nitrile, elastin, an elastomer, or a polymer. The seal may surround the objective lens and contact the top portion of the container. In some cases, a portion of the objective lens, including the front lens, is not covered by the seal. The front lens of the objective lens may be exposed to the container surrounding the substrate. In some cases, the front lens of the objective lens may be in contact with the substrate fluid.

[0326] The system (or any element thereof) can be maintained at a relative humidity of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at most 100%, at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or a relative humidity within a range defined by any two of the foregoing values. The system (or any element thereof) can be configured such that less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the volume of the layer of solution dispensed on the uncovered surface evaporates.

[0327] The system (or any element thereof) can be contained in a sealed container, housing or chamber that isolates the system (or any element thereof) from the external environment, thereby allowing temperature or humidity to be controlled. An environmental unit (e.g., humidifier, heater, heat exchanger, compressor, etc.) can be configured to regulate one or more operating conditions in each environment. In some cases, each environment can be regulated by an independent environmental unit. In some cases, a single environmental unit can regulate multiple environments. In some cases, multiple environmental units can regulate different environments individually or together. The environmental unit can use active or passive methods to regulate operating conditions. For example, a heating or cooling element can be used to control temperature. A humidifier or dehumidifier can be used to control humidity. In some cases, a part of the internal environment in the container or chamber can be further controlled from other parts of the internal environment. Different parts can have different local temperatures, pressures and / or humidity. For example, the internal environment can include a first internal environment and a second internal environment separated by a seal.

[0328] Alternatively or in combination, the systems or methods described herein can include a solution comprising an agent that can reduce evaporation. For example, the solution can include glycerol, which can prevent the solution from evaporating.

[0329] In some cases, the seal can include an immersion objective, which is described in more detail elsewhere herein. For example, the immersion objective can be part of a seal that separates the internal environment in the container into a first internal environment having 100% (or substantially 100%) humidity and a second internal environment having one or more of ambient temperature, ambient pressure, or ambient humidity. The immersion objective can be in contact with one or more of the detector and the imaging lens.

[0330] Substrate preparation and anti-contamination substrates

[0331] As mentioned above, substrate can include surface, and this surface includes the multiple adhesives coupled with it.In some cases, multiple adhesives can include multiple nucleic acid molecules (for example, directly coupled to surface or indirectly coupled to multiple nucleic acid molecules on surface via multiple joints, as described herein). The surface (for example, substrate and / or particle of basic plane, including substrate with multiple particles fixed thereon) of oligonucleotide (for example, nucleic acid molecule) coating can be used for various applications, including for capturing the specific sequence of nucleic acid molecule for use in, for example, by hybridization capture (gene array) carries out gene expression analysis, single nucleotide polymorphism (SNP) genotyping, capture the subset (for example, targeted capture or exon group sequencing) of sequencing library, capture cDNA from mRNA synthesis by oligo-dT, and for downstream analysis such as amplification on the surface of the nucleic acid molecule of next generation sequencing. The surface of oligonucleotide coating can be prepared before it is used for any such application and can be stored between its generation and final use (for example, during transportation, sample handling and preparation etc. from manufacturing site to operating site). The surface of oligonucleotide coating can be stored for at least 1 hour, can be stored for several months or even several years in some cases. During storage, the surface of the oligonucleotide coating may contact with one or more solutions or other materials that may contain nucleic acid molecules, which may be considered as pollutants. Pollutant nucleic acid molecules may hybridize with the oligonucleotides coupled to the surface, causing the efficiency of downstream analysis to decrease (for example, during the application period for those described herein) and / or the erroneous results in the downstream analysis. For example, the surface of the oligonucleotide coating prepared for sequencing analysis may be contaminated by irrelevant sequencing libraries during the surface treatment period before it is used for sequencing analysis (for example, before the substrate comprising the surface is placed in a sequencing instrument or an amplification process such as a clonal amplification process).

[0332] Can reduce the non-correlated interactions of the oligonucleotide (for example, bonding) that is coupled to substrate surface by the oligonucleotide sealing of comprising the sequence that is fully or partially complementary to the sequence of the oligonucleotide that is attached to surface.Blocking oligonucleotide can provide in solution and can be considered to " free " oligonucleotide.For example, blocking oligonucleotide can hybridize fully or partially with all or subset of the oligonucleotide that is coupled to substrate surface, thereby provide the partial double-stranded nucleic acid molecule that comprises the oligonucleotide of bonding and blocking oligonucleotide.The nucleic acid molecule of this partial double-strand can resist the hybridization of the nucleic acid molecule that may contact with the surface, comprise the potential contaminant nucleic acid molecule that may be irrelevant with any final analysis such as final nucleic acid sequencing.Can remove blocking oligonucleotide (for example, by applying suitable stimulation, for example chemical or thermal stimulation, or by enzymatic degradation) to provide the surface (for example, as described herein) that can be prepared for the oligonucleotide coated of analytical process.Surface can experience one or more washing processes (for example, one or more washing streams) to remove blocking oligonucleotide. Removal of the blocking oligonucleotide can provide the oligonucleotide coupled to the surface as a free oligonucleotide that can participate in various reactions, including capture of complementary or partially complementary nucleic acid molecules of interest.

[0333] The surface of oligonucleotide coating can store and continue any useful time amount.For example, the surface of oligonucleotide coating can store at least 1 hour, for example at least 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer.The surface of oligonucleotide coating can store under any useful condition.For example, the surface of oligonucleotide coating can store under standard temperature and pressure condition (for example, room temperature), for example, between approximately 18 ℃ to approximately 30 ℃, for example, between approximately 20 ℃ to approximately 25 ℃, and approximately 1 atmospheric pressure, store.The surface of oligonucleotide coating can be stored in dry environment (for example, in air or be rich in the environment of nitrogen or argon) or solution (for example, buffer solution, as saline sodium citrate).

[0334] The surface of oligonucleotide coating can be stored in packaging or container, and this packaging or container can comprise the surface of one or more such oligonucleotide coatings.For example, a plurality of oligonucleotide coatings can be provided in given packaging or container. The packaging or container comprising the surface of one or more oligonucleotide coatings can be rigid packaging or container or flexible packaging or container.For example, the surface of one or more oligonucleotide coatings, for example, one or more substantially flat substrates comprising the surface of one or more oligonucleotide coatings, can be provided in flexible packaging. Packaging or container can comprise for example glass, plastic polymer, metal (for example, metal foil) or any other material or be formed by it. The packaging or container comprising the surface of one or more oligonucleotide coatings can be sealed (for example, airtight seal). The packaging or container comprising the surface of one or more oligonucleotide coatings can be resealed after opening. For example, the surface of the first oligonucleotide coating can be removed from packaging or container, and the surface of the second oligonucleotide coating can be retained in packaging or container. The surface of the oligonucleotide coating can also be configured for packaging or container external storage for a period of time, for example, at least about 1 hour, 2 hours, 6 hours or longer time (for example, as described herein).

[0335] The surface of oligonucleotide coating can be prepared in manufacturing and / or transporting place.Alternatively, the surface of oligonucleotide coating can be prepared by the user, the user of for example sequencing instrument.In some cases, the surface of oligonucleotide coating can be prepared in manufacturing and / or transporting place, this surface comprises a plurality of blocking oligonucleotides, these a plurality of blocking oligonucleotides and a plurality of oligonucleotide couplings (for example, hybridization) that are coupled to the surface of oligonucleotide coating.Alternatively, the surface of oligonucleotide coating can be prepared by the user of for example sequencing instrument, this surface comprises a plurality of blocking oligonucleotides, these a plurality of blocking oligonucleotides and a plurality of oligonucleotide couplings (for example, hybridization) that are coupled to the surface of oligonucleotide coating.Can be removed by the user a plurality of blocking oligonucleotides that are coupled to the surface of oligonucleotide coating.For example, before the user used the surface of oligonucleotide coating, can be removed by the user a plurality of blocking oligonucleotides (for example, as described herein, for example, for sequencing application) that are coupled to the surface of oligonucleotide coating.

[0336] For one or more applications, the surface of the oligonucleotide coating can use one or more.For example, the surface of the oligonucleotide coating can be configured for single use.Alternatively, the surface of the oligonucleotide coating can be configured to multiple use, for identical and / or different applications.For example, the oligonucleotide coupling to the surface can " resupply " for subsequent applications, or the surface can be cleaned and new oligonucleotide can be coupled to the surface for subsequent applications.In another example, the surface of the oligonucleotide coating can comprise one or more different regions, and this region comprises one or more different oligonucleotides (for example, adhesive) (for example, as described herein) coupled therewith.One or more different oligonucleotides can be configured to one or more different applications.In one example, the surface of the oligonucleotide coating comprises the first multiple oligonucleotide coupled with the first region and the second multiple oligonucleotide coupled with the second region, wherein the first multiple oligonucleotide and the second multiple oligonucleotide have different nucleotide sequences.The first multiple oligonucleotide can be configured to hybridize at least in part with the first multiple blocking oligonucleotide, and the second multiple oligonucleotide can be configured to hybridize at least in part with t...

Claims

1. A method for scanning a surface, the method comprising: (a) scanning a scan field comprising a portion of a surface using a scanning system, wherein the scan field has an orientation relative to an axis of rotation of the surface; and (b) (i) rotating the surface about the rotation axis of the surface and (ii) rotating the scanning field about the rotation axis of the scanning field, so that the scanning field substantially maintains its orientation relative to the rotation axis of the surface before, during or after the surface is translated relative to the scanning field.

2. A scanning system comprising: a surface configured to rotate about an axis of rotation of the surface; a detector in optical communication with the surface, wherein the detector has a scanning field that includes a first portion of the surface; and an illumination source configured to illuminate an illumination area including a second portion of the surface, wherein the illumination area and the scanning field at least partially overlap, The detector is configured to maintain an orientation of the scan field relative to the axis of rotation of the surface during (i) rotation of the surface about the axis of rotation and (ii) translation of the surface relative to the scan field.

3. A method for carrying out a reaction on a surface, comprising: (i) dispensing a first solution onto the surface in a first dispensing pattern while the surface is rotated about an axis of rotation, and (ii) dispensing a second solution onto the surface in a second dispensing pattern while the surface rotates about the axis of rotation.

4. A method for sequencing a nucleic acid molecule, the method comprising: (i) providing an array of nucleic acid molecules on an uncovered surface; (ii) dispensing a layer of a solution over the uncovered surface at a rate of at least 1 nanoliter per second when measured at a temperature of 25 degrees Celsius, wherein the solution comprises a reagent comprising at least one nucleotide that is incorporated into a growing nucleic acid chain that is complementary to a nucleic acid molecule of the array of nucleic acid molecules; and (iii) detecting one or more signals indicative of incorporation of the nucleotide into the growing nucleic acid chain.

5. A method for processing a plurality of nucleic acid samples, comprising: (i) providing the plurality of nucleic acid samples, wherein the plurality of nucleic acid samples comprises a first nucleic acid sample comprising a first group of nucleic acid molecules and a second nucleic acid sample comprising a second group of nucleic acid molecules, wherein each sample in the plurality of nucleic acid samples has an identifiable sample source; (ii) loading the first nucleic acid sample into a first region of a substrate as a first array of the first group of nucleic acid molecules, and loading the second nucleic acid sample into a second region of the substrate as a second array of the second group of nucleic acid molecules, wherein the first region is different from the second region; (iii) dispensing a solution across the substrate, wherein the solution comprises a reagent sufficient to react with the nucleic acid molecules of the first array or the second array; (iv) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array; and (v) analyzing the first nucleic acid sample and the second nucleic acid sample based at least in part on (a) the one or more signals and (b) the locations from the first region and the second region from which the one or more signals are detected, and (1) determining a first subset of the nucleic acid molecules in the first array or the second array as originating from the first nucleic acid sample and (2) determining a second subset of the nucleic acid molecules in the first array or the second array as originating from the second nucleic acid sample.

6. A method for processing a plurality of nucleic acid samples, comprising: (i) providing the plurality of nucleic acid samples, wherein the plurality of nucleic acid samples comprises a first nucleic acid sample comprising a first group of nucleic acid molecules and a second nucleic acid sample comprising a second group of nucleic acid molecules; (ii) loading the first nucleic acid sample onto a substrate to associate the first set of nucleic acid molecules with a first array of individually addressable locations; (iii) imaging the substrate to identify the first array of individually addressable locations; (iv) loading the second nucleic acid sample onto a substrate to associate the second set of nucleic acid molecules with a second array of individually addressable locations; (v) imaging the substrate to identify the second array of individually addressable locations; (vi) dispensing a solution across the substrate, wherein the solution comprises a reagent sufficient to react with the nucleic acid molecules of the first array or the second array; (vii) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecules of the first array or the second array; and (viii) analyzing the first nucleic acid sample and the second nucleic acid sample based at least in part on (a) the one or more signals and (b) the positions from the first array of individually addressable locations and the second array of individually addressable locations from which the one or more signals are detected, and (1) determining a first subset of the nucleic acid molecules in the first array or the second array as originating from the first nucleic acid sample and (2) determining a second subset of the nucleic acid molecules in the first array or the second array as originating from the second nucleic acid sample.

7. A method for processing a plurality of nucleic acid samples, wherein each of the plurality of nucleic acid samples comprises a fluorescent dye; (i) providing the plurality of nucleic acid samples, wherein each of the plurality of nucleic acid samples comprises a fluorescent dye; (ii) dividing the plurality of nucleic acid samples into a first group of one or more samples and a second group of one or more samples; (iii) loading the first set of one or more samples onto a first set of areas on a substrate, wherein there is one sample per area in the first set of areas; (iv) imaging the substrate to identify (a) locations within the first set of regions and (b) locations within a second set of regions on the substrate, wherein the second set of regions is different from the first set of regions associated with the first set of one or more samples; (v) loading the second set of one or more samples onto the second set of areas on the substrate, wherein there is one sample per area in the second set of areas; (vi) imaging the substrate to identify (a) locations within the first set of regions and (b) locations within the second set of regions to which the second set of one or more samples are associated; (vii) dispensing a solution across the substrate, wherein the solution comprises reagents sufficient to react with nucleic acid molecules of the first set of one or more samples or the second set of one or more samples; (viii) detecting one or more signals indicative of a reaction between the reagent and the nucleic acid molecule; and (ix) analyzing each of the plurality of nucleic acid samples based at least in part on (a) the one or more signals and (b) the locations from the first set of regions and the second set of regions from which the one or more signals are detected.

8. A method for processing a biological analyte, comprising: (i) moving a substrate through a reel or moving a substrate along a reel, wherein a surface of the substrate comprises an array having the biological analyte immobilized thereon; (ii) contacting the surface of the substrate with a reservoir comprising a solution, wherein the solution comprises a plurality of probes; (iii) subjecting the biological analyte to conditions sufficient to effect a reaction between a probe in the plurality of probes and the biological analyte to couple the probe to the biological analyte; and (iv) detecting one or more signals from the probes coupled to the biological analyte, thereby analyzing the biological analyte, wherein the substrate is moved in the same direction through or along the reel in at least two consecutive cycles of (ii) to (iv).

9. A system for analyzing a biological analyte, comprising: a substrate containing a biological analyte, wherein the substrate is maintained at or above a first temperature, the first temperature being above an ambient temperature of an environment to which the substrate is exposed; and an optical imaging objective in optical communication with the substrate and exposed to the environment, wherein the optical imaging objective is subjected to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, wherein the optical imaging objective comprises a first optical element and a second optical element adjacent to the first optical element, wherein the second optical element is disposed farther from the substrate than the first optical element, wherein the first optical element is configured to be at least partially immersed in an immersion fluid in contact with the substrate, wherein the second optical element is in optical communication with the substrate via the first optical element, and wherein the first optical element is configured such that a second temperature of the second optical element is maintained at or below a predetermined threshold.

10. A method for analyzing a biological analyte, comprising: (i) providing a substrate comprising a biological analyte, wherein the substrate is maintained at a first temperature that is above the ambient temperature of an environment to which the substrate is exposed; (ii) providing an optical imaging objective in optical communication with the substrate and exposed to an environment, wherein the optical imaging objective is subjected to a temperature gradient between the first temperature of the substrate and the ambient temperature of the environment, wherein the optical imaging objective comprises a first optical element and a second optical element adjacent to the first optical element, wherein the second optical element is disposed further from the substrate than the first optical element, and wherein the first optical element is at least partially immersed in an immersion fluid in contact with the substrate; (iii) controlling or maintaining a second temperature of the first optical element to adjust the magnitude or position of the temperature gradient through the optical imaging objective so that a third temperature gradient through the optical element remains below a predetermined threshold; and (iv) detecting one or more signals from the biological analyte using the optical imaging objective during movement of the substrate relative to the optical imaging objective.

Images